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1
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Nievergelt P, Berliat F, McAuley KE, Dorgan CR, van Well RM, Thorn A, Spingler B. RNA oligomers at atomic resolution containing 1-methylpseudouridine, an essential building block of mRNA vaccines. ChemMedChem 2024; 19:e202300600. [PMID: 38235959 DOI: 10.1002/cmdc.202300600] [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: 11/01/2023] [Revised: 01/15/2024] [Accepted: 01/17/2024] [Indexed: 01/19/2024]
Abstract
All widely used mRNA vaccines against COVID-19 contain in their sequence 1-methylpseudouridine (m1Ψ) instead of uridine. In this publication, we report two high resolution crystal structures (at up to 1.01 and 1.32 Å, respectively) of one such double-stranded 12-mer RNA sequence crystallized in two crystal forms. The structures are compared with similar structures which do not contain this modification. Additionally, the X-ray structure of 1-methyl-pseudouridine itself was determined.
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Affiliation(s)
- Philipp Nievergelt
- Department of Chemistry, University of Zurich, 8057, Zurich, Switzerland
| | - Florian Berliat
- Department of Chemistry, University of Zurich, 8057, Zurich, Switzerland
| | | | - Colin R Dorgan
- Biosynth Limited, Compton, Berkshire, RG20 6NE, United Kingdom
| | | | - Andrea Thorn
- Institut für Nanostruktur und Festkörperphysik, Universität Hamburg, 22761, Hamburg, Germany
| | - Bernhard Spingler
- Department of Chemistry, University of Zurich, 8057, Zurich, Switzerland
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2
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Chen JL, Leeder WM, Morais P, Adachi H, Yu YT. Pseudouridylation-mediated gene expression modulation. Biochem J 2024; 481:1-16. [PMID: 38174858 DOI: 10.1042/bcj20230096] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/14/2023] [Revised: 12/13/2023] [Accepted: 12/19/2023] [Indexed: 01/05/2024]
Abstract
RNA-guided pseudouridylation, a widespread post-transcriptional RNA modification, has recently gained recognition for its role in cellular processes such as pre-mRNA splicing and the modulation of premature termination codon (PTC) readthrough. This review provides insights into its mechanisms, functions, and potential therapeutic applications. It examines the mechanisms governing RNA-guided pseudouridylation, emphasizing the roles of guide RNAs and pseudouridine synthases in catalyzing uridine-to-pseudouridine conversion. A key focus is the impact of RNA-guided pseudouridylation of U2 small nuclear RNA on pre-mRNA splicing, encompassing its influence on branch site recognition and spliceosome assembly. Additionally, the review discusses the emerging role of RNA-guided pseudouridylation in regulating PTC readthrough, impacting translation termination and genetic disorders. Finally, it explores the therapeutic potential of pseudouridine modifications, offering insights into potential treatments for genetic diseases and cancer and the development of mRNA vaccine.
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Affiliation(s)
- Jonathan L Chen
- Department of Biochemistry and Biophysics, Center for RNA Biology, University of Rochester Medical Center, Rochester, NY, U.S.A
| | | | | | - Hironori Adachi
- Department of Biochemistry and Biophysics, Center for RNA Biology, University of Rochester Medical Center, Rochester, NY, U.S.A
| | - Yi-Tao Yu
- Department of Biochemistry and Biophysics, Center for RNA Biology, University of Rochester Medical Center, Rochester, NY, U.S.A
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3
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Vögele J, Duchardt-Ferner E, Kruse H, Zhang Z, Sponer J, Krepl M, Wöhnert J. Structural and dynamic effects of pseudouridine modifications on noncanonical interactions in RNA. RNA (NEW YORK, N.Y.) 2023; 29:790-807. [PMID: 36868785 PMCID: PMC10187676 DOI: 10.1261/rna.079506.122] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 11/03/2022] [Accepted: 02/10/2023] [Indexed: 05/18/2023]
Abstract
Pseudouridine is the most frequently naturally occurring RNA modification, found in all classes of biologically functional RNAs. Compared to uridine, pseudouridine contains an additional hydrogen bond donor group and is therefore widely regarded as a structure stabilizing modification. However, the effects of pseudouridine modifications on the structure and dynamics of RNAs have so far only been investigated in a limited number of different structural contexts. Here, we introduced pseudouridine modifications into the U-turn motif and the adjacent U:U closing base pair of the neomycin-sensing riboswitch (NSR)-an extensively characterized model system for RNA structure, ligand binding, and dynamics. We show that the effects of replacing specific uridines with pseudouridines on RNA dynamics crucially depend on the exact location of the replacement site and can range from destabilizing to locally or even globally stabilizing. By using a combination of NMR spectroscopy, MD simulations and QM calculations, we rationalize the observed effects on a structural and dynamical level. Our results will help to better understand and predict the consequences of pseudouridine modifications on the structure and function of biologically important RNAs.
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Affiliation(s)
- Jennifer Vögele
- Institute of Molecular Biosciences and Center for Biomolecular Magnetic Resonance (BMRZ), Goethe-University Frankfurt, 60438 Frankfurt, Germany
| | - Elke Duchardt-Ferner
- Institute of Molecular Biosciences and Center for Biomolecular Magnetic Resonance (BMRZ), Goethe-University Frankfurt, 60438 Frankfurt, Germany
| | - Holger Kruse
- Institute of Biophysics of the Czech Academy of Sciences, 612 65 Brno, Czech Republic
| | - Zhengyue Zhang
- Institute of Biophysics of the Czech Academy of Sciences, 612 65 Brno, Czech Republic
- CEITEC-Central European Institute of Technology, Masaryk University, 625 00 Brno, Czech Republic
- National Centre for Biomolecular Research, Faculty of Science, Masaryk University, 625 00 Brno, Czech Republic
| | - Jiri Sponer
- Institute of Biophysics of the Czech Academy of Sciences, 612 65 Brno, Czech Republic
| | - Miroslav Krepl
- Institute of Biophysics of the Czech Academy of Sciences, 612 65 Brno, Czech Republic
| | - Jens Wöhnert
- Institute of Molecular Biosciences and Center for Biomolecular Magnetic Resonance (BMRZ), Goethe-University Frankfurt, 60438 Frankfurt, Germany
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4
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Ramakrishnan M, Rajan KS, Mullasseri S, Palakkal S, Kalpana K, Sharma A, Zhou M, Vinod KK, Ramasamy S, Wei Q. The plant epitranscriptome: revisiting pseudouridine and 2'-O-methyl RNA modifications. PLANT BIOTECHNOLOGY JOURNAL 2022; 20:1241-1256. [PMID: 35445501 PMCID: PMC9241379 DOI: 10.1111/pbi.13829] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 02/11/2022] [Revised: 04/11/2022] [Accepted: 04/18/2022] [Indexed: 06/01/2023]
Abstract
There is growing evidence that post-transcriptional RNA modifications are highly dynamic and can be used to improve crop production. Although more than 172 unique types of RNA modifications have been identified throughout the kingdom of life, we are yet to leverage upon the understanding to optimize RNA modifications in crops to improve productivity. The contributions of internal mRNA modifications such as N6-methyladenosine (m6 A) and 5-methylcytosine (m5 C) methylations to embryonic development, root development, leaf morphogenesis, flowering, fruit ripening and stress response are sufficiently known, but the roles of the two most abundant RNA modifications, pseudouridine (Ψ) and 2'-O-methylation (Nm), in the cell remain unclear due to insufficient advances in high-throughput technologies in plant development. Therefore, in this review, we discuss the latest methods and insights gained in mapping internal Ψ and Nm and their unique properties in plants and other organisms. In addition, we discuss the limitations that remain in high-throughput technologies for qualitative and quantitative mapping of these RNA modifications and highlight future challenges in regulating the plant epitranscriptome.
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Affiliation(s)
- Muthusamy Ramakrishnan
- Co‐Innovation Center for Sustainable Forestry in Southern ChinaNanjing Forestry UniversityNanjingJiangsuChina
- Bamboo Research InstituteNanjing Forestry UniversityNanjingJiangsuChina
| | - K. Shanmugha Rajan
- The Mina and Everard Goodman Faculty of Life Sciences and Advanced Materials and Nanotechnology InstituteBar‐Ilan University52900Ramat‐GanIsrael
- Department of Chemical and Structural BiologyWeizmann Institute7610001RehovotIsrael
| | - Sileesh Mullasseri
- School of Ocean Science and TechnologyKerala University of Fisheries and Ocean StudiesCochinIndia
| | - Sarin Palakkal
- The Institute for Drug ResearchSchool of PharmacyThe Hebrew University of JerusalemJerusalemIsrael
| | - Krishnan Kalpana
- Department of Plant PathologyAgricultural College and Research InstituteTamilnadu Agricultural University625 104MaduraiTamil NaduIndia
| | - Anket Sharma
- State Key Laboratory of Subtropical SilvicultureZhejiang A&F UniversityHangzhouZhejiangChina
| | - Mingbing Zhou
- State Key Laboratory of Subtropical SilvicultureZhejiang A&F UniversityHangzhouZhejiangChina
- Zhejiang Provincial Collaborative Innovation Center for Bamboo Resources and High‐Efficiency UtilizationZhejiang A&F UniversityHangzhouZhejiangChina
| | | | - Subbiah Ramasamy
- Cardiac Metabolic Disease LaboratoryDepartment of BiochemistrySchool of Biological SciencesMadurai Kamaraj UniversityMaduraiTamil NaduIndia
| | - Qiang Wei
- Co‐Innovation Center for Sustainable Forestry in Southern ChinaNanjing Forestry UniversityNanjingJiangsuChina
- Bamboo Research InstituteNanjing Forestry UniversityNanjingJiangsuChina
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5
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Westhof E. Pseudouridines or how to draw on weak energy differences. Biochem Biophys Res Commun 2020; 520:702-704. [PMID: 31761086 DOI: 10.1016/j.bbrc.2019.10.009] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/13/2019] [Accepted: 10/01/2019] [Indexed: 01/28/2023]
Abstract
In many RNA molecules, pseudouridines occur at conserved positions in functional sites. A great diversity of pseudouridine synthases guarantees the insertion of the modified base at precise locations. The accepted structural role of pseudouridines is a reduction of the RNA flexibility around the modification site. However, experiments rarely yield clear-cut evidence. The article "Dynamic stacking of an expected branch point adenosine in duplexes containing pseudouridine-modified or unmodified U2 snRNA sites" published in 2019 in Biochemical and Biophysical Research Communication by Kennedy et al. constitute a provocative case [1]. This example illustrates how a definite conformational state can be selected through small energy differences in a constrained environment.
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Affiliation(s)
- Eric Westhof
- Architecture et Réactivité de l'ARN, Université de Strasbourg, Institut de Biologie Moléculaire et Cellulaire du CNRS, 15 rue René Descartes, 67084, Strasbourg, France.
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6
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Rajan K, Doniger T, Cohen-Chalamish S, Chen D, Semo O, Aryal S, Glick Saar E, Chikne V, Gerber D, Unger R, Tschudi C, Michaeli S. Pseudouridines on Trypanosoma brucei spliceosomal small nuclear RNAs and their implication for RNA and protein interactions. Nucleic Acids Res 2019; 47:7633-7647. [PMID: 31147702 PMCID: PMC6698659 DOI: 10.1093/nar/gkz477] [Citation(s) in RCA: 33] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/21/2019] [Revised: 05/11/2019] [Accepted: 05/18/2019] [Indexed: 12/15/2022] Open
Abstract
The parasite Trypanosoma brucei, the causative agent of sleeping sickness, cycles between an insect and a mammalian host. Here, we investigated the presence of pseudouridines (Ψs) on the spliceosomal small nuclear RNAs (snRNAs), which may enable growth at the very different temperatures characterizing the two hosts. To this end, we performed the first high-throughput mapping of spliceosomal snRNA Ψs by small RNA Ψ-seq. The analysis revealed 42 Ψs on T. brucei snRNAs, which is the highest number reported so far. We show that a trypanosome protein analogous to human protein WDR79, is essential for guiding Ψ on snRNAs but not on rRNAs. snoRNA species implicated in snRNA pseudouridylation were identified by a genome-wide approach based on ligation of RNAs following in vivo UV cross-linking. snRNA Ψs are guided by single hairpin snoRNAs, also implicated in rRNA modification. Depletion of such guiding snoRNA by RNAi compromised the guided modification on snRNA and reduced parasite growth at elevated temperatures. We further demonstrate that Ψ strengthens U4/U6 RNA–RNA and U2B"/U2A’ proteins-U2 snRNA interaction at elevated temperatures. The existence of single hairpin RNAs that modify both the spliceosome and ribosome RNAs is unique for these parasites, and may be related to their ability to cycle between their two hosts that differ in temperature.
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Affiliation(s)
- K Shanmugha Rajan
- The Mina and Everard Goodman Faculty of Life Sciences and Advanced Materials and Nanotechnology Institute, Bar-Ilan University, Ramat-Gan 52900, Israel
| | - Tirza Doniger
- The Mina and Everard Goodman Faculty of Life Sciences and Advanced Materials and Nanotechnology Institute, Bar-Ilan University, Ramat-Gan 52900, Israel
| | - Smadar Cohen-Chalamish
- The Mina and Everard Goodman Faculty of Life Sciences and Advanced Materials and Nanotechnology Institute, Bar-Ilan University, Ramat-Gan 52900, Israel
| | - Dana Chen
- The Mina and Everard Goodman Faculty of Life Sciences and Advanced Materials and Nanotechnology Institute, Bar-Ilan University, Ramat-Gan 52900, Israel
| | - Oz Semo
- The Mina and Everard Goodman Faculty of Life Sciences and Advanced Materials and Nanotechnology Institute, Bar-Ilan University, Ramat-Gan 52900, Israel
| | - Saurav Aryal
- The Mina and Everard Goodman Faculty of Life Sciences and Advanced Materials and Nanotechnology Institute, Bar-Ilan University, Ramat-Gan 52900, Israel
| | | | - Vaibhav Chikne
- The Mina and Everard Goodman Faculty of Life Sciences and Advanced Materials and Nanotechnology Institute, Bar-Ilan University, Ramat-Gan 52900, Israel
| | - Doron Gerber
- The Mina and Everard Goodman Faculty of Life Sciences and Advanced Materials and Nanotechnology Institute, Bar-Ilan University, Ramat-Gan 52900, Israel
| | - Ron Unger
- The Mina and Everard Goodman Faculty of Life Sciences and Advanced Materials and Nanotechnology Institute, Bar-Ilan University, Ramat-Gan 52900, Israel
| | - Christian Tschudi
- Departmentof Epidemiology and Microbial Diseases, Yale School of Public Health, New Haven, CT 06536, USA
| | - Shulamit Michaeli
- The Mina and Everard Goodman Faculty of Life Sciences and Advanced Materials and Nanotechnology Institute, Bar-Ilan University, Ramat-Gan 52900, Israel
- To whom correspondence should be addressed. Tel:+972 3 5317522;
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7
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van der Feltz C, Hoskins AA. Structural and functional modularity of the U2 snRNP in pre-mRNA splicing. Crit Rev Biochem Mol Biol 2019; 54:443-465. [PMID: 31744343 DOI: 10.1080/10409238.2019.1691497] [Citation(s) in RCA: 39] [Impact Index Per Article: 6.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022]
Abstract
The U2 small nuclear ribonucleoprotein (snRNP) is an essential component of the spliceosome, the cellular machine responsible for removing introns from precursor mRNAs (pre-mRNAs) in all eukaryotes. U2 is an extraordinarily dynamic splicing factor and the most frequently mutated in cancers. Cryo-electron microscopy (cryo-EM) has transformed our structural and functional understanding of the role of U2 in splicing. In this review, we synthesize these and other data with respect to a view of U2 as an assembly of interconnected functional modules. These modules are organized by the U2 small nuclear RNA (snRNA) for roles in spliceosome assembly, intron substrate recognition, and protein scaffolding. We describe new discoveries regarding the structure of U2 components and how the snRNP undergoes numerous conformational and compositional changes during splicing. We specifically highlight large scale movements of U2 modules as the spliceosome creates and rearranges its active site. U2 serves as a compelling example for how cellular machines can exploit the modular organization and structural plasticity of an RNP.
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Affiliation(s)
| | - Aaron A Hoskins
- Department of Biochemistry, University of Wisconsin-Madison, Madison, WI, USA
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8
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Structures of SF3b1 reveal a dynamic Achilles heel of spliceosome assembly: Implications for cancer-associated abnormalities and drug discovery. BIOCHIMICA ET BIOPHYSICA ACTA-GENE REGULATORY MECHANISMS 2019; 1862:194440. [PMID: 31707043 DOI: 10.1016/j.bbagrm.2019.194440] [Citation(s) in RCA: 18] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 08/20/2019] [Accepted: 09/30/2019] [Indexed: 12/11/2022]
Abstract
The pre-mRNA splicing factor SF3b1 exhibits recurrent mutations among hematologic malignancies and cancers, and consequently is a major therapeutic target of clinically-advanced spliceosome inhibitors. In this review, we highlight and rigorously analyze emerging views of SF3b1 conformational transitions, including the human SF3b particle either in isolation or bound to spliceosome inhibitors, and human or yeast spliceosome assemblies. Among spliceosome states characterized to date, an SF3b1 α-helical superhelix significantly closes to surround a U2 small nuclear RNA duplex with the pre-mRNA branch point sequence. The SF3b1 torus is locally unwound at an active site adenosine, whereas protein cofactors appear to stabilize overall closure in the spliceosome. Network analyses demonstrates that the natural SF3b1 dynamics mimic its conformational change in the spliceosome, raising the possibility of conformational selection underpinning spliceosome assembly. These dynamic SF3b1 conformations have consequences for gatekeeping of spliceosome assembly and therapeutic targeting of its cancer-associated dysfunction.
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9
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Computational and NMR studies of RNA duplexes with an internal pseudouridine-adenosine base pair. Sci Rep 2019; 9:16278. [PMID: 31700156 PMCID: PMC6838189 DOI: 10.1038/s41598-019-52637-0] [Citation(s) in RCA: 24] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/14/2019] [Accepted: 10/03/2019] [Indexed: 01/28/2023] Open
Abstract
Pseudouridine (Ψ) is the most common chemical modification present in RNA. In general, Ψ increases the thermodynamic stability of RNA. However, the degree of stabilization depends on the sequence and structural context. To explain experimentally observed sequence dependence of the effect of Ψ on the thermodynamic stability of RNA duplexes, we investigated the structure, dynamics and hydration of RNA duplexes with an internal Ψ-A base pair in different nearest-neighbor sequence contexts. The structures of two RNA duplexes containing 5′-GΨC/3′-CAG and 5′-CΨG/3′-GAC motifs were determined using NMR spectroscopy. To gain insight into the effect of Ψ on duplex dynamics and hydration, we performed molecular dynamics (MD) simulations of RNA duplexes with 5′-GΨC/3′-CAG, 5′-CΨG/3′-GAC, 5′-AΨU/3′-UAA and 5′-UΨA/3′-AAU motifs and their unmodified counterparts. Our results showed a subtle impact from Ψ modification on the structure and dynamics of the RNA duplexes studied. The MD simulations confirmed the change in hydration pattern when U is replaced with Ψ. Quantum chemical calculations showed that the replacement of U with Ψ affected the intrinsic stacking energies at the base pair steps depending on the sequence context. The calculated intrinsic stacking energies help to explain the experimentally observed sequence dependent changes in the duplex stability from Ψ modification.
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10
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Bohnsack MT, Sloan KE. Modifications in small nuclear RNAs and their roles in spliceosome assembly and function. Biol Chem 2019; 399:1265-1276. [PMID: 29908124 DOI: 10.1515/hsz-2018-0205] [Citation(s) in RCA: 91] [Impact Index Per Article: 15.2] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/02/2018] [Accepted: 05/28/2018] [Indexed: 01/27/2023]
Abstract
Modifications in cellular RNAs have emerged as key regulators of all aspects of gene expression, including pre-mRNA splicing. During spliceosome assembly and function, the small nuclear RNAs (snRNAs) form numerous dynamic RNA-RNA and RNA-protein interactions, which are required for spliceosome assembly, correct positioning of the spliceosome on substrate pre-mRNAs and catalysis. The human snRNAs contain several base methylations as well as a myriad of pseudouridines and 2'-O-methylated nucleotides, which are largely introduced by small Cajal body-specific ribonucleoproteins (scaRNPs). Modified nucleotides typically cluster in functionally important regions of the snRNAs, suggesting that their presence could optimise the interactions of snRNAs with each other or with pre-mRNAs, or may affect the binding of spliceosomal proteins. snRNA modifications appear to play important roles in snRNP biogenesis and spliceosome assembly, and have also been proposed to influence the efficiency and fidelity of pre-mRNA splicing. Interestingly, alterations in the modification status of snRNAs have recently been observed in different cellular conditions, implying that some snRNA modifications are dynamic and raising the possibility that these modifications may fine-tune the spliceosome for particular functions. Here, we review the current knowledge on the snRNA modification machinery and discuss the timing, functions and dynamics of modifications in snRNAs.
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Affiliation(s)
- Markus T Bohnsack
- Department of Molecular Biology, University Medical Center Göttingen, Humboldtallee 23, D-37073 Göttingen, Germany.,Göttingen Centre for Molecular Biosciences, Georg August University, Justus-von-Liebig-Weg 11, D-37077 Göttingen, Germany
| | - Katherine E Sloan
- Department of Molecular Biology, University Medical Center Göttingen, Humboldtallee 23, D-37073 Göttingen, Germany
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11
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Kennedy SD, Bauer WJ, Wang W, Kielkopf CL. Dynamic stacking of an expected branch point adenosine in duplexes containing pseudouridine-modified or unmodified U2 snRNA sites. Biochem Biophys Res Commun 2019; 511:416-421. [PMID: 30797552 DOI: 10.1016/j.bbrc.2019.02.073] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/01/2019] [Accepted: 02/14/2019] [Indexed: 11/19/2022]
Abstract
The pre-mRNA branch point sequence (BPS) anneals with a pseudouridine-modified region of the U2 small nuclear (sn)RNA, and offers a 2' hydroxyl group of a bulged adenosine as the nucleophile for the first catalytic step of pre-mRNA splicing. To increase our structural understanding of branch site selection, we characterized a duplex containing a BPS sequence that is common among multicellular eukaryotes (5'-UACUGAC-3') and the complementary U2 snRNA site using NMR. A major conformation of the expected branch site adenosine stacked within the duplex and paired with the conserved pseudouridine of the U2 snRNA strand. In contrast, the guanosine preceding the branch site appeared flexible and had weak contacts with the surrounding nucleotides. Pseudouridine-modified and unmodified U2 snRNA-BPS-containing duplexes remained structurally similar. These results highlight the importance of auxiliary factors to achieve the active bulged conformation of the branch site nucleophile for the first step of pre-mRNA splicing.
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Affiliation(s)
- Scott D Kennedy
- Department of Biochemistry and Biophysics, Center for RNA Biology, University of Rochester School of Medicine and Dentistry, Rochester, NY, 14642, USA.
| | - William J Bauer
- Department of Biochemistry and Biophysics, Center for RNA Biology, University of Rochester School of Medicine and Dentistry, Rochester, NY, 14642, USA
| | - Wenhua Wang
- Department of Biochemistry and Biophysics, Center for RNA Biology, University of Rochester School of Medicine and Dentistry, Rochester, NY, 14642, USA
| | - Clara L Kielkopf
- Department of Biochemistry and Biophysics, Center for RNA Biology, University of Rochester School of Medicine and Dentistry, Rochester, NY, 14642, USA.
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12
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Roundtree IA, Evans ME, Pan T, He C. Dynamic RNA Modifications in Gene Expression Regulation. Cell 2017; 169:1187-1200. [PMID: 28622506 PMCID: PMC5657247 DOI: 10.1016/j.cell.2017.05.045] [Citation(s) in RCA: 2351] [Impact Index Per Article: 293.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/28/2017] [Revised: 05/08/2017] [Accepted: 05/26/2017] [Indexed: 12/14/2022]
Abstract
Over 100 types of chemical modifications have been identified in cellular RNAs. While the 5' cap modification and the poly(A) tail of eukaryotic mRNA play key roles in regulation, internal modifications are gaining attention for their roles in mRNA metabolism. The most abundant internal mRNA modification is N6-methyladenosine (m6A), and identification of proteins that install, recognize, and remove this and other marks have revealed roles for mRNA modification in nearly every aspect of the mRNA life cycle, as well as in various cellular, developmental, and disease processes. Abundant noncoding RNAs such as tRNAs, rRNAs, and spliceosomal RNAs are also heavily modified and depend on the modifications for their biogenesis and function. Our understanding of the biological contributions of these different chemical modifications is beginning to take shape, but it's clear that in both coding and noncoding RNAs, dynamic modifications represent a new layer of control of genetic information.
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Affiliation(s)
- Ian A Roundtree
- Department of Biochemistry and Molecular Biology, and Institute for Biophysical Dynamics, The University of Chicago, 929 East 57(th) Street, Chicago, IL 60637, USA; Medical Scientist Training Program, The University of Chicago, 924 East 57(th) Street, Chicago, IL 60637, USA
| | - Molly E Evans
- Department of Biochemistry and Molecular Biology, and Institute for Biophysical Dynamics, The University of Chicago, 929 East 57(th) Street, Chicago, IL 60637, USA
| | - Tao Pan
- Department of Biochemistry and Molecular Biology, and Institute for Biophysical Dynamics, The University of Chicago, 929 East 57(th) Street, Chicago, IL 60637, USA.
| | - Chuan He
- Department of Biochemistry and Molecular Biology, and Institute for Biophysical Dynamics, The University of Chicago, 929 East 57(th) Street, Chicago, IL 60637, USA; Department of Chemistry, The University of Chicago, 929 East 57(th) Street, Chicago, IL 60637, USA; Howard Hughes Medical Institute, The University of Chicago, 929 East 57(th) Street, Chicago, IL 60637, USA.
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13
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Tan J, Ho JXJ, Zhong Z, Luo S, Chen G, Roca X. Noncanonical registers and base pairs in human 5' splice-site selection. Nucleic Acids Res 2016; 44:3908-21. [PMID: 26969736 PMCID: PMC4856993 DOI: 10.1093/nar/gkw163] [Citation(s) in RCA: 34] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/09/2015] [Revised: 03/01/2016] [Accepted: 03/02/2016] [Indexed: 12/27/2022] Open
Abstract
Accurate recognition of splice sites is essential for pre-messenger RNA splicing. Mammalian 5' splice sites are mainly recognized by canonical base-pairing to the 5' end of U1 small nuclear RNA, yet we described multiple noncanonical base-pairing registers by shifting base-pair positions or allowing one-nucleotide bulges. By systematic mutational and suppressor U1 analyses, we prove three registers involving asymmetric loops and show that two-nucleotide bulges but not longer can form in this context. Importantly, we established that a noncanonical uridine-pseudouridine interaction in the 5' splice site/U1 helix contributes to the recognition of certain 5' splice sites. Thermal melting experiments support the formation of noncanonical registers and uridine-pseudouridine interactions. Overall, we experimentally validated or discarded the majority of predicted noncanonical registers, to derive a list of 5' splice sites using such alternative mechanisms that is much different from the original. This study allows not only the mechanistic understanding of the recognition of a wide diversity of mammalian 5' splice sites, but also the future development of better splice-site scoring methods that reliably predict the effects of disease-causing mutations at these sequences.
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Affiliation(s)
- Jiazi Tan
- School of Biological Sciences, Nanyang Technological University, 637551, Singapore
| | - Jia Xin Jessie Ho
- School of Biological Sciences, Nanyang Technological University, 637551, Singapore
| | - Zhensheng Zhong
- Division of Chemistry & Biological Chemistry, School of Physical and Mathematical Sciences, Nanyang Technological University, 637371, Singapore
| | - Shufang Luo
- School of Biological Sciences, Nanyang Technological University, 637551, Singapore
| | - Gang Chen
- Division of Chemistry & Biological Chemistry, School of Physical and Mathematical Sciences, Nanyang Technological University, 637371, Singapore
| | - Xavier Roca
- School of Biological Sciences, Nanyang Technological University, 637551, Singapore
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14
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Abstract
Nucleic acid crystallization buffers contain a large variety of chemicals fitting specific needs. Among them, anions are often solely considered for pH-regulating purposes and as cationic co-salts while their ability to directly bind to nucleic acid structures is rarely taken into account. Here we review current knowledge related to the use of anions in crystallization buffers along with data on their biological prevalence. Chloride ions are frequently identified in crystal structures but display low cytosolic concentrations. Hence, they are thought to be distant from nucleic acid structures in the cell. Sulfate ions are also frequently identified in crystal structures but their localization in the cell remains elusive. Nevertheless, the characterization of the binding properties of these ions is essential for better interpreting the solvent structure in crystals and consequently, avoiding mislabeling of electron densities. Furthermore, understanding the binding properties of these anions should help to get clues related to their potential effects in crowded cellular environments.
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Affiliation(s)
- Luigi D'Ascenzo
- Architecture et Réactivité de l'ARN, Institut de Biologie Moléculaire et Cellulaire du CNRS, UPR 9002 CNRS/Université de Strasbourg, 15, rue René Descartes, Strasbourg Cedex, 67084, France
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15
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Chawla M, Oliva R, Bujnicki JM, Cavallo L. An atlas of RNA base pairs involving modified nucleobases with optimal geometries and accurate energies. Nucleic Acids Res 2015; 43:6714-29. [PMID: 26117545 PMCID: PMC4538814 DOI: 10.1093/nar/gkv606] [Citation(s) in RCA: 47] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/31/2015] [Accepted: 05/28/2015] [Indexed: 01/08/2023] Open
Abstract
Posttranscriptional modifications greatly enhance the chemical information of RNA molecules, contributing to explain the diversity of their structures and functions. A significant fraction of RNA experimental structures available to date present modified nucleobases, with half of them being involved in H-bonding interactions with other bases, i.e. ‘modified base pairs’. Herein we present a systematic investigation of modified base pairs, in the context of experimental RNA structures. To this end, we first compiled an atlas of experimentally observed modified base pairs, for which we recorded occurrences and structural context. Then, for each base pair, we selected a representative for subsequent quantum mechanics calculations, to find out its optimal geometry and interaction energy. Our structural analyses show that most of the modified base pairs are non Watson–Crick like and are involved in RNA tertiary structure motifs. In addition, quantum mechanics calculations quantify and provide a rationale for the impact of the different modifications on the geometry and stability of the base pairs they participate in.
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Affiliation(s)
- Mohit Chawla
- King Abdullah University of Science and Technology (KAUST), Physical Sciences and Engineering Division, Kaust Catalysis Center, Thuwal 23955-6900, Saudi Arabia
| | - Romina Oliva
- Department of Sciences and Technologies, University Parthenope of Naples, Centro Direzionale Isola C4, I-80143, Naples, Italy
| | - Janusz M Bujnicki
- Laboratory of Bioinformatics and Protein Engineering, International Institute of Molecular and Cell Biology in Warsaw, ul. Ks. Trojdena 4, 02-109 Warsaw, Poland Laboratory of Bioinformatics, Institute of Molecular Biology and Biotechnology, Faculty of Biology, Adam Mickiewicz University, Umultowska 89, 61-614 Poznan, Poland
| | - Luigi Cavallo
- King Abdullah University of Science and Technology (KAUST), Physical Sciences and Engineering Division, Kaust Catalysis Center, Thuwal 23955-6900, Saudi Arabia
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16
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Adachi H, Yu YT. Insight into the mechanisms and functions of spliceosomal snRNA pseudouridylation. World J Biol Chem 2014; 5:398-408. [PMID: 25426264 PMCID: PMC4243145 DOI: 10.4331/wjbc.v5.i4.398] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/12/2013] [Revised: 05/20/2014] [Accepted: 09/10/2014] [Indexed: 02/05/2023] Open
Abstract
Pseudouridines (Ψs) are the most abundant and highly conserved modified nucleotides found in various stable RNAs of all organisms. Most Ψs are clustered in regions that are functionally important for pre-mRNA splicing. Ψ has an extra hydrogen bond donor that endows RNA molecules with distinct properties that contribute significantly to RNA-mediated cellular processes. Experimental data indicate that spliceosomal snRNA pseudouridylation can be catalyzed by both RNA-dependent and RNA-independent mechanisms. Recent work has also demonstrated that pseudouridylation can be induced at novel positions under stress conditions, suggesting a regulatory role for Ψ.
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17
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Montemayor EJ, Katolik A, Clark NE, Taylor AB, Schuermann JP, Combs DJ, Johnsson R, Holloway SP, Stevens SW, Damha MJ, Hart PJ. Structural basis of lariat RNA recognition by the intron debranching enzyme Dbr1. Nucleic Acids Res 2014; 42:10845-55. [PMID: 25123664 PMCID: PMC4176325 DOI: 10.1093/nar/gku725] [Citation(s) in RCA: 34] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022] Open
Abstract
The enzymatic processing of cellular RNA molecules requires selective recognition of unique chemical and topological features. The unusual 2',5'-phosphodiester linkages in RNA lariats produced by the spliceosome must be hydrolyzed by the intron debranching enzyme (Dbr1) before they can be metabolized or processed into essential cellular factors, such as snoRNA and miRNA. Dbr1 is also involved in the propagation of retrotransposons and retroviruses, although the precise role played by the enzyme in these processes is poorly understood. Here, we report the first structures of Dbr1 alone and in complex with several synthetic RNA compounds that mimic the branchpoint in lariat RNA. The structures, together with functional data on Dbr1 variants, reveal the molecular basis for 2',5'-phosphodiester recognition and explain why the enzyme lacks activity toward 3',5'-phosphodiester linkages. The findings illuminate structure/function relationships in a unique enzyme that is central to eukaryotic RNA metabolism and set the stage for the rational design of inhibitors that may represent novel therapeutic agents to treat retroviral infections and neurodegenerative disease.
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Affiliation(s)
- Eric J Montemayor
- Department of Biochemistry, The University of Texas Health Science Center, San Antonio, TX 78229, USA X-ray Crystallography Core Laboratory, The University of Texas Health Science Center, San Antonio, TX 78229, USA
| | - Adam Katolik
- Department of Chemistry, McGill University, Montreal, Quebec H3A 0B8, Canada
| | - Nathaniel E Clark
- Department of Biochemistry, The University of Texas Health Science Center, San Antonio, TX 78229, USA X-ray Crystallography Core Laboratory, The University of Texas Health Science Center, San Antonio, TX 78229, USA
| | - Alexander B Taylor
- Department of Biochemistry, The University of Texas Health Science Center, San Antonio, TX 78229, USA X-ray Crystallography Core Laboratory, The University of Texas Health Science Center, San Antonio, TX 78229, USA
| | - Jonathan P Schuermann
- Northeastern Collaborative Access Team, Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14853, USA
| | - D Joshua Combs
- Program in Cellular and Molecular Biology, University of Texas at Austin, Austin, TX 78212, USA
| | - Richard Johnsson
- Department of Chemistry, McGill University, Montreal, Quebec H3A 0B8, Canada
| | - Stephen P Holloway
- Department of Biochemistry, The University of Texas Health Science Center, San Antonio, TX 78229, USA
| | - Scott W Stevens
- Department of Molecular Biosciences, The University of Texas at Austin, Austin, TX 78712, USA Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, TX 78712, USA
| | - Masad J Damha
- Department of Chemistry, McGill University, Montreal, Quebec H3A 0B8, Canada
| | - P John Hart
- Department of Biochemistry, The University of Texas Health Science Center, San Antonio, TX 78229, USA X-ray Crystallography Core Laboratory, The University of Texas Health Science Center, San Antonio, TX 78229, USA Geriatric Research, Education, and Clinical Center, Department of Veterans Affairs, South Texas Veterans Health Care System, San Antonio, TX 78229, USA
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18
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Spenkuch F, Motorin Y, Helm M. Pseudouridine: still mysterious, but never a fake (uridine)! RNA Biol 2014; 11:1540-54. [PMID: 25616362 PMCID: PMC4615568 DOI: 10.4161/15476286.2014.992278] [Citation(s) in RCA: 166] [Impact Index Per Article: 15.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/26/2014] [Revised: 09/23/2014] [Accepted: 10/10/2014] [Indexed: 01/15/2023] Open
Abstract
Pseudouridine (Ψ) is the most abundant of >150 nucleoside modifications in RNA. Although Ψ was discovered as the first modified nucleoside more than half a century ago, neither the enzymatic mechanism of its formation, nor the function of this modification are fully elucidated. We present the consistent picture of Ψ synthases, their substrates and their substrate positions in model organisms of all domains of life as it has emerged to date and point out the challenges that remain concerning higher eukaryotes and the elucidation of the enzymatic mechanism.
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MESH Headings
- Escherichia coli/genetics
- Escherichia coli/metabolism
- Humans
- Intramolecular Transferases/genetics
- Intramolecular Transferases/metabolism
- Isoenzymes/genetics
- Isoenzymes/metabolism
- Nucleic Acid Conformation
- Pseudouridine/metabolism
- RNA/genetics
- RNA/metabolism
- RNA Processing, Post-Transcriptional
- RNA, Mitochondrial
- RNA, Ribosomal/genetics
- RNA, Ribosomal/metabolism
- RNA, Transfer, Amino Acid-Specific/chemistry
- RNA, Transfer, Amino Acid-Specific/genetics
- RNA, Transfer, Amino Acid-Specific/metabolism
- Ribonucleoproteins, Small Nuclear/genetics
- Ribonucleoproteins, Small Nuclear/metabolism
- Ribosomes/chemistry
- Ribosomes/metabolism
- Saccharomyces cerevisiae/genetics
- Saccharomyces cerevisiae/metabolism
- Uridine/metabolism
- RNA, Guide, CRISPR-Cas Systems
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Affiliation(s)
- Felix Spenkuch
- Institute of Pharmacy and Biochemistry; Johannes Gutenberg-University of Mainz; Mainz, Germany
| | - Yuri Motorin
- Laboratoire IMoPA; Ingénierie Moléculaire et Physiopathologie Articulaire; BioPôle de l'Université de Lorraine; Campus Biologie-Santé; Faculté de Médecine; Vandoeuvre-les-Nancy Cedex, France
| | - Mark Helm
- Institute of Pharmacy and Biochemistry; Johannes Gutenberg-University of Mainz; Mainz, Germany
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19
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Ge J, Yu YT. RNA pseudouridylation: new insights into an old modification. Trends Biochem Sci 2013; 38:210-8. [PMID: 23391857 PMCID: PMC3608706 DOI: 10.1016/j.tibs.2013.01.002] [Citation(s) in RCA: 199] [Impact Index Per Article: 16.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/16/2012] [Revised: 12/22/2012] [Accepted: 01/07/2013] [Indexed: 12/18/2022]
Abstract
Pseudouridine is the most abundant post-transcriptionally modified nucleotide in various stable RNAs of all organisms. Pseudouridine is derived from uridine via base-specific isomerization, resulting in an extra hydrogen-bond donor that distinguishes it from other nucleotides. In eukaryotes, uridine-to-pseudouridine isomerization is catalyzed primarily by box H/ACA RNPs, ribonucleoproteins that act as pseudouridylases. When introduced into RNA, pseudouridine contributes significantly to RNA-mediated cellular processes. It was recently discovered that pseudouridylation can be induced by stress, suggesting a regulatory role for pseudouridine. It has also been reported that pseudouridine can be artificially introduced into mRNA by box H/ACA RNPs and that such introduction can mediate nonsense-to-sense codon conversion, thus demonstrating a new means of generating coding or protein diversity.
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Affiliation(s)
- Junhui Ge
- Department of Biochemistry and Biophysics, Center for RNA Biology, University of Rochester Medical Center, 601 Elmwood Avenue, Rochester, NY 14642, USA
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20
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Popović M, Nelson JD, Schroeder KT, Greenbaum NL. Impact of base pair identity 5' to the spliceosomal branch site adenosine on branch site conformation. RNA (NEW YORK, N.Y.) 2012; 18:2093-2103. [PMID: 23002123 PMCID: PMC3479398 DOI: 10.1261/rna.035782.112] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 08/01/2012] [Accepted: 08/07/2012] [Indexed: 06/01/2023]
Abstract
The branch site helix from Saccharomyces cerevisiae with pseudouridine (ψ) incorporated in a phylogenetically conserved position of U2 snRNA features an extrahelical branch site adenosine (A) that forms a base triple interaction with the minor groove edge of a widely conserved purine(U2 strand)-pyrimidine(intron strand) (R(U2)-Y(intron)) base pair two positions upstream. In these studies, NMR spectra of a duplex in which 2-aminopurine (2ap), a fluorescent analog of adenine lacking the proposed hydrogen bond donor, was substituted for the branch site A, indicated that the substitution does not alter the extrahelical position of the branch site residue; thus, it appears that a hydrogen bond between the adenine amino group and the R-Y pair is not obligatory for stabilization of the extrahelical conformation. In contrast, reversal of the orientation of A(U2)-U(intron) to U(U2)-A(intron) resulted in an intrahelical position for the branch site A or 2ap. Fluorescence intensity of 2ap substituted for the branch site A with the original R(U2)-Y(intron) orientation (AU or GC) was high, consistent with an extrahelical position, whereas fluorescence in helices with the reversed R-Y orientation, or with a mismatched pair (A-U → G•A or U•C), was markedly quenched, implying that the residue was stacked in the helix. The A 5' to the branch site residue was not extrahelical in any of the duplexes. These findings suggest that the R(U2)-Y(intron) base pair orientation in the ψ-dependent branch site helix plays an important role in positioning the branch site A for recognition and/or function.
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Affiliation(s)
- Milena Popović
- Department of Chemistry and Biochemistry, Hunter College of the City University of New York, New York, New York 10065, USA
- Department of Chemistry and Biochemistry, The Florida State University, Tallahassee, Florida 32306, USA
| | - Joycelynn D. Nelson
- Department of Chemistry and Biochemistry, The Florida State University, Tallahassee, Florida 32306, USA
| | - Kersten T. Schroeder
- Department of Chemistry and Biochemistry, The Florida State University, Tallahassee, Florida 32306, USA
| | - Nancy L. Greenbaum
- Department of Chemistry and Biochemistry, Hunter College of the City University of New York, New York, New York 10065, USA
- The Graduate Center, City University of New York, New York, New York 10016, USA
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21
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Roca X, Akerman M, Gaus H, Berdeja A, Bennett CF, Krainer AR. Widespread recognition of 5' splice sites by noncanonical base-pairing to U1 snRNA involving bulged nucleotides. Genes Dev 2012; 26:1098-109. [PMID: 22588721 DOI: 10.1101/gad.190173.112] [Citation(s) in RCA: 77] [Impact Index Per Article: 5.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/24/2022]
Abstract
An established paradigm in pre-mRNA splicing is the recognition of the 5' splice site (5'ss) by canonical base-pairing to the 5' end of U1 small nuclear RNA (snRNA). We recently reported that a small subset of 5'ss base-pair to U1 in an alternate register that is shifted by 1 nucleotide. Using genetic suppression experiments in human cells, we now demonstrate that many other 5'ss are recognized via noncanonical base-pairing registers involving bulged nucleotides on either the 5'ss or U1 RNA strand, which we term "bulge registers." By combining experimental evidence with transcriptome-wide free-energy calculations of 5'ss/U1 base-pairing, we estimate that 10,248 5'ss (∼5% of human 5'ss) in 6577 genes use bulge registers. Several of these 5'ss occur in genes with mutations causing genetic diseases and are often associated with alternative splicing. These results call for a redefinition of an essential element for gene expression that incorporates these registers, with important implications for the molecular classification of splicing mutations and for alternative splicing.
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Affiliation(s)
- Xavier Roca
- Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724, USA
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22
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Yu AT, Ge J, Yu YT. Pseudouridines in spliceosomal snRNAs. Protein Cell 2011; 2:712-25. [PMID: 21976061 PMCID: PMC4722041 DOI: 10.1007/s13238-011-1087-1] [Citation(s) in RCA: 41] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/01/2011] [Accepted: 08/22/2011] [Indexed: 01/14/2023] Open
Abstract
Spliceosomal RNAs are a family of small nuclear RNAs (snRNAs) that are essential for pre-mRNA splicing. All vertebrate spliceosomal snRNAs are extensively pseudouridylated after transcription. Pseudouridines in spliceosomal snRNAs are generally clustered in regions that are functionally important during splicing. Many of these modified nucleotides are conserved across species lines. Recent studies have demonstrated that spliceosomal snRNA pseudouridylation is catalyzed by two different mechanisms: an RNA-dependent mechanism and an RNA-independent mechanism. The functions of the pseudouridines in spliceosomal snRNAs (U2 snRNA in particular) have also been extensively studied. Experimental data indicate that virtually all pseudouridines in U2 snRNA are functionally important. Besides the currently known pseudouridines (constitutive modifications), recent work has also indicated that pseudouridylation can be induced at novel positions under stress conditions, thus strongly suggesting that pseudouridylation is also a regulatory modification.
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Affiliation(s)
- Andrew T. Yu
- Department of Biochemistry and Biophysics, Center for RNA Biology, University of Rochester Medical Center, 601 Elmwood Avenue, Rochester, NY 14642 USA
| | - Junhui Ge
- Department of Pathology, Changzheng Hospital, Second Military Medical University, Shanghai, 200003 China
| | - Yi-Tao Yu
- Department of Biochemistry and Biophysics, Center for RNA Biology, University of Rochester Medical Center, 601 Elmwood Avenue, Rochester, NY 14642 USA
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23
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Wu G, Yu AT, Kantartzis A, Yu YT. Functions and mechanisms of spliceosomal small nuclear RNA pseudouridylation. WILEY INTERDISCIPLINARY REVIEWS. RNA 2011; 2:571-81. [PMID: 21957045 PMCID: PMC4161978 DOI: 10.1002/wrna.77] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/03/2023]
Abstract
Pseudouridines are the most abundant and highly conserved modified nucleotides identified in spliceosomal small nuclear RNAs (snRNAs). Most pseudouridines are also clustered in functionally important regions of spliceosomal snRNAs. Experiments carried out in several independent experimental systems show that the pseudouridines in spliceosomal snRNAs are functionally important for pre-messenger RNA (mRNA) splicing. Experimental data also indicate that spliceosomal snRNA pseudouridylation can be catalyzed by both RNA-dependent (box H/ACA Ribonucleoproteins) and RNA-independent (protein-only enzymes) mechanisms.
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Affiliation(s)
- Guowei Wu
- Department of Biochemistry and Biophysics, University of Rochester Medical Center, Rochester, NY, USA
| | - Andrew T. Yu
- Department of Biochemistry and Biophysics, University of Rochester Medical Center, Rochester, NY, USA
| | - Athena Kantartzis
- Department of Biochemistry and Biophysics, University of Rochester Medical Center, Rochester, NY, USA
| | - Yi-Tao Yu
- Department of Biochemistry and Biophysics, University of Rochester Medical Center, Rochester, NY, USA
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24
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Schellenberg MJ, Dul EL, MacMillan AM. Structural model of the p14/SF3b155 · branch duplex complex. RNA (NEW YORK, N.Y.) 2011; 17:155-65. [PMID: 21062891 PMCID: PMC3004057 DOI: 10.1261/rna.2224411] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 04/14/2010] [Accepted: 10/01/2010] [Indexed: 05/30/2023]
Abstract
Human p14 (SF3b14), a component of the spliceosomal U2 snRNP, interacts directly with the pre-mRNA branch adenosine within the context of the bulged duplex formed between the pre-mRNA branch region and U2 snRNA. This association occurs early in spliceosome assembly and persists within the fully assembled spliceosome. Analysis of the crystal structure of a complex containing p14 and a peptide derived from p14-associated SF3b155 combined with the results of cross-linking studies has suggested that the branch nucleotide interacts with a pocket on a non-canonical RNA binding surface formed by the complex. Here we report a structural model of the p14 · bulged duplex interaction based on a combination of X-ray crystallography of an adenine p14/SF3b155 peptide complex, biochemical comparison of a panel of disulfide cross-linked protein-RNA complexes, and small-angle X-ray scattering (SAXS). These studies reveal specific recognition of the branch adenosine within the p14 pocket and establish the orientation of the bulged duplex RNA bound on the protein surface. The intimate association of one surface of the bulged duplex with the p14/SF3b155 peptide complex described by this model buries the branch nucleotide at the interface and suggests that p14 · duplex interaction must be disrupted before the first step of splicing.
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Affiliation(s)
- Matthew J Schellenberg
- Department of Biochemistry, School of Molecular and Systems Medicine, University of Alberta, Edmonton, Alberta, Canada
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25
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Abstract
Post-transcriptional ribonucleotide modification is a phenomenon best studied in tRNA, where it occurs most frequently and in great chemical diversity. This paper reviews the intrinsic network of modifications in the structural core of the tRNA, which governs structural flexibility and rigidity to fine-tune the molecule to peak performance and to regulate its steady-state level. Structural effects of RNA modifications range from nanometer-scale rearrangements to subtle restrictions of conformational space on the angstrom scale. Structural stabilization resulting from nucleotide modification results in increased thermal stability and translates into protection against unspecific degradation by bases and nucleases. Several mechanisms of specific degradation of hypomodified tRNA, which were only recently discovered, provide a link between structural and metabolic stability.
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Affiliation(s)
- Yuri Motorin
- Laboratoire ARN-RNP Maturation-Structure-Fonction, Enzymologie Moléculaire et Structurale (AREMS), UMR 7214 CNRS-UHP Faculté des Sciences et Techniques, Université Henri Poincaré, Nancy 1, Bld des Aiguillettes, BP 70239, 54506 Vandoeuvre-les-Nancy, France
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26
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Kim NK, Theimer CA, Mitchell JR, Collins K, Feigon J. Effect of pseudouridylation on the structure and activity of the catalytically essential P6.1 hairpin in human telomerase RNA. Nucleic Acids Res 2010; 38:6746-56. [PMID: 20554853 PMCID: PMC2965242 DOI: 10.1093/nar/gkq525] [Citation(s) in RCA: 51] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022] Open
Abstract
Telomerase extends the 3'-ends of linear chromosomes by adding conserved telomeric DNA repeats and is essential for cell proliferation and genomic stability. Telomerases from all organisms contain a telomerase reverse transcriptase and a telomerase RNA (TER), which together provide the minimal functional elements for catalytic activity in vitro. The RNA component of many functional ribonucleoproteins contains modified nucleotides, including conserved pseudouridines (Ψs) that can have subtle effects on structure and activity. We have identified potential Ψ modification sites in human TER. Two of the predicted Ψs are located in the loop of the essential P6.1 hairpin from the CR4-CR5 domain that is critical for telomerase catalytic activity. We investigated the effect of P6.1 pseudouridylation on its solution NMR structure, thermodynamic stability of folding and telomerase activation in vitro. The pseudouridylated P6.1 has a significantly different loop structure and increase in stability compared to the unmodified P6.1. The extent of loop nucleotide interaction with adjacent residues more closely parallels the extent of loop nucleotide evolutionary sequence conservation in the Ψ-modified P6.1 structure. Pseudouridine-modification of P6.1 slightly attenuates telomerase activity but slightly increases processivity in vitro. Our results suggest that Ψs could have a subtle influence on human telomerase activity via impact on TER-TERT or TER-TER interactions.
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Affiliation(s)
- Nak-Kyoon Kim
- Department of Chemistry and Biochemistry, University of California, Los Angeles, CA 90095, USA
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27
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Invariant U2 snRNA nucleotides form a stem loop to recognize the intron early in splicing. Mol Cell 2010; 38:416-27. [PMID: 20471947 DOI: 10.1016/j.molcel.2010.02.036] [Citation(s) in RCA: 60] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/24/2009] [Revised: 09/10/2009] [Accepted: 02/16/2010] [Indexed: 12/16/2022]
Abstract
U2 snRNA-intron branchpoint pairing is a critical step in pre-mRNA recognition by the splicing apparatus, but the mechanism by which these two RNAs engage each other is unknown. Here, we identify a U2 snRNA structure, the branchpoint-interacting stem loop (BSL), which presents the U2 nucleotides that will contact the intron. We provide evidence that the BSL forms prior to interaction with the intron and is disrupted by the DExD/H protein Prp5p during engagement of the snRNA with the intron. In vitro splicing complex assembly in a BSL-destabilized mutant extract suggests that the BSL is required at a previously unrecognized step between commitment complex and prespliceosome formation. The extreme evolutionary conservation of the BSL suggests that it represents an ancient structural solution to the problem of intron branchpoint recognition by dynamic RNA elements that must serve multiple functions at other times during splicing.
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28
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Abstract
Spliceosomal snRNAs are extensively 2'-O-methylated and pseudouridylated. The modified nucleotides are relatively highly conserved across species, and are often clustered in regions of functional importance in pre-mRNA splicing. Over the past decade, the study of the mechanisms and functions of spliceosomal snRNA modifications has intensified. Two independent mechanisms behind these modifications, RNA-independent (protein-only) and RNA-dependent (RNA-guided), have been discovered. The role of spliceosomal snRNA modifications in snRNP biogenesis and spliceosome assembly has also been verified.
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Affiliation(s)
- John Karijolich
- Department of Biochemistry and Biophysics, University of Rochester Medical Center, Rochester, NY, USA
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29
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Abstract
Accuracy in the flow of genetic information from DNA to protein, or gene expression, is essential to the viability of an organisms. Pre-mRNA splicing and protein translation are two major steps in eukaryotic gene expression that necessitate the production of accurate gene products. Both processes occur in large complexes, consisting of both proteins and noncoding RNAs. Interestingly, the RNA components contain a large number of posttranscriptional modifications, including 2'-O-methylation and pseudouridylation, which are functionally important. In this chapter, we highlight the functional aspects of the modifications of spliceosomal snRNA and rRNA and provide a framework for understanding how posttranscriptional modifications are capable of influencing gene expression.
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30
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Ritchie DB, Schellenberg MJ, MacMillan AM. Spliceosome structure: piece by piece. BIOCHIMICA ET BIOPHYSICA ACTA-GENE REGULATORY MECHANISMS 2009; 1789:624-33. [PMID: 19733268 DOI: 10.1016/j.bbagrm.2009.08.010] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 05/12/2009] [Revised: 08/22/2009] [Accepted: 08/27/2009] [Indexed: 10/20/2022]
Abstract
Processing of pre-mRNAs by RNA splicing is an essential step in the maturation of protein coding RNAs in eukaryotes. Structural studies of the cellular splicing machinery, the spliceosome, are a major challenge in structural biology due to the size and complexity of the splicing ensemble. Specifically, the structural details of splice site recognition and the architecture of the spliceosome active site are poorly understood. X-ray and NMR techniques have been successfully used to address these questions defining the structure of individual domains, isolated splicing proteins, spliceosomal RNA fragments and recently the U1 snRNP multiprotein.RNA complex. These results combined with extant biochemical and genetic data have yielded important insights as well as posing fresh questions with respect to the regulation and mechanism of this critical gene regulatory process.
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Affiliation(s)
- Dustin B Ritchie
- Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2H7
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31
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Sperling J, Azubel M, Sperling R. Structure and function of the Pre-mRNA splicing machine. Structure 2009; 16:1605-15. [PMID: 19000813 DOI: 10.1016/j.str.2008.08.011] [Citation(s) in RCA: 59] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/01/2008] [Revised: 08/22/2008] [Accepted: 08/27/2008] [Indexed: 12/14/2022]
Abstract
Most eukaryotic pre-mRNAs contain non-coding sequences (introns) that must be removed in order to accurately place the coding sequences (exons) in the correct reading frame. This critical regulatory pre-mRNA splicing event is fundamental in development and cancer. It occurs within a mega-Dalton multicomponent machine composed of RNA and proteins, which undergoes dynamic changes in RNA-RNA, RNA-protein, and protein-protein interactions during the splicing reaction. Recent years have seen progress in functional and structural analyses of the splicing machine and its subcomponents, and this review is focused on structural aspects of the pre-mRNA splicing machine and their mechanistic implications on the splicing of multi-intronic pre-mRNAs. It brings together, in a comparative manner, structural information on spliceosomes and their intermediates in the stepwise assembly process in vitro, and on the preformed supraspliceosomes, which are isolated from living cell nuclei, with a view of portraying a consistent picture.
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Affiliation(s)
- Joseph Sperling
- Department of Organic Chemistry, The Weizmann Institute of Science, Rehovot 76100, Israel
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Urban A, Behm-Ansmant I, Branlant C, Motorin Y. RNA sequence and two-dimensional structure features required for efficient substrate modification by the Saccharomyces cerevisiae RNA:{Psi}-synthase Pus7p. J Biol Chem 2008; 284:5845-58. [PMID: 19114708 DOI: 10.1074/jbc.m807986200] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
The RNA:pseudouridine (Psi) synthase Pus7p of Saccharomyces cerevisiae is a multisite-specific enzyme that is able to modify U(13) in several yeast tRNAs, U(35) in the pre-tRNA(Tyr) (GPsiA), U(35) in U2 small nuclear RNA, and U(50) in 5 S rRNA. Pus7p belongs to the universally conserved TruD-like family of RNA:Psi-synthases found in bacteria, archaea, and eukarya. Although several RNA substrates for yeast Pus7p have been identified, specificity of their recognition and modification has not been studied. However, conservation of a 7-nt-long sequence, including the modified U residue, in all natural Pus7p substrates suggested the importance of these nucleotides for Pus7p recognition and/or catalysis. Using site-directed mutagenesis, we designed a set of RNA variants derived from the yeast tRNA(Asp)(GUC), pre-tRNA(Tyr)(GPsiA), and U2 small nuclear RNA and tested their ability to be modified by Pus7p in vitro. We demonstrated that the highly conserved U(-2) and A(+1) residues (nucleotide numbers refer to target U(0)) are crucial identity elements for efficient modification by Pus7p. Nucleotide substitutions at other surrounding positions (-4, -3, +2, +3) have only a moderate effect. Surprisingly, the identity of the nucleotide immediately 5' to the target U(0) residue (position -1) is not important for efficient modification. Alteration of tRNA three-dimensional structure had no detectable effect on Pus7p activity at position 13. However, our results suggest that the presence of at least one stem-loop structure including or close to the target U nucleotide is required for Pus7p-catalyzed modification.
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Affiliation(s)
- Alan Urban
- Laboratoire de Maturation des ARN et Enzymologie Moléculaire, UMR 7567, CNRS-UHP Nancy I, Nancy Université, 54506 Vandoeuvre-les-Nancy Cedex, France
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