|
1
|
Eltayeb A, Rubio-Casillas A, Uversky VN, Redwan EM. Intrinsic Factors Behind Long COVID: VI. Combined Impact of G3BPs and SARS-CoV-2 Nucleocapsid Protein on the Viral Persistence and Long COVID. J Cell Biochem 2025; 126:e70038. [PMID: 40415285 DOI: 10.1002/jcb.70038] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/27/2025] [Revised: 04/27/2025] [Accepted: 05/11/2025] [Indexed: 05/27/2025]
Abstract
The efficient transmission of SARS-CoV-2 caused the COVID-19 pandemic, which affected millions of people around the globe. Despite extensive efforts, specific therapeutic interventions and preventive measures against COVID-19 and its consequences, such as long COVID, have not yet been identified due to the lack of a comprehensive knowledge of the SARS-CoV-2 biology. Therefore, a deeper understanding of the sophisticated strategies employed by SARS-CoV-2 to bypass the host antiviral defense systems is needed. One of these strategies is the inhibition of the Ras GTPase-activating protein-binding protein (GAP SH3-binding protein or G3BP)-dependent host immune response by the SARS-CoV-2 nucleocapsid (N) protein. This inhibition disrupts the formation of stress granules (SGs), which are crucial for antiviral defense. By preventing SG formation, the virus enhances its replication and evades the host's immune response, leading to increased disease severity. Given the involvement of G3BP1 in SG formation and its ability to interact with viral proteins, along with the crucial role of the N protein in the replication of the virus, we hypothesize that these proteins may have a potential role in the pathogenesis of long COVID. Despite the current lack of direct evidence linking these proteins to long COVID, their interactions and functions suggest a possible connection that warrants further investigation.
Collapse
Affiliation(s)
- Ahmed Eltayeb
- Department of Biological Sciences, Faculty of Science, King Abdulaziz University, Jeddah, Saudi Arabia
| | - Alberto Rubio-Casillas
- Autlan Regional Hospital, Jalisco Health Services, Autlan, Jalisco, Mexico
- Biology Laboratory, Autlan Regional Preparatory School, University of Guadalajara, Autlan, Jalisco, Mexico
| | - Vladimir N Uversky
- Department of Molecular Medicine and USF Health Byrd Alzheimer's Research Institute, Morsani College of Medicine, University of South Florida, Tampa, Florida, USA
| | - Elrashdy M Redwan
- Department of Biological Sciences, Faculty of Science, King Abdulaziz University, Jeddah, Saudi Arabia
- Centre of Excellence in Bionanoscience Research, King Abdulaziz University, Jeddah, Saudi Arabia
- Protein Research Department, Therapeutic and Protective Proteins Laboratory, Genetic Engineering and Biotechnology Research Institute, City for Scientific Research and Technology Applications, New Borg EL-Arab, Alexandria, Egypt
| |
Collapse
|
|
2
|
Ranjan A, Mattijssen S, Charlly N, Gallardo IC, Pitman L, Coleman J, Conte M, Maraia R. The short conserved region-2 of LARP4 interacts with ribosome-associated RACK1 and promotes translation. Nucleic Acids Res 2025; 53:gkaf053. [PMID: 39898547 PMCID: PMC11788930 DOI: 10.1093/nar/gkaf053] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/08/2024] [Revised: 01/14/2025] [Accepted: 01/22/2025] [Indexed: 02/04/2025] Open
Abstract
LARP4 interacts with poly(A)-binding protein (PABP) to protect messenger RNAs (mRNAs) from deadenylation and decay, and recent data indicate it can direct the translation of functionally related mRNA subsets. LARP4 was known to bind RACK1, a ribosome-associated protein, although the specific regions involved and relevance had been undetermined. Here, through a combination of in-cell and in vitro methodologies, we identified positions 615-625 in conserved region-2 (CR2) of LARP4 (and 646-656 in LARP4B) as directly binding RACK1. Consistent with these results, AlphaFold2-Multimer predicted high-confidence interaction of CR2 with RACK1 propellers 5 and 6. CR2 mutations strongly decreased LARP4 association with cellular RACK1 and ribosomes by multiple assays, whereas PABP association was less affected, consistent with independent interactions. The CR2 mutations decreased LARP4's ability to stabilize a β-globin mRNA reporter containing an AU-rich element (ARE) to higher degree than β-globin and GFP (green fluorescent protein) mRNAs lacking the ARE. We show LARP4 robustly increases translation of β-glo-ARE mRNA, whereas the LARP4 CR2 mutant is impaired. Analysis of nanoLuc-ARE mRNA for production of luciferase activity confirmed LARP4 promotes translation efficiency, while CR2 mutations are disabling. Thus, LARP4 CR2-mediated interaction with RACK1 can promote translational efficiency of some mRNAs.
Collapse
Affiliation(s)
- Amitabh Ranjan
- Section on Molecular and Cellular Biology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, 20892, United States
| | - Sandy Mattijssen
- Section on Molecular and Cellular Biology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, 20892, United States
| | - Nithin Charlly
- Section on Molecular and Cellular Biology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, 20892, United States
| | - Isabel Cruz Gallardo
- Randall Centre for Cell and Molecular Biophysics, King’s College London, London SE1 1UL, United Kingdom
| | - Leah F Pitman
- Section on Molecular and Cellular Biology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, 20892, United States
- Messenger RNA Regulation and Decay Section, Center for Cancer Research, National Cancer Institute, Frederick, MD, United States 21702
| | - Jennifer C Coleman
- Randall Centre for Cell and Molecular Biophysics, King’s College London, London SE1 1UL, United Kingdom
| | - Maria R Conte
- Randall Centre for Cell and Molecular Biophysics, King’s College London, London SE1 1UL, United Kingdom
| | - Richard J Maraia
- Section on Molecular and Cellular Biology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, 20892, United States
| |
Collapse
|
|
3
|
Ranjan A, Mattijssen S, Charlly N, Gallardo IC, Pitman LF, Coleman JC, Conte MR, Maraia RJ. The short conserved region-2 of LARP4 interacts with ribosome-associated RACK1 and promotes translation. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2024:2024.11.01.621267. [PMID: 39554137 PMCID: PMC11565960 DOI: 10.1101/2024.11.01.621267] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/19/2024]
Abstract
LARP4 interacts with poly(A)-binding protein (PABP) to protect mRNAs from deadenylation and decay, and recent data indicate it can direct the translation of functionally related mRNA subsets. LARP4 was known to bind RACK1, a ribosome-associated protein, although the specific regions involved, and relevance had been undetermined. Here, yeast two-hybrid domain mapping followed by other methods identified positions 615-625 in conserved region-2 (CR2) of LARP4 (and LARP4B) as directly binding RACK1 region 200-317. Consistent with these results, AlphaFold2-multimer predicted high confidence interaction of CR2 with RACK1 propellers 5-6. CR2 mutations strongly decreased LARP4 association with cellular RACK1 and ribosomes by multiple assays, whereas less effect was observed for PABP association, consistent with independent interactions. CR2 mutations decreased LARP4 ability to optimally stabilize a β-globin mRNA reporter containing an AU-rich element (ARE) more significantly than a β-globin and other reporters lacking this element. While polysome profiles indicate the β-glo-ARE mRNA is inefficiently translated, consistent with published data, we show that LARP4 increases its translation whereas the LARP4-CR2 mutant is impaired. Analysis of nanoLuc-ARE mRNA for production of luciferase activity confirmed LARP4 promotes translation efficiency while CR2 mutations are disabling. Thus, LARP4 CR2-mediated interaction with RACK1 can promote translational efficiency of some mRNAs.
Collapse
Affiliation(s)
- Amitabh Ranjan
- Section on Molecular and Cellular Biology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD
| | - Sandy Mattijssen
- Section on Molecular and Cellular Biology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD
| | - Nithin Charlly
- Section on Molecular and Cellular Biology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD
| | - Isabel Cruz Gallardo
- Randall Centre for Cell and Molecular Biophysics, King’s College London, London SE1 1UL, UK
| | - Leah F. Pitman
- Section on Molecular and Cellular Biology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD
- Messenger RNA Regulation and Decay Section, Center for Cancer Research, National Cancer Institute, Frederick, MD, United States
| | - Jennifer C. Coleman
- Randall Centre for Cell and Molecular Biophysics, King’s College London, London SE1 1UL, UK
| | - Maria R. Conte
- Randall Centre for Cell and Molecular Biophysics, King’s College London, London SE1 1UL, UK
| | - Richard J. Maraia
- Section on Molecular and Cellular Biology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD
| |
Collapse
|
|
4
|
Proteostasis Deregulation in Neurodegeneration and Its Link with Stress Granules: Focus on the Scaffold and Ribosomal Protein RACK1. Cells 2022; 11:cells11162590. [PMID: 36010666 PMCID: PMC9406587 DOI: 10.3390/cells11162590] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/21/2022] [Revised: 08/09/2022] [Accepted: 08/17/2022] [Indexed: 12/12/2022] Open
Abstract
The role of protein misfolding, deposition, and clearance has been the dominant topic in the last decades of investigation in the field of neurodegeneration. The impairment of protein synthesis, along with RNA metabolism and RNA granules, however, are significantly emerging as novel potential targets for the comprehension of the molecular events leading to neuronal deficits. Indeed, defects in ribosome activity, ribosome stalling, and PQC—all ribosome-related processes required for proteostasis regulation—can contribute to triggering stress conditions and promoting the formation of stress granules (SGs) that could evolve in the formation of pathological granules, usually occurring during neurodegenerating effects. In this review, the interplay between proteostasis, mRNA metabolism, and SGs has been explored in a neurodegenerative context with a focus on Alzheimer’s disease (AD), although some defects in these same mechanisms can also be found in frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS), which are discussed here. Finally, we highlight the role of the receptor for activated C kinase 1 (RACK1) in these pathologies and note that, besides its well characterized function as a scaffold protein, it has an important role in translation and can associate to stress granules (SGs) determining cell fate in response to diverse stress stimuli.
Collapse
|
|
5
|
O'Reilly CL, Uranga S, Fluckey JD. Culprits or consequences: Understanding the metabolic dysregulation of muscle in diabetes. World J Biol Chem 2021; 12:70-86. [PMID: 34630911 PMCID: PMC8473417 DOI: 10.4331/wjbc.v12.i5.70] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/31/2021] [Revised: 06/21/2021] [Accepted: 08/03/2021] [Indexed: 02/06/2023] Open
Abstract
The prevalence of type 2 diabetes (T2D) continues to rise despite the amount of research dedicated to finding the culprits of this debilitating disease. Skeletal muscle is arguably the most important contributor to glucose disposal making it a clear target in insulin resistance and T2D research. Within skeletal muscle there is a clear link to metabolic dysregulation during the progression of T2D but the determination of culprits vs consequences of the disease has been elusive. Emerging evidence in skeletal muscle implicates influential cross talk between a key anabolic regulatory protein, the mammalian target of rapamycin (mTOR) and its associated complexes (mTORC1 and mTORC2), and the well-described canonical signaling for insulin-stimulated glucose uptake. This new understanding of cellular signaling crosstalk has blurred the lines of what is a culprit and what is a consequence with regard to insulin resistance. Here, we briefly review the most recent understanding of insulin signaling in skeletal muscle, and how anabolic responses favoring anabolism directly impact cellular glucose disposal. This review highlights key cross-over interactions between protein and glucose regulatory pathways and the implications this may have for the design of new therapeutic targets for the control of glucoregulatory function in skeletal muscle.
Collapse
Affiliation(s)
| | - Selina Uranga
- Health and Kinesiology, Texas A&M University, TX 77843, United States
| | - James D Fluckey
- Health and Kinesiology, Texas A&M University, TX 77843, United States
| |
Collapse
|
|
6
|
Rasputin a decade on and more promiscuous than ever? A review of G3BPs. BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR CELL RESEARCH 2018; 1866:360-370. [PMID: 30595162 PMCID: PMC7114234 DOI: 10.1016/j.bbamcr.2018.09.001] [Citation(s) in RCA: 48] [Impact Index Per Article: 6.9] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 05/24/2018] [Revised: 08/29/2018] [Accepted: 09/04/2018] [Indexed: 12/12/2022]
Abstract
Ras-GTPase-activating protein (SH3 domain)-binding proteins (G3BPs, also known as Rasputin) are a family of RNA binding proteins that regulate gene expression in response to environmental stresses by controlling mRNA stability and translation. G3BPs appear to facilitate this activity through their role in stress granules for which they are considered a core component, however, it should be noted that not all stress granules contain G3BPs and this appears to be contextual depending on the environmental stress and the cell type. Although the role of G3BPs in stress granules appears to be one of its major roles, data also strongly suggests that they interact with mRNAs outside of stress granules to regulate gene expression. G3BPs have been implicated in several diseases including cancer progression, invasion, and metastasis as well as virus survival. There is now a body of evidence that suggests targeting of G3BPs could be explored as a form of cancer therapeutic. This review discusses the important discoveries and advancements made in the field of G3BPs biology over the last two decades including their roles in RNA stability, translational control of cellular transcripts, stress granule formation, cancer progression and its interactions with viruses during infection. An emerging theme for G3BPs is their ability to regulate gene expression in response to environmental stimuli, disease progression and virus infection making it an intriguing target for disease therapies.
Triage of many cellular mRNA occurs via stress granules in a G3BP-dependant manner. G3BPs control intra cellular responses to viral infection. Transcript stability, degradation and translation are controlled by G3BPs. G3BPs can control cancer progression.
Collapse
|
|
7
|
Synaptic Paths to Neurodegeneration: The Emerging Role of TDP-43 and FUS in Synaptic Functions. Neural Plast 2018; 2018:8413496. [PMID: 29755516 PMCID: PMC5925147 DOI: 10.1155/2018/8413496] [Citation(s) in RCA: 51] [Impact Index Per Article: 7.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/15/2017] [Revised: 02/08/2018] [Accepted: 02/27/2018] [Indexed: 12/13/2022] Open
Abstract
TAR DNA-binding protein-43 KDa (TDP-43) and fused in sarcoma (FUS) as the defining pathological hallmarks for amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), coupled with ALS-FTD-causing mutations in both genes, indicate that their dysfunctions damage the motor system and cognition. On the molecular level, TDP-43 and FUS participate in the biogenesis and metabolism of coding and noncoding RNAs as well as in the transport and translation of mRNAs as part of cytoplasmic mRNA-ribonucleoprotein (mRNP) granules. Intriguingly, many of the RNA targets of TDP-43 and FUS are involved in synaptic transmission and plasticity, indicating that synaptic dysfunction could be an early event contributing to motor and cognitive deficits in ALS and FTD. Furthermore, the ability of the low-complexity prion-like domains of TDP-43 and FUS to form liquid droplets suggests a potential mechanism for mRNP assembly and conversion. This review will discuss the role of TDP-43 and FUS in RNA metabolism, with an emphasis on the involvement of this process in synaptic function and neuroprotection. This will be followed by a discussion of the potential phase separation mechanism for forming RNP granules and pathological inclusions.
Collapse
|
|
8
|
Opitz N, Schmitt K, Hofer-Pretz V, Neumann B, Krebber H, Braus GH, Valerius O. Capturing the Asc1p/ Receptor for Activated C Kinase 1 (RACK1) Microenvironment at the Head Region of the 40S Ribosome with Quantitative BioID in Yeast. Mol Cell Proteomics 2017; 16:2199-2218. [PMID: 28982715 DOI: 10.1074/mcp.m116.066654] [Citation(s) in RCA: 56] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/21/2016] [Revised: 09/29/2017] [Indexed: 12/13/2022] Open
Abstract
The Asc1 protein of Saccharomyces cerevisiae is a scaffold protein at the head region of ribosomal 40S that links mRNA translation to cellular signaling. In this study, proteins that colocalize with Asc1p were identified with proximity-dependent Biotin IDentification (BioID), an in vivo labeling technique described here for the first time for yeast. Biotinylated Asc1p-birA*-proximal proteins were identified and quantitatively verified against controls applying SILAC and mass spectrometry. The mRNA-binding proteins Sro9p and Gis2p appeared together with Scp160p, each providing ribosomes with nuclear transcripts. The cap-binding protein eIF4E (Cdc33p) and the eIF3/a-subunit (Rpg1p) were identified reflecting the encounter of proteins involved in the initiation of mRNA translation at the head region of ribosomal 40S. Unexpectedly, a protein involved in ribosome preservation (the clamping factor Stm1p), the deubiquitylation complex Ubp3p-Bre5p, the RNA polymerase II degradation factor 1 (Def1p), and transcription factors (Spt5p, Mbf1p) colocalize with Asc1p in exponentially growing cells. For Asc1R38D, K40Ep, a variant considered to be deficient in binding to ribosomes, BioID revealed its predominant ribosome localization. Glucose depletion replaced most of the Asc1p colocalizing proteins for additional ribosomal proteins, suggesting a ribosome aggregation process during early nutrient limitation, possibly concomitant with ribosomal subunit clamping. Overall, the characterization of the Asc1p microenvironment with BioID confirmed and substantiated our recent findings that the β-propeller broadly contributes to signal transduction influencing phosphorylation of colocalizing proteins (e.g. of Bre5p), and by that might affect nuclear gene transcription and the fate of ribosomes.
Collapse
Affiliation(s)
- Nadine Opitz
- From the ‡Department of Molecular Microbiology and Genetics, Institute of Microbiology and Genetics, Göttingen Center for Molecular Biosciences (GZMB), Georg-August-University Göttingen, 37077 Göttingen, Germany
| | - Kerstin Schmitt
- From the ‡Department of Molecular Microbiology and Genetics, Institute of Microbiology and Genetics, Göttingen Center for Molecular Biosciences (GZMB), Georg-August-University Göttingen, 37077 Göttingen, Germany
| | - Verena Hofer-Pretz
- From the ‡Department of Molecular Microbiology and Genetics, Institute of Microbiology and Genetics, Göttingen Center for Molecular Biosciences (GZMB), Georg-August-University Göttingen, 37077 Göttingen, Germany
| | - Bettina Neumann
- §Department of Molecular Genetics, Institute of Microbiology and Genetics, GZMB, Georg-August-University Göttingen, 37077 Göttingen, Germany
| | - Heike Krebber
- §Department of Molecular Genetics, Institute of Microbiology and Genetics, GZMB, Georg-August-University Göttingen, 37077 Göttingen, Germany
| | - Gerhard H Braus
- From the ‡Department of Molecular Microbiology and Genetics, Institute of Microbiology and Genetics, Göttingen Center for Molecular Biosciences (GZMB), Georg-August-University Göttingen, 37077 Göttingen, Germany
| | - Oliver Valerius
- From the ‡Department of Molecular Microbiology and Genetics, Institute of Microbiology and Genetics, Göttingen Center for Molecular Biosciences (GZMB), Georg-August-University Göttingen, 37077 Göttingen, Germany;
| |
Collapse
|
|
9
|
Abrakhi S, Kretov DA, Desforges B, Dobra I, Bouhss A, Pastré D, Hamon L. Nanoscale Analysis Reveals the Maturation of Neurodegeneration-Associated Protein Aggregates: Grown in mRNA Granules then Released by Stress Granule Proteins. ACS NANO 2017; 11:7189-7200. [PMID: 28657719 DOI: 10.1021/acsnano.7b03071] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/27/2023]
Abstract
TDP-43 and FUS are two mRNA-binding proteins associated with neurodegenerative diseases that form cytoplasmic inclusions with prion-like properties in affected neurons. Documenting the early stages of the formation of TDP-43 or FUS protein aggregates and the role of mRNA stress granules that are considered as critical intermediates for protein aggregation is therefore of interest to understand disease propagation. Here, we developed a single molecule approach via atomic force microscopy (AFM), which provides structural information out of reach by fluorescence microscopy. In addition, the aggregation process can be probed in the test tube without separating the interacting partners, which would affect the thermodynamic equilibrium. The results demonstrate that isolated mRNA molecules serve as crucibles to promote TDP-43 and FUS multimerization. Their subsequent merging results in the formation of mRNA granules containing TDP-43 and FUS aggregates. Interestingly, TDP-43 or FUS protein aggregates can be released from mRNA granules by either YB-1 or G3BP1, two stress granule proteins that compete for the binding to mRNA with TDP-43 and FUS. Altogether, the results indicate that age-related successive assembly/disassembly of stress granules in neurons, regulated by mRNA-binding proteins such as YB-1 and G3BP1, could be a source of protein aggregation.
Collapse
Affiliation(s)
- Sanae Abrakhi
- SABNP, Univ Evry, INSERM U1204, Université Paris-Saclay , 91025 Evry, France
| | - Dmitry A Kretov
- SABNP, Univ Evry, INSERM U1204, Université Paris-Saclay , 91025 Evry, France
- Institute of Protein Research, Russian Academy of Sciences , Pushchino, Moscow Region 142290, Russia
| | - Bénédicte Desforges
- SABNP, Univ Evry, INSERM U1204, Université Paris-Saclay , 91025 Evry, France
| | - Ioana Dobra
- SABNP, Univ Evry, INSERM U1204, Université Paris-Saclay , 91025 Evry, France
| | - Ahmed Bouhss
- SABNP, Univ Evry, INSERM U1204, Université Paris-Saclay , 91025 Evry, France
| | - David Pastré
- SABNP, Univ Evry, INSERM U1204, Université Paris-Saclay , 91025 Evry, France
| | - Loic Hamon
- SABNP, Univ Evry, INSERM U1204, Université Paris-Saclay , 91025 Evry, France
| |
Collapse
|
|
10
|
Kershner L, Welshhans K. RACK1 is necessary for the formation of point contacts and regulates axon growth. Dev Neurobiol 2017; 77:1038-1056. [PMID: 28245531 DOI: 10.1002/dneu.22491] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/27/2016] [Revised: 02/17/2017] [Accepted: 02/19/2017] [Indexed: 11/08/2022]
Abstract
Receptor for activated C kinase 1 (RACK1) is a multifunctional ribosomal scaffolding protein that can interact with multiple signaling molecules concurrently through its seven WD40 repeats. We recently found that RACK1 is localized to mammalian growth cones, prompting an investigation into its role during neural development. Here, we show for the first time that RACK1 localizes to point contacts within mouse cortical growth cones. Point contacts are adhesion sites that link the actin network within growth cones to the extracellular matrix, and are necessary for appropriate axon guidance. Our experiments show that RACK1 is necessary for point contact formation. Brain-derived neurotrophic factor (BDNF) stimulates an increase in point contact density, which was eliminated by RACK1 shRNA or overexpression of a nonphosphorylatable mutant form of RACK1. We also found that axonal growth requires both RACK1 expression and phosphorylation. We have previously shown that the local translation of β-actin mRNA within growth cones is necessary for appropriate axon guidance and is dependent on RACK1. Thus, we examined the location of members of the local translation complex relative to point contacts. Indeed, both β-actin mRNA and RACK1 colocalize with point contacts, and this colocalization increases following BDNF stimulation. This implies the novel finding that local translation is regulated at point contacts. Taken together, these data suggest that point contacts are a targeted site of local translation within growth cones, and RACK1 is a critical member of the point contact complex and necessary for appropriate neural development. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 77: 1038-1056, 2017.
Collapse
Affiliation(s)
- Leah Kershner
- Department of Biological Sciences, Kent State University, Kent, Ohio, 44242
| | - Kristy Welshhans
- Department of Biological Sciences, Kent State University, Kent, Ohio, 44242.,School of Biomedical Sciences, Kent State University, Kent, Ohio, 44242
| |
Collapse
|
|
11
|
Gallo S, Manfrini N. Working hard at the nexus between cell signaling and the ribosomal machinery: An insight into the roles of RACK1 in translational regulation. ACTA ACUST UNITED AC 2015; 3:e1120382. [PMID: 26824030 DOI: 10.1080/21690731.2015.1120382] [Citation(s) in RCA: 21] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/20/2015] [Revised: 10/19/2015] [Accepted: 11/09/2015] [Indexed: 02/08/2023]
Abstract
RACK1 is a ribosome-associated protein which functions as a receptor for activated PKCs. It also acts as a scaffold for many other proteins involved in diverse signaling pathways, e.g. Src, JNK, PDE4D and FAK signaling. With such a broad interactome, RACK1 has been suggested to function as a linker between cell signaling and the translation machinery. Accordingly, RACK1 modulates translation at different levels in several model organisms. For instance, it regulates ribosome stalling and mRNA quality control in yeasts and promotes translation efficiency downstream of specific cellular stimuli in mammals. However, the molecular mechanism by which RACK1 exerts these roles is widely uncharacterized. Moreover, the full list of ribosome-recruited RACK1 interactors still needs characterization. Here we discuss in vivo and in vitro findings to better delineate the roles of RACK1 in regulating ribosome function and translation.
Collapse
Affiliation(s)
- Simone Gallo
- Molecular Histology and Cell Growth Unit; National Institute of Molecular Genetics - INGM "Romeo and Enrica Invernizzi" ; Milan, Italy
| | - Nicola Manfrini
- Molecular Histology and Cell Growth Unit; National Institute of Molecular Genetics - INGM "Romeo and Enrica Invernizzi" ; Milan, Italy
| |
Collapse
|
|
12
|
Stavraka C, Blagden S. The La-Related Proteins, a Family with Connections to Cancer. Biomolecules 2015; 5:2701-22. [PMID: 26501340 PMCID: PMC4693254 DOI: 10.3390/biom5042701] [Citation(s) in RCA: 93] [Impact Index Per Article: 9.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/06/2015] [Revised: 09/21/2015] [Accepted: 10/07/2015] [Indexed: 01/09/2023] Open
Abstract
The evolutionarily-conserved La-related protein (LARP) family currently comprises Genuine La, LARP1, LARP1b, LARP4, LARP4b, LARP6 and LARP7. Emerging evidence suggests each LARP has a distinct role in transcription and/or mRNA translation that is attributable to subtle sequence variations within their La modules and specific C-terminal domains. As emerging research uncovers the function of each LARP, it is evident that La, LARP1, LARP6, LARP7 and possibly LARP4a and 4b are dysregulated in cancer. Of these, LARP1 is the first to be demonstrated to drive oncogenesis. Here, we review the role of each LARP and the evidence linking it to malignancy. We discuss a future strategy of targeting members of this protein family as cancer therapy.
Collapse
Affiliation(s)
- Chara Stavraka
- Ovarian Cancer Research Centre, Institute for Reproductive and Developmental Biology, Imperial College, Du Cane Road, London W12 0HS, UK.
| | - Sarah Blagden
- Ovarian Cancer Research Centre, Institute for Reproductive and Developmental Biology, Imperial College, Du Cane Road, London W12 0HS, UK.
- Department of Oncology, University of Oxford, Churchill Hospital, Old Road, Oxford OX3 7LE, UK.
| |
Collapse
|
|
13
|
Küspert M, Murakawa Y, Schäffler K, Vanselow JT, Wolf E, Juranek S, Schlosser A, Landthaler M, Fischer U. LARP4B is an AU-rich sequence associated factor that promotes mRNA accumulation and translation. RNA (NEW YORK, N.Y.) 2015; 21:1294-305. [PMID: 26001795 PMCID: PMC4478348 DOI: 10.1261/rna.051441.115] [Citation(s) in RCA: 39] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 02/18/2015] [Accepted: 04/02/2015] [Indexed: 05/23/2023]
Abstract
mRNAs are key molecules in gene expression and subject to diverse regulatory events. Regulation is accomplished by distinct sets of trans-acting factors that interact with mRNAs and form defined mRNA-protein complexes (mRNPs). The resulting "mRNP code" determines the fate of any given mRNA and thus controlling gene expression at the post-transcriptional level. The La-related protein 4B (LARP4B) belongs to an evolutionarily conserved family of RNA-binding proteins characterized by the presence of a La-module implicated in direct RNA binding. Biochemical experiments have shown previously direct interactions of LARP4B with factors of the translation machinery. This finding along with the observation of an association with actively translating ribosomes suggested that LARP4B is a factor contributing to the mRNP code. To gain insight into the function of LARP4B in vivo we tested its mRNA association at the transcriptome level and its impact on the proteome. PAR-CLIP analyses allowed us to identify the in vivo RNA targets of LARP4B. We show that LARP4B binds to a distinct set of cellular mRNAs by contacting their 3' UTRs. Biocomputational analysis combined with in vitro binding assays identified the LARP4B-binding motif on mRNA targets. The reduction of cellular LARP4B levels leads to a marked destabilization of its mRNA targets and consequently their reduced translation. Our data identify LARP4B as a component of the mRNP code that influences the expression of its mRNA targets by affecting their stability.
Collapse
Affiliation(s)
- Maritta Küspert
- Biozentrum Universität Würzburg, Am Hubland, D-97074 Würzburg, Germany
| | | | - Katrin Schäffler
- Biozentrum Universität Würzburg, Am Hubland, D-97074 Würzburg, Germany
| | - Jens T Vanselow
- Rudolf-Virchow-Zentrum für Experimentelle Biomedizin, Universität Würzburg, D-97080 Würzburg, Germany
| | - Elmar Wolf
- Biozentrum Universität Würzburg, Am Hubland, D-97074 Würzburg, Germany
| | - Stefan Juranek
- Biozentrum Universität Würzburg, Am Hubland, D-97074 Würzburg, Germany
| | - Andreas Schlosser
- Rudolf-Virchow-Zentrum für Experimentelle Biomedizin, Universität Würzburg, D-97080 Würzburg, Germany
| | | | - Utz Fischer
- Biozentrum Universität Würzburg, Am Hubland, D-97074 Würzburg, Germany Rudolf-Virchow-Zentrum für Experimentelle Biomedizin, Universität Würzburg, D-97080 Würzburg, Germany Department of Radiation Medicine and Applied Sciences, University of California at San Diego, San Diego, California 92037, USA
| |
Collapse
|
|
14
|
Smith R, Rathod RJ, Rajkumar S, Kennedy D. Nervous translation, do you get the message? A review of mRNPs, mRNA-protein interactions and translational control within cells of the nervous system. Cell Mol Life Sci 2014; 71:3917-37. [PMID: 24952431 PMCID: PMC11113408 DOI: 10.1007/s00018-014-1660-x] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/14/2014] [Revised: 05/22/2014] [Accepted: 05/30/2014] [Indexed: 01/01/2023]
Abstract
In neurons, translation of a message RNA can occur metres away from its transcriptional origin and in normal cells this is orchestrated with perfection. The life of an mRNA will see it pass through multiple steps of processing in the nucleus and the cytoplasm before it reaches its final destination. Processing of mRNA is determined by a myriad of RNA-binding proteins in multi-protein complexes called messenger ribonucleoproteins; however, incorrect processing and delivery of mRNA can cause several human neurological disorders. This review takes us through the life of mRNA from the nucleus to its point of translation in the cytoplasm. The review looks at the various cis and trans factors that act on the mRNA and discusses their roles in different cells of the nervous system and human disorders.
Collapse
Affiliation(s)
- Ross Smith
- School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, QLD, 4072, Australia,
| | | | | | | |
Collapse
|
|
15
|
Kimura S, Sakakibara Y, Sato K, Ote M, Ito H, Koganezawa M, Yamamoto D. TheDrosophilalingerer protein cooperates with Orb2 in long-term memory formation. J Neurogenet 2014; 29:8-17. [DOI: 10.3109/01677063.2014.917644] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022]
|
|
16
|
Proteome analysis of the HIV-1 Gag interactome. Virology 2014; 460-461:194-206. [PMID: 25010285 DOI: 10.1016/j.virol.2014.04.038] [Citation(s) in RCA: 43] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/20/2014] [Revised: 02/06/2014] [Accepted: 04/19/2014] [Indexed: 11/22/2022]
Abstract
Human immunodeficiency virus Gag drives assembly of virions in infected cells and interacts with host factors which facilitate or restrict viral replication. Although several Gag-binding proteins have been characterized, understanding of virus-host interactions remains incomplete. In a series of six affinity purification screens, we have identified protein candidates for interaction with HIV-1 Gag. Proteins previously found in virions or identified in siRNA screens for host factors influencing HIV-1 replication were recovered. Helicases, translation factors, cytoskeletal and motor proteins, factors involved in RNA degradation and RNA interference were enriched in the interaction data. Cellular networks of cytoskeleton, SR proteins and tRNA synthetases were identified. Most prominently, components of cytoplasmic RNA transport granules were co-purified with Gag. This study provides a survey of known Gag-host interactions and identifies novel Gag binding candidates. These factors are associated with distinct molecular functions and cellular pathways relevant in host-pathogen interactions.
Collapse
|
|
17
|
Volta V, Beugnet A, Gallo S, Magri L, Brina D, Pesce E, Calamita P, Sanvito F, Biffo S. RACK1 depletion in a mouse model causes lethality, pigmentation deficits and reduction in protein synthesis efficiency. Cell Mol Life Sci 2013; 70:1439-50. [PMID: 23212600 PMCID: PMC11113757 DOI: 10.1007/s00018-012-1215-y] [Citation(s) in RCA: 59] [Impact Index Per Article: 4.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/12/2012] [Revised: 11/02/2012] [Accepted: 11/08/2012] [Indexed: 10/27/2022]
Abstract
The receptor for activated C-kinase 1 (RACK1) is a conserved structural protein of 40S ribosomes. Strikingly, deletion of RACK1 in yeast homolog Asc1 is not lethal. Mammalian RACK1 also interacts with many nonribosomal proteins, hinting at several extraribosomal functions. A knockout mouse for RACK1 has not previously been described. We produced the first RACK1 mutant mouse, in which both alleles of RACK1 gene are defective in RACK1 expression (ΔF/ΔF), in a pure C57 Black/6 background. In a sample of 287 pups, we observed no ΔF/ΔF mice (72 expected). Dissection and genotyping of embryos at various stages showed that lethality occurs at gastrulation. Heterozygotes (ΔF/+) have skin pigmentation defects with a white belly spot and hypopigmented tail and paws. ΔF/+ have a transient growth deficit (shown by measuring pup size at P11). The pigmentation deficit is partly reverted by p53 deletion, whereas the lethality is not. ΔF/+ livers have mild accumulation of inactive 80S ribosomal subunits by polysomal profile analysis. In ΔF/+ fibroblasts, protein synthesis response to extracellular and pharmacological stimuli is reduced. These results highlight the role of RACK1 as a ribosomal protein converging signaling to the translational apparatus.
Collapse
Affiliation(s)
- Viviana Volta
- Laboratory of Molecular Histology and Cell Growth, Division of Oncology, San Raffaele Scientific Institute, Via Olgettina 58, 20132 Milan, Italy
| | - Anne Beugnet
- Laboratory of Molecular Histology and Cell Growth, Division of Oncology, San Raffaele Scientific Institute, Via Olgettina 58, 20132 Milan, Italy
| | - Simone Gallo
- Laboratory of Molecular Histology and Cell Growth, Division of Oncology, San Raffaele Scientific Institute, Via Olgettina 58, 20132 Milan, Italy
| | - Laura Magri
- Laboratory of Molecular Histology and Cell Growth, Division of Oncology, San Raffaele Scientific Institute, Via Olgettina 58, 20132 Milan, Italy
| | - Daniela Brina
- Laboratory of Molecular Histology and Cell Growth, Division of Oncology, San Raffaele Scientific Institute, Via Olgettina 58, 20132 Milan, Italy
| | - Elisa Pesce
- Laboratory of Molecular Histology and Cell Growth, Division of Oncology, San Raffaele Scientific Institute, Via Olgettina 58, 20132 Milan, Italy
- Environmental and Life Science Department (DISAV), University of Eastern Piedmont, Alessandria, Italy
| | - Piera Calamita
- Laboratory of Molecular Histology and Cell Growth, Division of Oncology, San Raffaele Scientific Institute, Via Olgettina 58, 20132 Milan, Italy
| | - Francesca Sanvito
- Department of Pathology, San Raffaele Scientific Institute, Milan, Italy
| | - Stefano Biffo
- Laboratory of Molecular Histology and Cell Growth, Division of Oncology, San Raffaele Scientific Institute, Via Olgettina 58, 20132 Milan, Italy
- Environmental and Life Science Department (DISAV), University of Eastern Piedmont, Alessandria, Italy
| |
Collapse
|
|
18
|
Das A, Morales R, Banday M, Garcia S, Hao L, Cross GA, Estevez AM, Bellofatto V. The essential polysome-associated RNA-binding protein RBP42 targets mRNAs involved in Trypanosoma brucei energy metabolism. RNA (NEW YORK, N.Y.) 2012; 18:1968-1983. [PMID: 22966087 PMCID: PMC3479388 DOI: 10.1261/rna.033829.112] [Citation(s) in RCA: 41] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 04/12/2012] [Accepted: 08/02/2012] [Indexed: 05/29/2023]
Abstract
RNA-binding proteins that target mRNA coding regions are emerging as regulators of post-transcriptional processes in eukaryotes. Here we describe a newly identified RNA-binding protein, RBP42, which targets the coding region of mRNAs in the insect form of the African trypanosome, Trypanosoma brucei. RBP42 is an essential protein and associates with polysome-bound mRNAs in the cytoplasm. A global survey of RBP42-bound mRNAs was performed by applying HITS-CLIP technology, which captures protein-RNA interactions in vivo using UV light. Specific RBP42-mRNA interactions, as well as mRNA interactions with a known RNA-binding protein, were purified using specific antibodies. Target RNA sequences were identified and quantified using high-throughput RNA sequencing. Analysis revealed that RBP42 bound mainly within the coding region of mRNAs that encode proteins involved in cellular energy metabolism. Although the mechanism of RBP42's function is unclear at present, we speculate that RBP42 plays a critical role in modulating T. brucei energy metabolism.
Collapse
Affiliation(s)
- Anish Das
- Department of Microbiology and Molecular Genetics, UMDNJ-NJ Medical School, Newark, New Jersey 07103, USA
| | - Rachel Morales
- Department of Microbiology and Molecular Genetics, UMDNJ-NJ Medical School, Newark, New Jersey 07103, USA
| | - Mahrukh Banday
- Department of Microbiology and Molecular Genetics, UMDNJ-NJ Medical School, Newark, New Jersey 07103, USA
| | - Stacey Garcia
- Department of Microbiology and Molecular Genetics, UMDNJ-NJ Medical School, Newark, New Jersey 07103, USA
| | - Li Hao
- Center for Genome Informatics, UMDNJ-NJ Medical School, Newark, New Jersey 07103, USA
| | | | - Antonio M. Estevez
- Instituto de Parasitologia y Biomedicina “Lopez-Neyra,” CSIC Avda. Del Comocimiento s/n. Armilla, 18100 Granada, Spain
| | - Vivian Bellofatto
- Department of Microbiology and Molecular Genetics, UMDNJ-NJ Medical School, Newark, New Jersey 07103, USA
| |
Collapse
|
|
19
|
The ezrin metastatic phenotype is associated with the initiation of protein translation. Neoplasia 2012; 14:297-310. [PMID: 22577345 DOI: 10.1593/neo.11518] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/27/2011] [Revised: 03/13/2012] [Accepted: 03/16/2012] [Indexed: 12/14/2022] Open
Abstract
We previously associated the cytoskeleton linker protein, Ezrin, with the metastatic phenotype of pediatric sarcomas, including osteosarcoma and rhabdomyosarcoma. These studies have suggested that Ezrin contributes to the survival of cancer cells after their arrival at secondary metastatic locations. To better understand this role in metastasis, we undertook two noncandidate analyses of Ezrin function including a microarray subtraction of high-and low-Ezrin-expressing cells and a proteomic approach to identify proteins that bound the N-terminus of Ezrin in tumor lysates. Functional analyses of these data led to a novel and unifying hypothesis that Ezrin contributes to the efficiency of metastasis through regulation of protein translation. In support of this hypothesis, we found Ezrin to be part of the ribonucleoprotein complex to facilitate the expression of complex messenger RNA in cells and to bind with poly A binding protein 1 (PABP1; PABPC1). The relevance of these findings was supported by our identification of Ezrin and components of the translational machinery in pseudopodia of highly metastatic cells during the process of cell invasion. Finally, two small molecule inhibitors recently shown to inhibit the Ezrin metastatic phenotype disrupted the Ezrin/PABP1 association. Taken together, these results provide a novel mechanistic basis by which Ezrin may contribute to metastasis.
Collapse
|
|
20
|
Culver BP, Savas JN, Park SK, Choi JH, Zheng S, Zeitlin SO, Yates JR, Tanese N. Proteomic analysis of wild-type and mutant huntingtin-associated proteins in mouse brains identifies unique interactions and involvement in protein synthesis. J Biol Chem 2012; 287:21599-614. [PMID: 22556411 DOI: 10.1074/jbc.m112.359307] [Citation(s) in RCA: 105] [Impact Index Per Article: 8.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Huntington disease is a neurodegenerative disorder caused by a CAG repeat amplification in the gene huntingtin (HTT) that is reflected by a polyglutamine expansion in the Htt protein. Nearly 20 years of research have uncovered roles for Htt in a wide range of cellular processes, and many of these discoveries stemmed from the identification of Htt-interacting proteins. However, no study has employed an impartial and comprehensive strategy to identify proteins that differentially associate with full-length wild-type and mutant Htt in brain tissue, the most relevant sample source to the disease condition. We analyzed Htt affinity-purified complexes from wild-type and HTT mutant juvenile mouse brain from two different biochemical fractions by tandem mass spectrometry. We compared variations in protein spectral counts relative to Htt to identify those proteins that are the most significantly contrasted between wild-type and mutant Htt purifications. Previously unreported Htt interactions with Myo5a, Prkra (PACT), Gnb2l1 (RACK1), Rps6, and Syt2 were confirmed by Western blot analysis. Gene Ontology analysis of these and other Htt-associated proteins revealed a statistically significant enrichment for proteins involved in translation among other categories. Furthermore, Htt co-sedimentation with polysomes in cytoplasmic mouse brain extracts is dependent upon the presence of intact ribosomes. Finally, wild-type or mutant Htt overexpression inhibits cap-dependent translation of a reporter mRNA in an in vitro system. Cumulatively, these data support a new role for Htt in translation and provide impetus for further study into the link between protein synthesis and Huntington disease pathogenesis.
Collapse
Affiliation(s)
- Brady P Culver
- Department of Microbiology, New York University School of Medicine, New York, New York 10016, USA
| | | | | | | | | | | | | | | |
Collapse
|
|
21
|
Kobayashi T, Winslow S, Sunesson L, Hellman U, Larsson C. PKCα binds G3BP2 and regulates stress granule formation following cellular stress. PLoS One 2012; 7:e35820. [PMID: 22536444 PMCID: PMC3335008 DOI: 10.1371/journal.pone.0035820] [Citation(s) in RCA: 41] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/15/2011] [Accepted: 03/26/2012] [Indexed: 01/17/2023] Open
Abstract
Protein kinase C (PKC) isoforms regulate a number of processes crucial for the fate of a cell. In this study we identify previously unrecognized interaction partners of PKCα and a novel role for PKCα in the regulation of stress granule formation during cellular stress. Three RNA-binding proteins, cytoplasmic poly(A)(+) binding protein (PABPC1), IGF-II mRNA binding protein 3 (IGF2BP3), and RasGAP binding protein 2 (G3BP2) all co-precipitate with PKCα. RNase treatment abolished the association with IGF2BP3 and PABPC1 whereas the PKCα-G3BP2 interaction was largely resistant to this. Furthermore, interactions between recombinant PKCα and G3BP2 indicated that the interaction is direct and PKCα can phosphorylate G3BP2 in vitro. The binding is mediated via the regulatory domain of PKCα and the C-terminal RNA-binding domain of G3BP2. Both proteins relocate to and co-localize in stress granules, but not to P-bodies, when cells are subjected to stress. Heat shock-induced stress granule assembly and phosphorylation of eIF2α are suppressed following downregulation of PKCα by siRNA. In conclusion this study identifies novel interaction partners of PKCα and a novel role for PKCα in regulation of stress granules.
Collapse
Affiliation(s)
- Tamae Kobayashi
- Center for Molecular Pathology, Lund University, Malmö, Sweden
| | - Sofia Winslow
- Center for Molecular Pathology, Lund University, Malmö, Sweden
| | - Lovisa Sunesson
- Center for Molecular Pathology, Lund University, Malmö, Sweden
| | - Ulf Hellman
- Ludwig Institute for Cancer Research Ltd., Uppsala University, Uppsala, Sweden
| | - Christer Larsson
- Center for Molecular Pathology, Lund University, Malmö, Sweden
- * E-mail:
| |
Collapse
|
|
22
|
Ceci M, Welshhans K, Ciotti MT, Brandi R, Parisi C, Paoletti F, Pistillo L, Bassell GJ, Cattaneo A. RACK1 is a ribosome scaffold protein for β-actin mRNA/ZBP1 complex. PLoS One 2012; 7:e35034. [PMID: 22523568 PMCID: PMC3327689 DOI: 10.1371/journal.pone.0035034] [Citation(s) in RCA: 43] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/28/2011] [Accepted: 03/08/2012] [Indexed: 12/24/2022] Open
Abstract
In neurons, specific mRNAs are transported in a translationally repressed manner along dendrites or axons by transport ribonucleic-protein complexes called RNA granules. ZBP1 is one RNA binding protein present in transport RNPs, where it transports and represses the translation of cotransported mRNAs, including β-actin mRNA. The release of β-actin mRNA from ZBP1 and its subsequent translation depends on the phosphorylation of ZBP1 by Src kinase, but little is known about how this process is regulated. Here we demonstrate that the ribosomal-associated protein RACK1, another substrate of Src, binds the β-actin mRNA/ZBP1 complex on ribosomes and contributes to the release of β-actin mRNA from ZBP1 and to its translation. We identify the Src binding and phosphorylation site Y246 on RACK1 as the critical site for the binding to the β-actin mRNA/ZBP1 complex. Based on these results we propose RACK1 as a ribosomal scaffold protein for specific mRNA-RBP complexes to tightly regulate the translation of specific mRNAs.
Collapse
Affiliation(s)
- Marcello Ceci
- European Brain Research Institute (EBRI), Rome, Italy
| | - Kristy Welshhans
- Department of Biological Sciences, Kent State University, Kent, Ohio, United States of America
| | | | | | - Chiara Parisi
- European Brain Research Institute (EBRI), Rome, Italy
| | | | | | - Gary J. Bassell
- Departments of Cell Biology, Neurology, Emory University School of Medicine, Atlanta, Georgia, United States of America
| | - Antonino Cattaneo
- European Brain Research Institute (EBRI), Rome, Italy
- Scuola Normale Superiore di Pisa, Pisa, Italy
- * E-mail:
| |
Collapse
|
|
23
|
Adams DR, Ron D, Kiely PA. RACK1, A multifaceted scaffolding protein: Structure and function. Cell Commun Signal 2011; 9:22. [PMID: 21978545 PMCID: PMC3195729 DOI: 10.1186/1478-811x-9-22] [Citation(s) in RCA: 349] [Impact Index Per Article: 24.9] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/07/2011] [Accepted: 10/06/2011] [Indexed: 12/17/2022] Open
Abstract
The Receptor for Activated C Kinase 1 (RACK1) is a member of the tryptophan-aspartate repeat (WD-repeat) family of proteins and shares significant homology to the β subunit of G-proteins (Gβ). RACK1 adopts a seven-bladed β-propeller structure which facilitates protein binding. RACK1 has a significant role to play in shuttling proteins around the cell, anchoring proteins at particular locations and in stabilising protein activity. It interacts with the ribosomal machinery, with several cell surface receptors and with proteins in the nucleus. As a result, RACK1 is a key mediator of various pathways and contributes to numerous aspects of cellular function. Here, we discuss RACK1 gene and structure and its role in specific signaling pathways, and address how posttranslational modifications facilitate subcellular location and translocation of RACK1. This review condenses several recent studies suggesting a role for RACK1 in physiological processes such as development, cell migration, central nervous system (CN) function and circadian rhythm as well as reviewing the role of RACK1 in disease.
Collapse
Affiliation(s)
- David R Adams
- Department of Life Sciences, and Materials and Surface Science Institute, University of Limerick, Limerick, Ireland.
| | | | | |
Collapse
|
|
24
|
Gonçalves KDA, Bressan GC, Saito A, Morello LG, Zanchin NIT, Kobarg J. Evidence for the association of the human regulatory protein Ki-1/57 with the translational machinery. FEBS Lett 2011; 585:2556-60. [PMID: 21771594 DOI: 10.1016/j.febslet.2011.07.010] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/09/2011] [Revised: 07/04/2011] [Accepted: 07/04/2011] [Indexed: 02/06/2023]
Abstract
Ki-1/57 is a cytoplasmic and nuclear protein of 57 kDa first identified in malignant cells from Hodgkin's lymphoma. Based on yeast-two hybrid protein interaction we found out that Ki-1/57 interacts with adaptor protein RACK1 (receptor of activated kinase 1), CIRP (cold-inducible RNA-binding protein), RPL38 (ribosomal protein L38) and FXR1 (fragile X mental retardation-related protein 1). Since these proteins are involved in the regulation of translation we suspected that Ki-1/57 may have a role in it. We show by immunoprecipitation the association of Ki-1/57 with FMRP. Confocal microscopy revealed that Ki-1/57 colocalizes with FMRP/FXR1/2 to stress granules. Furthermore Ki-1/57 cosediments with free ribosomal particles and enhances translation, when tethered to a reporter mRNA, suggesting that Ki-1/57 may be involved in translational regulation.
Collapse
Affiliation(s)
- Kaliandra de Almeida Gonçalves
- Laboratório Nacional de Biociências (LNBio), Centro Nacional de Pesquisa em Energia e Materiais (CNPEM), Campinas, São Paulo, Brazil
| | | | | | | | | | | |
Collapse
|
|
25
|
Yang R, Gaidamakov SA, Xie J, Lee J, Martino L, Kozlov G, Crawford AK, Russo AN, Conte MR, Gehring K, Maraia RJ. La-related protein 4 binds poly(A), interacts with the poly(A)-binding protein MLLE domain via a variant PAM2w motif, and can promote mRNA stability. Mol Cell Biol 2011; 31:542-56. [PMID: 21098120 PMCID: PMC3028612 DOI: 10.1128/mcb.01162-10] [Citation(s) in RCA: 85] [Impact Index Per Article: 6.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/04/2010] [Revised: 11/05/2010] [Accepted: 11/12/2010] [Indexed: 12/19/2022] Open
Abstract
The conserved RNA binding protein La recognizes UUU-3'OH on its small nuclear RNA ligands and stabilizes them against 3'-end-mediated decay. We report that newly described La-related protein 4 (LARP4) is a factor that can bind poly(A) RNA and interact with poly(A) binding protein (PABP). Yeast two-hybrid analysis and reciprocal immunoprecipitations (IPs) from HeLa cells revealed that LARP4 interacts with RACK1, a 40S ribosome- and mRNA-associated protein. LARP4 cosediments with 40S ribosome subunits and polyribosomes, and its knockdown decreases translation. Mutagenesis of the RNA binding or PABP interaction motifs decrease LARP4 association with polysomes. Several translation and mRNA metabolism-related proteins use a PAM2 sequence containing a critical invariant phenylalanine to make direct contact with the MLLE domain of PABP, and their competition for the MLLE is thought to regulate mRNA homeostasis. Unlike all ∼150 previously analyzed PAM2 sequences, LARP4 contains a variant PAM2 (PAM2w) with tryptophan in place of the phenylalanine. Binding and nuclear magnetic resonance (NMR) studies have shown that a peptide representing LARP4 PAM2w interacts with the MLLE of PABP within the affinity range measured for other PAM2 motif peptides. A cocrystal of PABC bound to LARP4 PAM2w shows tryptophan in the pocket in PABC-MLLE otherwise occupied by phenylalanine. We present evidence that LARP4 expression stimulates luciferase reporter activity by promoting mRNA stability, as shown by mRNA decay analysis of luciferase and cellular mRNAs. We propose that LARP4 activity is integrated with other PAM2 protein activities by PABP as part of mRNA homeostasis.
Collapse
Affiliation(s)
- Ruiqing Yang
- Intramural Research Program on Genomics of Differentiation, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, Randall Division of Cell and Molecular Biophysics, King's College London, London, United Kingdom, Department of Biochemistry, McGill University, Montreal, QC, Canada, Commissioned Corps, U.S. Public Health Service, Washington, DC
| | - Sergei A. Gaidamakov
- Intramural Research Program on Genomics of Differentiation, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, Randall Division of Cell and Molecular Biophysics, King's College London, London, United Kingdom, Department of Biochemistry, McGill University, Montreal, QC, Canada, Commissioned Corps, U.S. Public Health Service, Washington, DC
| | - Jingwei Xie
- Intramural Research Program on Genomics of Differentiation, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, Randall Division of Cell and Molecular Biophysics, King's College London, London, United Kingdom, Department of Biochemistry, McGill University, Montreal, QC, Canada, Commissioned Corps, U.S. Public Health Service, Washington, DC
| | - Joowon Lee
- Intramural Research Program on Genomics of Differentiation, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, Randall Division of Cell and Molecular Biophysics, King's College London, London, United Kingdom, Department of Biochemistry, McGill University, Montreal, QC, Canada, Commissioned Corps, U.S. Public Health Service, Washington, DC
| | - Luigi Martino
- Intramural Research Program on Genomics of Differentiation, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, Randall Division of Cell and Molecular Biophysics, King's College London, London, United Kingdom, Department of Biochemistry, McGill University, Montreal, QC, Canada, Commissioned Corps, U.S. Public Health Service, Washington, DC
| | - Guennadi Kozlov
- Intramural Research Program on Genomics of Differentiation, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, Randall Division of Cell and Molecular Biophysics, King's College London, London, United Kingdom, Department of Biochemistry, McGill University, Montreal, QC, Canada, Commissioned Corps, U.S. Public Health Service, Washington, DC
| | - Amanda K. Crawford
- Intramural Research Program on Genomics of Differentiation, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, Randall Division of Cell and Molecular Biophysics, King's College London, London, United Kingdom, Department of Biochemistry, McGill University, Montreal, QC, Canada, Commissioned Corps, U.S. Public Health Service, Washington, DC
| | - Amy N. Russo
- Intramural Research Program on Genomics of Differentiation, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, Randall Division of Cell and Molecular Biophysics, King's College London, London, United Kingdom, Department of Biochemistry, McGill University, Montreal, QC, Canada, Commissioned Corps, U.S. Public Health Service, Washington, DC
| | - Maria R. Conte
- Intramural Research Program on Genomics of Differentiation, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, Randall Division of Cell and Molecular Biophysics, King's College London, London, United Kingdom, Department of Biochemistry, McGill University, Montreal, QC, Canada, Commissioned Corps, U.S. Public Health Service, Washington, DC
| | - Kalle Gehring
- Intramural Research Program on Genomics of Differentiation, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, Randall Division of Cell and Molecular Biophysics, King's College London, London, United Kingdom, Department of Biochemistry, McGill University, Montreal, QC, Canada, Commissioned Corps, U.S. Public Health Service, Washington, DC
| | - Richard J. Maraia
- Intramural Research Program on Genomics of Differentiation, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, Randall Division of Cell and Molecular Biophysics, King's College London, London, United Kingdom, Department of Biochemistry, McGill University, Montreal, QC, Canada, Commissioned Corps, U.S. Public Health Service, Washington, DC
| |
Collapse
|
|
26
|
Schäffler K, Schulz K, Hirmer A, Wiesner J, Grimm M, Sickmann A, Fischer U. A stimulatory role for the La-related protein 4B in translation. RNA (NEW YORK, N.Y.) 2010; 16:1488-99. [PMID: 20573744 PMCID: PMC2905749 DOI: 10.1261/rna.2146910] [Citation(s) in RCA: 50] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/09/2023]
Abstract
La-related proteins (LARPs) belong to an evolutionarily conserved family of factors with predicted roles in RNA metabolism. Here, we have analyzed the cellular interactions and function of LARP4B, a thus far uncharacterized member of the LARP family. We show that LARP4B is a cytosolic protein that accumulates upon arsenite treatment in cellular stress granules. Biochemical experiments further uncovered an interaction of LARP4B with the cytosolic poly(A) binding protein 1 (PABPC1) and the receptor for activated C Kinase (RACK1), a component of the 40S ribosomal subunit. Under physiological conditions, LARP4B co-sedimented with polysomes in cellular extracts, suggesting a role in translation. In agreement with this notion, overexpression of LARP4B stimulated protein synthesis, whereas knockdown of the factor by RNA interference impaired translation of a large number of cellular mRNAs. In sum, we identified LARP4B as a stimulatory factor of translation. We speculate that LARP4B exerts its function by bridging mRNA factors of the 3' end with initiating ribosomes.
Collapse
Affiliation(s)
- Katrin Schäffler
- Department of Biochemistry, Theodor Boveri-Institute, University of Wuerzburg, Wuerzburg D-97074, Germany
| | | | | | | | | | | | | |
Collapse
|
|
27
|
Gonçalves KA, Borges JC, Silva JC, Papa PF, Bressan GC, Torriani IL, Kobarg J. Solution structure of the human signaling protein RACK1. BMC STRUCTURAL BIOLOGY 2010; 10:15. [PMID: 20529362 PMCID: PMC2896345 DOI: 10.1186/1472-6807-10-15] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 02/17/2010] [Accepted: 06/08/2010] [Indexed: 01/09/2023]
Abstract
Background The adaptor protein RACK1 (receptor of activated kinase 1) was originally identified as an anchoring protein for protein kinase C. RACK1 is a 36 kDa protein, and is composed of seven WD repeats which mediate its protein-protein interactions. RACK1 is ubiquitously expressed and has been implicated in diverse cellular processes involving: protein translation regulation, neuropathological processes, cellular stress, and tissue development. Results In this study we performed a biophysical analysis of human RACK1 with the aim of obtaining low resolution structural information. Small angle X-ray scattering (SAXS) experiments demonstrated that human RACK1 is globular and monomeric in solution and its low resolution structure is strikingly similar to that of an homology model previously calculated by us and to the crystallographic structure of RACK1 isoform A from Arabidopsis thaliana. Both sedimentation velocity and sedimentation equilibrium analytical ultracentrifugation techniques showed that RACK1 is predominantly a monomer of around 37 kDa in solution, but also presents small amounts of oligomeric species. Moreover, hydrodynamic data suggested that RACK1 has a slightly asymmetric shape. The interaction of RACK1 and Ki-1/57 was tested by sedimentation equilibrium. The results suggested that the association between RACK1 and Ki-1/57(122-413) follows a stoichiometry of 1:1. The binding constant (KB) observed for RACK1-Ki-1/57(122-413) interaction was of around (1.5 ± 0.2) × 106 M-1 and resulted in a dissociation constant (KD) of (0.7 ± 0.1) × 10-6 M. Moreover, the fluorescence data also suggests that the interaction may occur in a cooperative fashion. Conclusion Our SAXS and analytical ultracentrifugation experiments indicated that RACK1 is predominantly a monomer in solution. RACK1 and Ki-1/57(122-413) interact strongly under the tested conditions.
Collapse
Affiliation(s)
- Kaliandra A Gonçalves
- Laboratório Nacional de Biociências (LNBio), Centro de Pesquisa em Energia e Materiais (CNPEM), Campinas, SP, Brazil
| | | | | | | | | | | | | |
Collapse
|
|
28
|
Han J, Miyamae Y, Shigemori H, Isoda H. Neuroprotective effect of 3,5-di-O-caffeoylquinic acid on SH-SY5Y cells and senescence-accelerated-prone mice 8 through the up-regulation of phosphoglycerate kinase-1. Neuroscience 2010; 169:1039-45. [PMID: 20570715 DOI: 10.1016/j.neuroscience.2010.05.049] [Citation(s) in RCA: 109] [Impact Index Per Article: 7.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/08/2010] [Revised: 05/19/2010] [Accepted: 05/21/2010] [Indexed: 02/06/2023]
Abstract
As aged population dramatically increases in these decades, efforts should be made on the intervention for curing age-associated neurologic degenerative diseases such as Alzheimer's disease (AD). Caffeoylquinic acid (CQA), an antioxidant component and its derivatives are natural functional compounds isolated from a variety of plants. In this study, we determined the neuroprotective effect of 3,5-di-O-CQA on Abeta(1-42) treated SH-SY5Y cells using MTT assay. To investigate the possible neuroprotective mechanism of 3,5-di-O-CQA, we performed proteomics analysis, real-time PCR analysis and measurement of the intracellular ATP level. In addition, we carried out the measurement of escape latency time to find the hidden platform in Morris water maze (MWM), real-time PCR using senescence-accelerated-prone mice (SAMP) 8 and senescence-accelerated-resistant mice (SAMR) 1 mice. Results showed that 3,5-di-O-CQA had neuroprotective effect on Abeta (1-42) treated cells. The mRNA expression of glycolytic enzyme (phosphoglycerate kinase-1; PGK1) and intracellular ATP level were increased in 3,5-di-O-CQA treated SH-SY5Y cells. We also found that 3,5-di-O-CQA administration induced the improvement of spatial learning and memory on SAMP8 mice, and the overexpression of PGK1 mRNA. These findings suggest that 3,5-di-O-CQA has a neuroprotective effect on neuron through the upregulation of PGK1 expression and ATP production activation.
Collapse
Affiliation(s)
- J Han
- Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan
| | | | | | | |
Collapse
|
|
29
|
Annenkov A. The insulin-like growth factor (IGF) receptor type 1 (IGF1R) as an essential component of the signalling network regulating neurogenesis. Mol Neurobiol 2009; 40:195-215. [PMID: 19714501 DOI: 10.1007/s12035-009-8081-0] [Citation(s) in RCA: 51] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/10/2009] [Accepted: 08/14/2009] [Indexed: 02/07/2023]
Abstract
The insulin-like growth factor receptor type 1 (IGF1R) signalling pathway is activated in the mammalian nervous system from early developmental stages. Its major effect on developing neural cells is to promote their growth and survival. This pathway can integrate its action with signalling pathways of growth and morphogenetic factors that induce cell fate specification and selective expansion of specified neural cell subsets. This suggests that during developmental and adult neurogenesis cellular responses to many signalling factors, including ligands of Notch, sonic hedgehog, fibroblast growth factor family members, ligands of the epidermal growth factor receptor, bone morphogenetic proteins and Wingless and Int-1, may be modified by co-activation of the IGF1R. Modulation of cell migration is another possible role that IGF1R activation may play in neurogenesis. Here, I briefly overview neurogenesis and discuss a role for IGF1R-mediated signalling in the developing and mature nervous system with emphasis on crosstalk between the signalling pathways of the IGF1R and other factors regulating neural cell development and migration. Studies on neural as well as on non-neural cells are highlighted because it may be interesting to test in neurogenic paradigms some of the models based on the information obtained in studies on non-neural cell types.
Collapse
Affiliation(s)
- Alexander Annenkov
- William Harvey Research Institute, Queen Mary University of London, Charterhouse Square, UK.
| |
Collapse
|
|
30
|
Grosso S, Volta V, Vietri M, Gorrini C, Marchisio PC, Biffo S. Eukaryotic ribosomes host PKC activity. Biochem Biophys Res Commun 2008; 376:65-9. [PMID: 18768137 DOI: 10.1016/j.bbrc.2008.08.118] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/18/2008] [Accepted: 08/20/2008] [Indexed: 10/21/2022]
Abstract
PKC isoform betaII modulates translation and can be recruited on ribosomes via its scaffold RACK1 (receptor for activated protein kinase C 1), which resides on the 40S ribosomal subunit. However, whether a PKC activity exists on the ribosome is not yet demonstrated. We purified native ribosomes by two different techniques, which avoid stripping of initiation factors and other associated proteins. In both cases, purified ribosomes are able to phosphorylate a specific PKC substrate, MARCKS (Myristoylated Alanine-Rich C-Kinase Substrate). MARCKS phosphorylation is switched on by treatment with PKC agonist PMA (Phorbol 12-Myristate 13-Acetate). Consistently, the broad PKC inhibitor BMI (Bisindolyl Maleimide I) abrogates MARCKS phosphorylation. These data show that native ribosomes host active PKC and hence allow the phosphorylation of ribosome-associated substrates like initiation factors and mRNA binding proteins.
Collapse
Affiliation(s)
- Stefano Grosso
- Laboratory of Molecular Histology and Cell Growth, DIBIT, Via Olgettina 58, HSR, 20132 Milano, Italy
| | | | | | | | | | | |
Collapse
|
|
31
|
Regmi S, Rothberg KG, Hubbard JG, Ruben L. The RACK1 signal anchor protein from Trypanosoma brucei associates with eukaryotic elongation factor 1A: a role for translational control in cytokinesis. Mol Microbiol 2008; 70:724-45. [PMID: 18786142 PMCID: PMC2581647 DOI: 10.1111/j.1365-2958.2008.06443.x] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 09/02/2008] [Indexed: 01/05/2023]
Abstract
RACK1 is a WD-repeat protein that forms signal complexes at appropriate locations in the cell. RACK1 homologues are core components of ribosomes from yeast, plants and mammals. In contrast, a cryo-EM analysis of trypanosome ribosomes failed to detect RACK1, thus eliminating an important translational regulatory mechanism. Here we report that TbRACK1 from Trypanosoma brucei associates with eukaryotic translation elongation factor-1a (eEF1A) as determined by tandem MS of TAP-TbRACK1 affinity eluates, co-sedimentation in a sucrose gradient, and co-precipitation assays. Consistent with these observations, sucrose gradient purified 80S monosomes and translating polysomes each contained TbRACK1. When RNAi was used to deplete cells of TbRACK1, a shift in the polysome profile was observed, while the phosphorylation of a ribosomal protein increased. Under these conditions, cell growth became hypersensitive to the translational inhibitor anisomycin. The kinetoplasts and nuclei were misaligned in the postmitotic cells, resulting in partial cleavage furrow ingression during cytokinesis. Overall, these findings identify eEF1A as a novel TbRACK1 binding partner and establish TbRACK1 as a component of the trypanosome translational apparatus. The synergy between anisomycin and TbRACK1 RNAi suggests that continued translation is required for complete ingression of the cleavage furrow.
Collapse
Affiliation(s)
- Sandesh Regmi
- Department of Biological Sciences, Southern Methodist UniversityDallas, TX 75275, USA
| | - Karen G Rothberg
- Department of Biological Sciences, Southern Methodist UniversityDallas, TX 75275, USA
| | - James G Hubbard
- Department of Biological Sciences, Southern Methodist UniversityDallas, TX 75275, USA
| | - Larry Ruben
- Department of Biological Sciences, Southern Methodist UniversityDallas, TX 75275, USA
| |
Collapse
|
|
32
|
Grosso S, Volta V, Sala LA, Vietri M, Marchisio PC, Ron D, Biffo S. PKCbetaII modulates translation independently from mTOR and through RACK1. Biochem J 2008; 415:77-85. [PMID: 18557705 DOI: 10.1042/bj20080463] [Citation(s) in RCA: 54] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022]
Abstract
RACK1 (receptor for activated C kinase 1) is an abundant scaffolding protein, which binds active PKCbetaII (protein kinase C betaII) increasing its activity in vitro. RACK1 has also been described as a component of the small ribosomal subunit, in proximity to the mRNA exit channel. In the present study we tested the hypothesis that PKCbetaII plays a specific role in translational control and verified whether it may associate with the ribosomal machinery. We find that specific inhibition of PKCbetaI/II reduces translation as well as global PKC inhibition, but without affecting phosphorylation of mTOR (mammalian target of rapamycin) targets. These results suggest that PKCbetaII acts as a specific PKC isoform affecting translation in an mTOR-independent fashion, possibly close to the ribosomal machinery. Using far-Western analysis, we found that PKCbetaII binds ribosomes in vitro. Co-immunoprecipitation studies indicate that a small but reproducible pool of PKCbetaII is associated with membranes containing ribosomes, suggesting that in vivo PKCbetaII may also physically interact with the ribosomal machinery. Polysomal profiles show that stimulation of PKC results in an increased polysomes/80S ratio, associated with a shift of PKCbetaII to the heavier part of the gradient. A RACK1-derived peptide that inhibits the binding of active PKCbetaII to RACK1 reduces the polysomes/80S ratio and methionine incorporation, suggesting that binding of PKCbetaII to RACK1 is important for PKC-mediated translational control. Finally, down-regulation of RACK1 by siRNA (small interfering RNA) impairs the PKC-mediated increase of translation. Taken together the results of the present study show that PKCbetaII can act as a specific PKC isoform regulating translation, in an mTOR-independent fashion, possibly close to the ribosomal machinery.
Collapse
Affiliation(s)
- Stefano Grosso
- Molecular Histology and Cell Growth, HSR, 20132 Milan, Italy
| | | | | | | | | | | | | |
Collapse
|
|
33
|
Aranda-Orgillés B, Trockenbacher A, Winter J, Aigner J, Köhler A, Jastrzebska E, Stahl J, Müller EC, Otto A, Wanker EE, Schneider R, Schweiger S. The Opitz syndrome gene product MID1 assembles a microtubule-associated ribonucleoprotein complex. Hum Genet 2008; 123:163-76. [PMID: 18172692 PMCID: PMC3774420 DOI: 10.1007/s00439-007-0456-6] [Citation(s) in RCA: 51] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/24/2007] [Accepted: 12/15/2007] [Indexed: 01/31/2023]
Abstract
Opitz BBB/G syndrome (OS) is a heterogenous malformation syndrome mainly characterised by hypertelorism and hypospadias. In addition, patients may present with several other defects of the ventral midline such as cleft lip and palate and congenital heart defects. The syndrome-causing gene encodes the X-linked E3 ubiquitin ligase MID1 that mediates ubiquitin-specific modification and degradation of the catalytic subunit of the translation regulator protein phosphatase 2A (PP2A). Here, we show that the MID1 protein also associates with elongation factor 1alpha (EF-1alpha) and several other proteins involved in mRNA transport and translation, including RACK1, Annexin A2, Nucleophosmin and proteins of the small ribosomal subunits. Mutant MID1 proteins as found in OS patients lose the ability to interact with EF-1alpha. The composition of the MID1 protein complex was determined by several independent methods: (1) yeast two-hybrid screening and (2) immunofluorescence, (3) a biochemical approach involving affinity purification of the complex, (4) co-fractionation in a microtubule assembly assay and (5) immunoprecipitation. Moreover, we show that the cytoskeleton-bound MID1/translation factor complex specifically associates with G- and U-rich RNAs and incorporates MID1 mRNA, thus forming a microtubule-associated ribonucleoprotein (RNP) complex. Our data suggest a novel function of the OS gene product in directing translational control to the cytoskeleton. The dysfunction of this mechanism would lead to malfunction of microtubule-associated protein translation and to the development of OS.
Collapse
Affiliation(s)
- Beatriz Aranda-Orgillés
- />Max-Planck Institute for Molecular Genetics, Ihnestr. 73, 14195 Berlin, Germany
- />Department of Biology, Chemistry and Pharmacy, Free University Berlin, Thielallee 63, 14195 Berlin, Germany
| | - Alexander Trockenbacher
- />Max-Planck Institute for Molecular Genetics, Ihnestr. 73, 14195 Berlin, Germany
- />Institute of Biochemistry, Center for Molecular Biosciences Innsbruck (CMBI), University Innsbruck, Peter-Mayr-Str. 1a, 6020 Innsbruck, Austria
| | - Jennifer Winter
- />Max-Planck Institute for Molecular Genetics, Ihnestr. 73, 14195 Berlin, Germany
| | - Johanna Aigner
- />Max-Planck Institute for Molecular Genetics, Ihnestr. 73, 14195 Berlin, Germany
| | - Andrea Köhler
- />Institute of Biochemistry, Center for Molecular Biosciences Innsbruck (CMBI), University Innsbruck, Peter-Mayr-Str. 1a, 6020 Innsbruck, Austria
| | - Ewa Jastrzebska
- />Max-Planck Institute for Molecular Genetics, Ihnestr. 73, 14195 Berlin, Germany
- />Department of Dermatology, Charité, Schumannstr. 21-22, 10117 Berlin, Germany
| | - Joachim Stahl
- />Max-Delbrueck Center of Molecular Medicine, Robert-Roessle-Str. 10, 13125 Berlin, Germany
| | - Eva-Christina Müller
- />Max-Delbrueck Center of Molecular Medicine, Robert-Roessle-Str. 10, 13125 Berlin, Germany
| | - Albrecht Otto
- />Max-Delbrueck Center of Molecular Medicine, Robert-Roessle-Str. 10, 13125 Berlin, Germany
| | - Erich E. Wanker
- />Max-Delbrueck Center of Molecular Medicine, Robert-Roessle-Str. 10, 13125 Berlin, Germany
| | - Rainer Schneider
- />Max-Planck Institute for Molecular Genetics, Ihnestr. 73, 14195 Berlin, Germany
- />Institute of Biochemistry, Center for Molecular Biosciences Innsbruck (CMBI), University Innsbruck, Peter-Mayr-Str. 1a, 6020 Innsbruck, Austria
| | - Susann Schweiger
- />Max-Planck Institute for Molecular Genetics, Ihnestr. 73, 14195 Berlin, Germany
- />Medical School, Division of Pathology and Neuroscience, University of Dundee, DD1 9SY Dundee, UK
| |
Collapse
|
|
34
|
Atlas R, Behar L, Sapoznik S, Ginzburg I. Dynamic association with polysomes during P19 neuronal differentiation and an untranslated-region-dependent translation regulation of the tau mRNA by the tau mRNA-associated proteins IMP1, HuD, and G3BP1. J Neurosci Res 2007; 85:173-83. [PMID: 17086542 DOI: 10.1002/jnr.21099] [Citation(s) in RCA: 36] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/10/2022]
Abstract
Regulation of mRNA translation is a key step in mediating neuronal polarity during differentiation, insofar as neuronal polarity is partially determined by local translation of specific mRNA molecules as dendrites and axons are emanating. The multiplicity of mRNA-binding proteins in neurons plays an essential role in controlling mRNA translation. These proteins are associated with ribosomes and translation factors, thereby regulating both temporally and spatially the translation process. In a previous study, we have shown an association among the tau mRNA-binding proteins HuD, IMP1, and G3BP1 with translating polysomes in P19 neurons. In the present study, we determined the dynamics of the association among G3BP1, IMP1, and HuD with polysomes through P19 neuronal differentiation as well as the functional effect of these proteins on tau mRNA translation. We show a novel, differentiation-dependent association of these proteins with polysomes. In addition, we show a strong, negative effect on translation of the tau mRNA by IMP1, G3BP1, and HuD proteins in HEK-293 cells. To our knowledge this is the first observation of a direct translational role of G3BP1 for any mRNA and the first report of a translation inhibition by IMP1 and HuD on the tau mRNA in a cell system. The translation inhibition is shown to be mediated by the tau mRNA 3'untranslated regions (UTRs), thus giving a new, translational role for these sequences, which were previously implicated in mRNA stabilization. We also define a novel mechanism for IMP1 binding to tau mRNA, which suggests a conformational binding, which is not sequence dependent.
Collapse
Affiliation(s)
- Roee Atlas
- Department of Neurobiology, Weizmann Institute of Science, Rehovot, Israel
| | | | | | | |
Collapse
|
|
35
|
Bjørndal B, Myklebust LM, Rosendal KR, Myromslien FD, Lorens JB, Nolan G, Bruland O, Lillehaug JR. RACK1 regulates Ki-Ras-mediated signaling and morphological transformation of NIH 3T3 cells. Int J Cancer 2007; 120:961-9. [PMID: 17149700 DOI: 10.1002/ijc.22373] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/07/2022]
Abstract
Activating Ras mutations are involved in a significant fraction of human tumors. A suppressor screen using a retroviral mouse fibroblast cDNA library was performed to identify novel factors in Ras-mediated transformation. We identified a novel potent inhibitor of Ras-mediated morphological transformation encoded by a truncated version of the receptor for activated C-kinase (RACK1). The truncated protein, designated RACK1DeltaWD1, lacked the N-terminal 49 amino acids encoding the first of the 7 WD40 repeats in RACK1. RACK1DeltaWD1 expression restored contact inhibition, stress fiber formation and reduced ERK phosphorylation in Ki-Ras transformed NIH 3T3 cells. We demonstrate that truncated RACK1 is involved in complexes consisting of wild-type RACK1 and protein kinase C isoforms alpha, betaI and delta, compromising the transduction of an activated Ras signal to the Raf-MEK-ERK pathway. The cellular localization of RACK1DeltaWD1 differed from wtRACK1, indicating that signaling complexes containing the truncated version of RACK1 are incorrectly localized. Notably, 12-O-tetradecanoyl-13-phorbol acetate (TPA) mediated intracellular translocation of RACK1-interacting PKC alpha and delta was abrogated in RACK1DeltaWD1-expressing cells. Our data support a model where RACK1 acts as a key factor in Ki-Ras-mediated morphological transformation.
Collapse
Affiliation(s)
- Bodil Bjørndal
- Department of Molecular Biology, University of Bergen, Bergen, Norway
| | | | | | | | | | | | | | | |
Collapse
|
|
36
|
Solomon S, Xu Y, Wang B, David MD, Schubert P, Kennedy D, Schrader JW. Distinct structural features of caprin-1 mediate its interaction with G3BP-1 and its induction of phosphorylation of eukaryotic translation initiation factor 2alpha, entry to cytoplasmic stress granules, and selective interaction with a subset of mRNAs. Mol Cell Biol 2007; 27:2324-42. [PMID: 17210633 PMCID: PMC1820512 DOI: 10.1128/mcb.02300-06] [Citation(s) in RCA: 208] [Impact Index Per Article: 11.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/22/2023] Open
Abstract
Caprin-1 is a ubiquitously expressed, well-conserved cytoplasmic phosphoprotein that is needed for normal progression through the G(1)-S phase of the cell cycle and occurs in postsynaptic granules in dendrites of neurons. We demonstrate that Caprin-1 colocalizes with RasGAP SH3 domain binding protein-1 (G3BP-1) in cytoplasmic RNA granules associated with microtubules and concentrated in the leading and trailing edge of migrating cells. Caprin-1 exhibits a highly conserved motif, F(M/I/L)Q(D/E)Sx(I/L)D that binds to the NTF-2-like domain of G3BP-1. The carboxy-terminal region of Caprin-1 selectively bound mRNA for c-Myc or cyclin D2, this binding being diminished by mutation of the three RGG motifs and abolished by deletion of the RGG-rich region. Overexpression of Caprin-1 induced phosphorylation of eukaryotic translation initiation factor 2alpha (eIF-2alpha) through a mechanism that depended on its ability to bind mRNA, resulting in global inhibition of protein synthesis. However, cells lacking Caprin-1 exhibited no changes in global rates of protein synthesis, suggesting that physiologically, the effects of Caprin-1 on translation were limited to restricted subsets of mRNAs. Overexpression of Caprin-1 induced the formation of cytoplasmic stress granules (SG). Its ability to bind RNA was required to induce SG formation but not necessarily its ability to enter SG. The ability of Caprin-1 or G3BP-1 to induce SG formation or enter them did not depend on their association with each other. The Caprin-1/G3BP-1 complex is likely to regulate the transport and translation of mRNAs of proteins involved with synaptic plasticity in neurons and cellular proliferation and migration in multiple cell types.
Collapse
Affiliation(s)
- Samuel Solomon
- The Biomedical Research Centre, University of British Columbia, Vancouver, B.C. V6T 1Z3, Canada
| | | | | | | | | | | | | |
Collapse
|
|
37
|
Abstract
The transport of messenger RNAs (mRNAs) in neurons serves many purposes. During development, trafficking of mRNAs to both axonal and dendritic growth cones regulates neuronal growth. After synapse formation, mRNAs continue to be transported to dendrites both as a mechanism for the localization of proteins to specific compartments and as a substrate for local translational regulation of synaptic plasticity. Finally, activity-dependent mRNAs are transported quickly to dendrites after transcription. Determining how mRNAs are transported and specifically translated in these different paradigms is a major unanswered question. Addressing this question is also complicated by the presence of many other RNA processing and storage centers that may not be involved in transport but share components with the transport structures. In the present review, we will discuss several recent studies addressing mechanisms of mRNA transport in neurons, as well as proteomic characterization of mRNA transporting structures in neurons. We define two types of RNA transport structures in neurons, transport particles and RNA granules and distinguish them by the presence or absence of ribosomes. We will present a number of different molecular models for how mRNAs are repressed during transport, and how these may affect the regulation of local translation in neurons.
Collapse
Affiliation(s)
- Wayne S Sossin
- Department of Neurology and Neurosurgery, McGill University, Montreal Neurological Institute, BT 110, 3801 University Street, Montreal, Quebec, Canada H3A 2B4.
| | | |
Collapse
|
|
38
|
Nery FC, Bressan GC, Alborghetti MR, Passos DO, Kuniyoshi TM, Ramos CHI, Oyama S, Kobarg J. A spectroscopic analysis of the interaction between the human regulatory proteins RACK1 and Ki-1/57. Biol Chem 2006; 387:577-82. [PMID: 16740129 DOI: 10.1515/bc.2006.074] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/15/2022]
Abstract
Ki-1/57 is a 57-kDa cytoplasmic and nuclear protein associated with protein kinase activity and is hyper-phosphorylated on Ser/Thr residues upon cellular activation. In previous studies we identified the receptor of activated kinase-1 (RACK1), a signaling adaptor protein that binds activated PKC, as a protein that interacts with Ki-1/57. Here we demonstrate that the far-UV circular dichroism spectrum of the WD repeat-containing RACK1 protein shows an unusual positive ellipticity at 229 nm, which in other proteins of the WD family has been attributed to surface tryptophans that are quenchable by N-bromosuccinimide (NBS). As well as NBS, in vitro binding of 6xHis-Ki-1/57(122-413) and 6xHis-Ki-1/57(264-413) can also quench the positive ellipticity of the RACK1 spectrum. We generated a model of RACK1 by homology modeling using a G protein beta subunit as template. Our model suggests the family-typical seven-bladed beta-propeller, with an aromatic cluster around the central tunnel that contains four Trp residues (17, 83, 150, 170), which are likely involved in the interaction with Ki-1/57.
Collapse
Affiliation(s)
- Flávia C Nery
- Centro de Biologia Molecular Estrutural, Laboratório Nacional de Luz Síncrotron, Rua Giuseppe Máximo Scolfaro 10.000, C.P. 6192, 13084-971 Campinas SP, Brazil
| | | | | | | | | | | | | | | |
Collapse
|
|
39
|
Rothberg KG, Burdette DL, Pfannstiel J, Jetton N, Singh R, Ruben L. The RACK1 homologue from Trypanosoma brucei is required for the onset and progression of cytokinesis. J Biol Chem 2006; 281:9781-90. [PMID: 16469736 PMCID: PMC1997280 DOI: 10.1074/jbc.m600133200] [Citation(s) in RCA: 55] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
The receptor for activated C kinase 1 (RACK1) is a conserved scaffold protein that helps regulate a range of cell activities including cell growth, shape, and protein translation. We report that a homologue of RACK1 is required for cytokinesis in pathogenic Trypanosoma brucei. The protein, referred to as TRACK, is comprised of WD repeat elements and can complement cpc2 null mutants of Schizosaccharomyces pombe. TRACK is expressed throughout the trypanosome life cycle and is distributed predominantly in a perinuclear region and the cytoplasm but not along the endoplasmic reticulum, mitochondrion, or cleavage furrow of dividing cells. When tetracycline-inducible RNA interference (RNAi) is used to deplete the cellular content of TRACK, the cells remain metabolically active, but growth is inhibited. In bloodstream forms, growth arrest is due to a delay in the onset of cytokinesis. By contrast, procyclic forms are able to initiate cytokinesis in the absence of TRACK but arrest midway through cell cleavage. The RNAi cells undergo multiple rounds of partial cytokinesis and accumulate nuclei and cytoplasmic extensions with attached flagella. The TRACK RNAi construct is also inducible within infected mice. Under these conditions parasites are eliminated from peripheral blood within 3 days post-infection. Taken as a whole, these data indicate that trypanosomes utilize a RACK1 homologue to regulate the final stages of mitosis. Moreover, disrupting the interaction between TRACK and its partners might be targeted in the design of novel therapies.
Collapse
Affiliation(s)
- Karen G Rothberg
- Department of Biological Sciences, Southern Methodist University, Dallas, Texas 75275, USA
| | | | | | | | | | | |
Collapse
|
|
40
|
Sklan EH, Podoly E, Soreq H. RACK1 has the nerve to act: structure meets function in the nervous system. Prog Neurobiol 2006; 78:117-34. [PMID: 16457939 DOI: 10.1016/j.pneurobio.2005.12.002] [Citation(s) in RCA: 101] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/02/2005] [Revised: 11/20/2005] [Accepted: 12/07/2005] [Indexed: 11/26/2022]
Abstract
The receptor for activated protein kinase C 1 (RACK1) is an intracellular adaptor protein. Accumulating evidence attributes to this member of the tryptophan-aspartate (WD) repeat family the role of regulating several major nervous system pathways. Structurally, RACK1 is a seven-bladed-beta-propeller, interacting with diverse proteins having distinct structural folds. When bound to the IP3 receptor, RACK1 regulates intracellular Ca2+ levels, potentially contributing to processes such as learning, memory and synaptic plasticity. By binding to the NMDA receptor, it dictates neuronal excitation and sensitivity to ethanol. When bound to the stress-induced acetylcholinesterase variant AChE-R, RACK1 is implicated in stress responses and behavior, compatible with reports of RACK1 modulations in brain ageing and in various neurodegenerative diseases. This review sheds new light on both the virtues and the variety of neuronal RACK1 interactions and their physiological consequences.
Collapse
Affiliation(s)
- Ella H Sklan
- The Department of Biological Chemistry, The Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem 91904, Israel
| | | | | |
Collapse
|
|
41
|
Ashique AM, Kharazia V, Yaka R, Phamluong K, Peterson AS, Ron D. Localization of the scaffolding protein RACK1 in the developing and adult mouse brain. Brain Res 2006; 1069:31-8. [PMID: 16414032 DOI: 10.1016/j.brainres.2005.11.018] [Citation(s) in RCA: 31] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/06/2005] [Revised: 11/08/2005] [Accepted: 11/09/2005] [Indexed: 11/20/2022]
Abstract
RACK1 is a multifunctional scaffolding protein known to be involved in the regulation of various signaling cascades in the central nervous system (CNS). In order to gain insight into the neurological functions of RACK1, we examined the expression of RACK1 mRNA and protein during gestation and in the adult mouse brain. Several expression patterns were observed. At embryonic day 11.5 (E11.5), RACK1 is expressed in a high-dorsal to low-ventral gradient throughout the brain. At E13.5, RACK1 is most abundant in the telencephalon. In the developing cortical primordium, RACK1 protein is expressed in a high-rostromidline to low-caudolateral gradient that appears to be regulated post-transcriptionally. At E18.5, RACK1 is expressed most abundantly in layers 1-4 of the cortex, striatum, hippocampus, dentate gyrus and specific thalamic nuclei. In the adult mouse, RACK1 is ubiquitously expressed in neuronal perikarya in most brain regions, with relatively higher levels in hippocampus, olfactory bulb, cortex and cerebellum. Subcellular staining was detected mainly in the cell bodies and extending into dendrites, whereas RACK1 was not present significantly in axonal fibers or nuclei. We also determined brain regions in which RACK1 interacts with one of its binding partners, the betaII isoform of protein kinase C (betaIIPKC). We found that betaIIPKC had a much more restricted expression pattern than RACK1 and overlapped with the scaffolding protein only in certain regions, including the CA1 area of the hippocampus, cerebellum and striatum. Our results suggest an important role for RACK1 during CNS development and support multiple functions of the protein in the adult brain.
Collapse
Affiliation(s)
- A M Ashique
- Ernest Gallo Clinic and Research Center, University of California San Francisco, 5858 Horton St., Suite 200, Emeryville, CA 94608, USA
| | | | | | | | | | | |
Collapse
|
|
42
|
Hofmann I, Casella M, Schnölzer M, Schlechter T, Spring H, Franke WW. Identification of the junctional plaque protein plakophilin 3 in cytoplasmic particles containing RNA-binding proteins and the recruitment of plakophilins 1 and 3 to stress granules. Mol Biol Cell 2006; 17:1388-98. [PMID: 16407409 PMCID: PMC1382326 DOI: 10.1091/mbc.e05-08-0708] [Citation(s) in RCA: 88] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
Recent studies on the subcellular distribution of cytoplasmic plaque proteins of intercellular junctions have revealed that a number of such proteins can also occur in the cyto- and the nucleoplasm. This occurrence in different, and distant locations suggest that some plaque proteins play roles in cytoplasmic and nuclear processes in addition to their involvement in cell-cell adhesive interactions. Plakophilin (PKP) 3, a member of the arm-repeat family of proteins, occurs, in a diversity of cell types, both as an architectural component in plaques of desmosomes and dispersed in cytoplasmic particles. In immuno-selection experiments using PKP3-specific antibodies, we have identified by mass spectrometric analysis the following RNA-binding proteins: Poly (A) binding protein (PABPC1), fragile-X-related protein (FXR1), and ras-GAP-SH3-binding protein (G3BP). Moreover, the RNA-binding proteins codistributed after sucrose gradient centrifugation in PKP3-containing fractions corresponding to 25-35 S and 45-55 S. When cells are exposed to environmental stress (e.g., heat shock or oxidative stress) proteins FXR1, G3BP, and PABPC1 are found, together with PKP3 or PKP1, in "stress granules" known to accumulate stalled translation initiation complexes. Moreover, the protein eIF-4E and the ribosomal protein S6 are also detected in PKP3 particles. Our results show that cytoplasmic PKP3 is constitutively associated with RNA-binding proteins and indicate an involvement in processes of translation and RNA metabolism.
Collapse
Affiliation(s)
- Ilse Hofmann
- Division of Cell Biology, German Cancer Research Center, D-69120 Heidelberg, Germany.
| | | | | | | | | | | |
Collapse
|
|
43
|
Elvira G, Wasiak S, Blandford V, Tong XK, Serrano A, Fan X, del Rayo Sánchez-Carbente M, Servant F, Bell AW, Boismenu D, Lacaille JC, McPherson PS, DesGroseillers L, Sossin WS. Characterization of an RNA granule from developing brain. Mol Cell Proteomics 2005; 5:635-51. [PMID: 16352523 DOI: 10.1074/mcp.m500255-mcp200] [Citation(s) in RCA: 226] [Impact Index Per Article: 11.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
In brain, mRNAs are transported from the cell body to the processes, allowing for local protein translation at sites distant from the nucleus. Using subcellular fractionation, we isolated a fraction from rat embryonic day 18 brains enriched for structures that resemble amorphous collections of ribosomes. This fraction was enriched for the mRNA encoding beta-actin, an mRNA that is transported in dendrites and axons of developing neurons. Abundant protein components of this fraction, determined by tandem mass spectrometry, include ribosomal proteins, RNA-binding proteins, microtubule-associated proteins (including the motor protein dynein), and several proteins described only as potential open reading frames. The conjunction of RNA-binding proteins, transported mRNA, ribosomal machinery, and transporting motor proteins defines these structures as RNA granules. Expression of a subset of the identified proteins in cultured hippocampal neurons confirmed that proteins identified in the proteomics were present in neurites associated with ribosomes and mRNAs. Moreover many of the expressed proteins co-localized together. Time lapse video microscopy indicated that complexes containing one of these proteins, the DEAD box 3 helicase, migrated in dendrites of hippocampal neurons at the same speed as that reported for RNA granules. Although the speed of the granules was unchanged by activity or the neurotrophin brain-derived neurotrophic factor, brain-derived neurotrophic factor, but not activity, increased the proportion of moving granules. These studies define the isolation and composition of RNA granules expressed in developing brain.
Collapse
Affiliation(s)
- George Elvira
- Département de Biochimie, Université de Montréal, 2900 Edouard-Montpetit, Montreal, Quebec H3C3J7, Canada
| | | | | | | | | | | | | | | | | | | | | | | | | | | |
Collapse
|
|
44
|
Abdelmegeed MA, Carruthers NJ, Woodcroft KJ, Kim SK, Novak RF. Acetoacetate induces CYP2E1 protein and suppresses CYP2E1 mRNA in primary cultured rat hepatocytes. J Pharmacol Exp Ther 2005; 315:203-13. [PMID: 15980059 DOI: 10.1124/jpet.105.084608] [Citation(s) in RCA: 29] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022] Open
Abstract
The ketone body acetoacetate (AA) in the absence of insulin or in the presence of diabetic insulin levels decreases CYP2E1 mRNA expression in a concentration- and time-dependent manner in primary cultured rat hepatocytes. AA activates p70 ribosomal S6 kinase (p70S6K) and protein kinase C (PKC) by approximately 2- to 2.5-fold, respectively, following 6-h treatment. The AA-mediated activation of p70S6K, but not PKC, was abolished by inhibition of PI 3-K with LY294002 [2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one] or wortmannin, in agreement with p70S6K being downstream of phosphatidylinositol 3-kinase (PI 3-K). Inhibition of PI 3-K, mTOR with rapamycin, or PKC with bisindolylmaleimide ameliorated the AA-mediated down-regulation of CYP2E1 mRNA expression. Neither the mitogen-activated protein kinase kinase inhibitor PD98059 (2'-amino-3'-methoxyflavone) nor the p38 mitogen-activated protein kinase inhibitor SB203580 [4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole] ameliorated the AA-mediated suppression of CYP2E1 mRNA expression. Heterogeneous nuclear RNA analysis revealed that AA suppressed CYP2E1 gene transcription by approximately 50% and that inhibition of PI 3-K and PKC diminished this AA-mediated effect on transcription. CYP2E1 mRNA half-life slightly increased from approximately 24 h in untreated hepatocytes to approximately 32 h in AA-treated cells. Interestingly, AA increased CYP2E1 protein levels by approximately 2- and 2.5-fold at 24 and 48 h, respectively. DL-beta-hydroxybutyrate was without effect. Polysomal distribution studies revealed that AA increased the proportion of RNA associated with the actively translated polysomal fractions versus the 40S to 60S untranslated fractions by approximately 40%. CYP2E1 protein half-life increased from approximately 8 h in untreated hepatocytes to approximately 24 in AA-treated cells. These data show that AA decreases CYP2E1 mRNA expression through inhibition of gene transcription while simultaneously elevating CYP2E1 protein levels through increased translation and decreased protein degradation.
Collapse
Affiliation(s)
- Mohamed A Abdelmegeed
- Institute of Environmental Health Sciences, Wayne State University, Detroit, MI 48201-2675, USA
| | | | | | | | | |
Collapse
|
|
45
|
Yu Y, Ji H, Doudna JA, Leary JA. Mass spectrometric analysis of the human 40S ribosomal subunit: native and HCV IRES-bound complexes. Protein Sci 2005; 14:1438-46. [PMID: 15883184 PMCID: PMC2253395 DOI: 10.1110/ps.041293005] [Citation(s) in RCA: 84] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022]
Abstract
Hepatitis C virus uses an internal ribosome entry site (IRES) in the viral RNA to directly recruit human 40S ribosome subunits during cap-independent translation initiation. Although IRES-mediated translation initiation is not subject to many of the regulatory mechanisms that control cap-dependent translation initiation, it is unknown whether other noncanonical protein factors are involved in this process. Thus, a global protein composition analysis of native and IRES-bound 40S ribosomal complexes has been conducted to facilitate an understanding of the IRES ribosome recruitment mechanism. A combined top-down and bottom-up mass spectrometry approach was used to identify both the proteins and their posttranslational modifications (PTMs) in the native 40S subunit and the IRES recruited translation initiation complex. Thirty-one out of a possible 32 ribosomal proteins were identified by combining top-down and bottom-up mass spectrometry techniques. Proteins were found to contain PTMs, including loss of methionine, acetylation, methylation, and disulfide bond formation. In addition to the 40S ribosomal proteins, RACK1 was consistently identified in the 40S fraction, indicating that this protein is associated with the 40S subunit. Similar methodology was then applied to the hepatitis C virus IRES-bound 40S complex. Two 40S ribosomal proteins, RS25 and RS29, were found to contain different PTMs than those in the native 40S subunit. In addition, RACK1, eukaryotic initiation factor 3 proteins and nucleolin were identified in the IRES-mediated translation initiation complex.
Collapse
Affiliation(s)
- Yonghao Yu
- Department of Chemistry, University of California, Berkeley 94720, USA
| | | | | | | |
Collapse
|
|
46
|
Nilsson J, Sengupta J, Frank J, Nissen P. Regulation of eukaryotic translation by the RACK1 protein: a platform for signalling molecules on the ribosome. EMBO Rep 2005; 5:1137-41. [PMID: 15577927 PMCID: PMC1299186 DOI: 10.1038/sj.embor.7400291] [Citation(s) in RCA: 226] [Impact Index Per Article: 11.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/16/2004] [Accepted: 09/30/2004] [Indexed: 11/08/2022] Open
Abstract
The receptor for activated C-kinase (RACK1) is a scaffold protein that is able to interact simultaneously with several signalling molecules. It binds to protein kinases and membrane-bound receptors in a regulated fashion. Interestingly, RACK1 is also a constituent of the eukaryotic ribosome, and a recent cryo-electron microscopy study localized it to the head region of the 40S subunit in the vicinity of the messenger RNA (mRNA) exit channel. RACK1 recruits activated protein kinase C to the ribosome, which leads to the stimulation of translation through the phosphorylation of initiation factor 6 and, potentially, of mRNA-associated proteins. RACK1 therefore links signal-transduction pathways directly to the ribosome, which allows translation to be regulated in response to cell stimuli. In addition, the fact that RACK1 associates with membrane-bound receptors indicates that it promotes the docking of ribosomes at sites where local translation is required, such as focal adhesions.
Collapse
Affiliation(s)
- Jakob Nilsson
- Department of Molecular Biology, University of Aarhus, Gustav Wieds Vej 10C, DK-8000 Aarhus C, Denmark
| | - Jayati Sengupta
- Health Research, Inc., State University of New York at Albany, Empire State Plaza, Albany, New York 12201-0509, USA
| | - Joachim Frank
- Health Research, Inc., State University of New York at Albany, Empire State Plaza, Albany, New York 12201-0509, USA
- Howard Hughes Medical Institute, Wadsworth Center, New York State Department of Health, State University of New York at Albany, Empire State Plaza, Albany, New York 12201-0509, USA
- Department of Biomedical Sciences, State University of New York at Albany, Empire State Plaza, Albany, New York 12201-0509, USA
| | - Poul Nissen
- Department of Molecular Biology, University of Aarhus, Gustav Wieds Vej 10C, DK-8000 Aarhus C, Denmark
- Tel: +45 89425025; Fax: +45 86123178;
| |
Collapse
|
|
47
|
Chang IF, Szick-Miranda K, Pan S, Bailey-Serres J. Proteomic characterization of evolutionarily conserved and variable proteins of Arabidopsis cytosolic ribosomes. PLANT PHYSIOLOGY 2005; 137:848-62. [PMID: 15734919 PMCID: PMC1065386 DOI: 10.1104/pp.104.053637] [Citation(s) in RCA: 119] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/16/2004] [Revised: 11/16/2004] [Accepted: 11/23/2004] [Indexed: 05/18/2023]
Abstract
Analysis of 80S ribosomes of Arabidopsis (Arabidopsis thaliana) by use of high-speed centrifugation, sucrose gradient fractionation, one- and two-dimensional gel electrophoresis, liquid chromatography purification, and mass spectrometry (matrix-assisted laser desorption/ionization time-of-flight and electrospray ionization) identified 74 ribosomal proteins (r-proteins), of which 73 are orthologs of rat r-proteins and one is the plant-specific r-protein P3. Thirty small (40S) subunit and 44 large (60S) subunit r-proteins were confirmed. In addition, an ortholog of the mammalian receptor for activated protein kinase C, a tryptophan-aspartic acid-domain repeat protein, was found to be associated with the 40S subunit and polysomes. Based on the prediction that each r-protein is present in a single copy, the mass of the Arabidopsis 80S ribosome was estimated as 3.2 MD (1,159 kD 40S; 2,010 kD 60S), with the 4 single-copy rRNAs (18S, 26S, 5.8S, and 5S) contributing 53% of the mass. Despite strong evolutionary conservation in r-protein composition among eukaryotes, Arabidopsis 80S ribosomes are variable in composition due to distinctions in mass or charge of approximately 25% of the r-proteins. This is a consequence of amino acid sequence divergence within r-protein gene families and posttranslational modification of individual r-proteins (e.g. amino-terminal acetylation, phosphorylation). For example, distinct types of r-proteins S15a and P2 accumulate in ribosomes due to evolutionarily divergence of r-protein genes. Ribosome variation is also due to amino acid sequence divergence and differential phosphorylation of the carboxy terminus of r-protein S6. The role of ribosome heterogeneity in differential mRNA translation is discussed.
Collapse
Affiliation(s)
- Ing-Feng Chang
- Center for Plant Cell Biology, Department of Botany and Plant Sciences, University of California, Riverside, California 92521-0124, USA.
| | | | | | | |
Collapse
|
|
48
|
Chen ML, Chen CH. Microarray analysis of differentially expressed genes in rat frontal cortex under chronic risperidone treatment. Neuropsychopharmacology 2005; 30:268-77. [PMID: 15536490 DOI: 10.1038/sj.npp.1300612] [Citation(s) in RCA: 38] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
Abstract
Long-term administration of antipsychotic drugs can induce differential expression of a variety of genes in the brain, which may underscore the molecular mechanism of the clinical efficacy and/or side effects of antipsychotic drugs. We used cDNA microarray analysis to screen differentially expressed genes in rat frontal cortex under 4 weeks' treatment of risperidone (1 mg/kg). Using real-time quantitative PCR, we were able to verify eight genes, whose expression were significantly upregulated in rat frontal cortex under chronic risperidone treatment when compared with control animals. These genes include receptor for activated protein kinase C, amida, cathepsin D, calpain 2, calcium-independent receptor for alpha-latrotoxin, monoamine oxidase B, polyubiquitin, and kinesin light chain. In view of the physiological function of these genes, the results of our study suggest that chronic risperidone treatment may affect the neurotransmission, synaptic plasticity, and proteolysis of brain cells. This study also demonstrates that cDNA microarray analysis is useful for uncovering genes that are regulated by chronic antipsychotic drugs treatment, which may help bring new insight into the molecular mechanism of antipsychotic drugs.
Collapse
Affiliation(s)
- Mao-Liang Chen
- Institute of Medical Sciences, Tzu-Chi University, Hualien City, Taiwan
| | | |
Collapse
|
|
49
|
Angenstein F, Evans AM, Ling SC, Settlage RE, Ficarro S, Carrero-Martinez FA, Shabanowitz J, Hunt DF, Greenough WT. Proteomic Characterization of Messenger Ribonucleoprotein Complexes Bound to Nontranslated or Translated Poly(A) mRNAs in the Rat Cerebral Cortex. J Biol Chem 2005; 280:6496-503. [PMID: 15596439 DOI: 10.1074/jbc.m412742200] [Citation(s) in RCA: 35] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/01/2023] Open
Abstract
Receptor-triggered control of local postsynaptic protein synthesis plays a crucial role for enabling long lasting changes in synaptic functions, but signaling pathways that link receptor stimulation with translational control remain poorly known. Among the putative regulatory factors are mRNA-binding proteins (messenger ribonucleoprotein, mRNP), which control the fate of cytosolic localized mRNAs. Based on the assumption that a subset of mRNA is maintained in an inactive state, mRNP-mRNA complexes were separated into polysome-bound (translated) and polysome-free (nontranslated) fractions by sucrose density centrifugation. Poly(A) mRNA-mRNP complexes were purified from a postmitochondrial extract of rat cerebral cortex by oligo(dT)-cellulose affinity chromatography. The mRNA processing proteins were characterized, from solution, by a nanoflow reverse phase-high pressure liquid chromatography-mu-electrospray ionization mass spectrometry. The majority of detected mRNA-binding proteins was found in both fractions. However, a small number of proteins appeared to be fraction-specific. This subset of proteins is by far the most interesting because the proteins are potentially involved in controlling an activity-dependent onset of translation. They include transducer proteins, kinases, and anchor proteins. This study of the mRNP proteome is the first step in allowing future experimentation to characterize individual proteins responsible for mRNA processing and translation in dendrites.
Collapse
Affiliation(s)
- Frank Angenstein
- Beckman Institute/Neuronal Pattern Analysis, University of Illinois, Urbana, Illinois 61801, USA.
| | | | | | | | | | | | | | | | | |
Collapse
|
|
50
|
Gerbasi VR, Weaver CM, Hill S, Friedman DB, Link AJ. Yeast Asc1p and mammalian RACK1 are functionally orthologous core 40S ribosomal proteins that repress gene expression. Mol Cell Biol 2004; 24:8276-87. [PMID: 15340087 PMCID: PMC515043 DOI: 10.1128/mcb.24.18.8276-8287.2004] [Citation(s) in RCA: 139] [Impact Index Per Article: 6.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022] Open
Abstract
Translation of mRNA into protein is a fundamental step in eukaryotic gene expression requiring the large (60S) and small (40S) ribosome subunits and associated proteins. By modern proteomic approaches, we previously identified a novel 40S-associated protein named Asc1p in budding yeast and RACK1 in mammals. The goals of this study were to establish Asc1p or RACK1 as a core conserved eukaryotic ribosomal protein and to determine the role of Asc1p or RACK1 in translational control. We provide biochemical, evolutionary, genetic, and functional evidence showing that Asc1p or RACK1 is indeed a conserved core component of the eukaryotic ribosome. We also show that purified Asc1p-deficient ribosomes have increased translational activity compared to that of wild-type yeast ribosomes. Further, we demonstrate that asc1Delta null strains have increased levels of specific proteins in vivo and that this molecular phenotype is complemented by either Asc1p or RACK1. Our data suggest that one of Asc1p's or RACK1's functions is to repress gene expression.
Collapse
Affiliation(s)
- Vincent R Gerbasi
- Department of Microbiology and Immunology, Vanderbilt University School of Medicine, 1161 21st Ave. South, Nashville, TN 37232-2363, USA
| | | | | | | | | |
Collapse
|