• RNA
• snRNP
• splice site
• spliceosome
• splicing

• Spliceosomes/metabolism
• Spliceosomes/genetics
• Humans
• RNA Splice Sites
• RNA Splicing
• Saccharomyces cerevisiae/genetics
• Saccharomyces cerevisiae/metabolism
• Introns
• Ribonucleoprotein, U2 Small Nuclear/metabolism
• Ribonucleoprotein, U2 Small Nuclear/genetics
• RNA, Small Nuclear/metabolism
• RNA, Small Nuclear/genetics
• RNA, Small Nuclear/chemistry
• Ribonucleoprotein, U1 Small Nuclear/metabolism
• Ribonucleoprotein, U1 Small Nuclear/genetics
• Ribonucleoprotein, U1 Small Nuclear/chemistry
• Protein Binding

• C-terminal domain of RNA polymerase II
• OTS964
• ULM–UHM interaction
• cell cycle progression
• constitutive splicing
• histone transcription

• Alternative polyadenylation
• Alternative splicing
• Congenital heart defects
• Heart development
• RNA binding protein
• mRNA

• Female
• Pregnancy
• Infant, Newborn
• Humans
• Heart
• Heart Defects, Congenital/genetics
• RNA, Messenger/metabolism
• RNA
• RNA-Binding Proteins/genetics
• RNA-Binding Proteins/metabolism

• R01 HL150589 NHLBI NIH HHS
• R01 HL157780 NHLBI NIH HHS

• Ovarian cancer (OC)
• Oxidative phosphorylation
• PABPC1
• PUF60
• mRNA decay

• Female
• Humans
• Cell Line, Tumor
• Cell Movement
• Cell Proliferation
• Gene Expression Regulation, Neoplastic
• Ovarian Neoplasms/drug therapy
• Ovarian Neoplasms/pathology
• Oxidative Phosphorylation
• Up-Regulation

• 202140129 Shanghai Municipal Health Commission
• 81974407,81672587 National Natural Science Foundation of China

• CP: Molecular biology
• RBM17
• SAB30BP
• SF3B1
• SF3b155
• SPF45
• U2 snRNP
• U2AF heterodimer
• U2AF-homology motif
• UHM
• UHM-ligand motif
• ULM
• polypyrimidine tract
• pre-mRNA splicing
• short intron

• Humans
• Introns/genetics
• Splicing Factor U2AF/genetics
• Splicing Factor U2AF/metabolism
• RNA Splicing/genetics
• RNA Splicing Factors/genetics
• RNA Splicing Factors/metabolism
• Spliceosomes/metabolism
• Ribonucleoprotein, U2 Small Nuclear/genetics
• Transcription Factors/metabolism
• RNA Precursors/metabolism
• Nuclear Proteins/genetics
• Nuclear Proteins/metabolism

• FUBP1-interacting repressor (FIR)
• RPB6 of RNA polymerase (RNAP)
• aberrant RNA splicing
• ribosomal RNA (rRNA)
• transcription factor IIH (TFIIH) P62 PH (pleckstrin homology)

• Humans
• RNA Splicing Factors/genetics
• RNA, Messenger/genetics
• RNA, Messenger/metabolism
• Repressor Proteins/genetics
• Alternative Splicing
• Neoplasms/drug therapy
• Neoplasms/genetics
• Transcription Factor TFIIH/genetics
• Transcription Factor TFIIH/metabolism
• DNA-Binding Proteins/genetics
• DNA-Binding Proteins/metabolism
• RNA-Binding Proteins/metabolism

• RNA-binding proteins
• alternative splicing
• gene regulation
• high-throughput screening
• neurogenesis
• single-cell profiling

• Alternative Splicing
• Animals
• Exons/genetics
• Mammals
• Mice
• Neurogenesis/genetics
• Neurons
• RNA Splicing
• RNA-Binding Proteins/genetics

• ZIA BC012019 Intramural NIH HHS

• Cell Membrane/metabolism
• Cytoplasm/metabolism
• Diffusion
• Escherichia coli/metabolism
• Escherichia coli Proteins/chemistry

• European Commission Directorate-General for Research and Innovation
• H2020 European Research Council
• NWO

Human pre-mRNA introns vary in size from under fifty to over a million nucleotides. We searched for essential factors involved in the splicing of human short introns by screening siRNAs against 154 human nuclear proteins. The splicing activity was assayed with a model HNRNPH1 pre-mRNA containing short 56-nucleotide intron. We identify a known alternative splicing regulator SPF45 (RBM17) as a constitutive splicing factor that is required to splice out this 56-nt intron. Whole-transcriptome sequencing of SPF45-deficient cells reveals that SPF45 is essential in the efficient splicing of many short introns. To initiate the spliceosome assembly on a short intron with the truncated poly-pyrimidine tract, the U2AF-homology motif (UHM) of SPF45 competes out that of U2AF⁶⁵ (U2AF2) for binding to the UHM-ligand motif (ULM) of the U2 snRNP protein SF3b155 (SF3B1). We propose that splicing in a distinct subset of human short introns depends on SPF45 but not U2AF heterodimer.

The length distribution of human pre-mRNA introns is very extensive. The authors demonstrate that splicing in a subset of short introns is dependent on SPF45 (RBM17), which replaces authentic U2AF-heterodimer on the truncated poly-pyrimidine tracts and interacts with the U2 snRNP protein SF3b155.

• Allostery
• BPS, branch-point sequence
• Bact, activated B spliceosome assembly
• Cryo-EM structure
• Cryo-EM, cryo-electron microscopy
• DOPE, discrete optimized protein energy
• NMA, normal mode analysis
• PDB, protein data bank
• Protein dynamics
• RMSD, root mean square deviation
• RRM, RNA recognition motif
• SF3b complex
• SF3b1
• SF3b1SF3b6−bound, SF3b1 bound to SF3b6
• SF3b1iso, SF3b1 in isolation
• SIP, square inner product
• Spliceosome

Interactions between U2AF homology motifs (UHMs) and U2AF ligand motifs (ULMs) play a crucial role in early spliceosome assembly in eukaryotic gene regulation. UHM-ULM interactions mediate heterodimerization of the constitutive splicing factors U2AF65 and U2AF35 and between other splicing factors that regulate spliceosome assembly at the 3′ splice site, where UHM domains of alternative splicing factors, such as SPF45 and PUF60, contribute to alternative splicing regulation. Here, we performed high-throughput screening using fluorescence polarization assays with hit validation by NMR and identified phenothiazines as general inhibitors of UHM-ULM interactions. NMR studies show that these compounds occupy the tryptophan binding pocket of UHM domains. Co-crystal structures of the inhibitors with the PUF60 UHM domain and medicinal chemistry provide structure-activity-relationships and reveal functional groups important for binding. These inhibitors inhibit early spliceosome assembly on pre-mRNA substrates in vitro. Our data show that spliceosome assembly can be inhibited by targeting UHM-ULM interactions by small molecules, thus extending the toolkit of splicing modulators for structural and biochemical studies of the spliceosome and splicing regulation.

So far only a few compounds have been reported as splicing modulators. Here, the authors combine high-throughput screening, chemical synthesis, NMR, X-ray crystallography with functional studies and develop phenothiazines as inhibitors for the U2AF Homology Motif (UHM) domains of proteins that regulate splicing and show that they inhibit early spliceosome assembly on pre-mRNA substrates in vitro.

• Conformational analysis
• Drug discovery
• FBDD
• Hit validation
• SBDD

• Calorimetry, Differential Scanning
• Crystallography, X-Ray
• Drug Design
• Drug Discovery/methods
• High-Throughput Screening Assays
• Ligands
• Molecular Docking Simulation
• Nuclear Magnetic Resonance, Biomolecular/methods
• Protein Binding
• Quantitative Structure-Activity Relationship
• Small Molecule Libraries

• PUF60
• RNA-seq
• coloboma
• nonsense-mediated mRNA decay
• vertebral abnormality

U2AF65 (U2AF2) and PUF60 (PUF60) are splicing factors important for recruitment of the U2 small nuclear ribonucleoprotein to lariat branch points and selection of 3′ splice sites (3′ss). Both proteins preferentially bind uridine-rich sequences upstream of 3′ss via their RNA recognition motifs (RRMs). Here, we examined 36 RRM substitutions reported in cancer patients to identify variants that alter 3′ss selection, RNA binding and protein properties. Employing PUF60- and U2AF65-dependent 3′ss previously identified by RNA-seq of depleted cells, we found that 43% (10/23) and 15% (2/13) of independent RRM mutations in U2AF65 and PUF60, respectively, conferred splicing defects. At least three RRM mutations increased skipping of internal U2AF2 (~9%, 2/23) or PUF60 (~8%, 1/13) exons, indicating that cancer-associated RRM mutations can have both cis- and trans-acting effects on splicing. We also report residues required for correct folding/stability of each protein and map functional RRM substitutions on to existing high-resolution structures of U2AF65 and PUF60. These results identify new RRM residues critical for 3′ss selection and provide relatively simple tools to detect clonal RRM mutations that enhance the mRNA isoform diversity.

• 3′ splice site
• Functional genomics
• PUF60
• SF3B4
• U2AF2
• cancer
• differential scanning fluorimetry
• driver mutation
• exon inclusion
• gel shift assay
• lariat branch point
• leukemia
• mRNA
• pre-mRNA splicing

• high-throughput screening
• cancer genomics
• focal adhesion
• cell signalling

• 18kk0305007h9902 Japan Agency for Medical Research and Development (AMED)
• Grant-in-Aid 26460667 for priority areas in cancer research from “the Ministry of Education, Science, Sports and Culture of Japan” (KAKENHI) “Chiba Foundation for Health Promotion & Disease Prevention” and was partly supported by Extramural Collaborative Research Grant of Cancer Research Institute, Kanazawa University to K.M.

The HIV-1 protein Rev is essential for virus replication and ensures the expression of partially spliced and unspliced transcripts. We identified a ULM (UHM ligand motif) motif in the Arginine-Rich Motif (ARM) of the Rev protein. ULMs (UHM ligand motif) mediate protein interactions during spliceosome assembly by binding to UHM (U2AF homology motifs) domains. Using NMR, biophysical methods and crystallography we show that the Rev ULM binds to the UHMs of U2AF65 and SPF45. The highly conserved Trp45 in the Rev ULM is crucial for UHM binding in vitro, for Rev co-precipitation with U2AF65 in human cells and for proper processing of HIV transcripts. Thus, Rev-ULM interactions with UHM splicing factors contribute to the regulation of HIV-1 transcript processing, also at the splicing level. The Rev ULM is an example of viral mimicry of host short linear motifs that enables the virus to interfere with the host molecular machinery.

• DEAD-box protein
• UHM
• ULM
• branchpoint
• pre-mRNA splicing
• pre-spliceosome

• Adenosine Triphosphate/metabolism
• Amino Acid Motifs
• Binding Sites
• Conserved Sequence
• Crystallography, X-Ray
• DEAD-box RNA Helicases/metabolism
• Humans
• Models, Molecular
• Mutation
• Protein Binding
• RNA Splicing
• RNA-Binding Proteins/chemistry
• RNA-Binding Proteins/genetics
• RNA-Binding Proteins/metabolism
• Ribonucleoprotein, U2 Small Nuclear/chemistry
• Ribonucleoprotein, U2 Small Nuclear/genetics
• Ribonucleoprotein, U2 Small Nuclear/metabolism
• Saccharomyces cerevisiae/genetics
• Saccharomyces cerevisiae/metabolism
• Saccharomyces cerevisiae Proteins/chemistry
• Saccharomyces cerevisiae Proteins/genetics
• Saccharomyces cerevisiae Proteins/metabolism

• R01 GM040478 NIGMS NIH HHS
• R01 GM070503 NIGMS NIH HHS
• R01 GM117005 NIGMS NIH HHS

PUF60 is a splicing factor that binds uridine (U)-rich tracts and facilitates association of the U2 small nuclear ribonucleoprotein with primary transcripts. PUF60 deficiency (PD) causes a developmental delay coupled with intellectual disability and spinal, cardiac, ocular and renal defects, but PD pathogenesis is not understood. Using RNA-Seq, we identify human PUF60-regulated exons and show that PUF60 preferentially acts as their activator. PUF60-activated internal exons are enriched for Us upstream of their 3′ splice sites (3′ss), are preceded by longer AG dinucleotide exclusion zones and more distant branch sites, with a higher probability of unpaired interactions across a typical branch site location as compared to control exons. In contrast, PUF60-repressed exons show U-depletion with lower estimates of RNA single-strandedness. We also describe PUF60-regulated, alternatively spliced isoforms encoding other U-bound splicing factors, including PUF60 partners, suggesting that they are co-regulated in the cell, and identify PUF60-regulated exons derived from transposed elements. PD-associated amino-acid substitutions, even within a single RNA recognition motif (RRM), altered selection of competing 3′ss and branch points of a PUF60-dependent exon and the 3′ss choice was also influenced by alternative splicing of PUF60. Finally, we propose that differential distribution of RNA processing steps detected in cells lacking PUF60 and the PUF60-paralog RBM39 is due to the RBM39 RS domain interactions. Together, these results provide new insights into regulation of exon usage by the 3′ss organization and reveal that germline mutation heterogeneity in RRMs can enhance phenotypic variability at the level of splice-site and branch-site selection.

• X-ray crystallography
• protein–protein interaction
• isothermal titration calorimetry (ITC)
• RNA splicing
• protein structure
• RNA-binding protein
• protein domain
• gene regulation

• Dynamics
• HuR
• Ion-mobility
• NMR
• RNA
• RRM
• Structure

• Binding Sites
• Dimerization
• ELAV-Like Protein 1/metabolism
• Humans
• Mass Spectrometry
• Nuclear Magnetic Resonance, Biomolecular
• Protein Binding
• RNA/chemistry
• RNA/metabolism
• RNA-Binding Proteins/chemistry
• Ribonucleoside Diphosphate Reductase
• Tumor Suppressor Proteins/chemistry

Overexpression of alternative splicing of far upstream element binding protein 1 (FUBP1) interacting repressor (FIR; poly(U) binding splicing factor 60 [PUF60]) and cyclin E were detected in esophageal squamous cell carcinomas (ESCC). Accordingly, the expression of FBW7 was examined by which cyclin E is degraded as a substrate via the proteasome system. Expectedly, FBW7 expression was decreased significantly in ESCC. Conversely, c-myc gene transcriptional repressor FIR (alias PUF60; U2AF-related protein) and its alternative splicing variant form (FIRΔexon2) were overexpressed in ESCC. Further, anticancer drugs (cis-diaminedichloroplatinum/cisplatin [CDDP] or 5-fluorouracil [5-FU]) and knockdown of FIR by small interfering RNA (siRNA) increased cyclin E while knockdown of FIRΔexon2 by siRNA decreased cyclin E expression in ESCC cell lines (TE1, TE2, and T.Tn) or cervical SCC cells (HeLa cells). Especially, knockdown of SAP155 (SF3b1), a splicing factor required for proper alternative splicing of FIR pre-mRNA, decreased cyclin E. Therefore, disturbed alternative splicing of FIR generated FIR/FIRΔexon2 with cyclin E overexpression in esophageal cancers, indicating that SAP155 siRNA potentially rescued FBW7 function by reducing expression of FIR and/or FIRΔexon2. Remarkably, Three-dimensional structure analysis revealed the hypothetical inhibitory mechanism of FBW7 function by FIR/FIRΔexon2, a novel mechanism of cyclin E overexpression by FIR/FIRΔexon2-FBW7 interaction was discussed. Clinically, elevated FIR expression potentially is an indicator of the number of lymph metastases and anti-FIR/FIRΔexon2 antibodies in sera as cancer diagnosis, indicating chemical inhibitors of FIR/FIRΔexon2-FBW7 interaction could be potential candidate drugs for cancer therapy. In conclusion, elevated cyclin E expression was, in part, induced owing to potential FIR/FIRΔexon2–FBW7 interaction in ESCC.

• FBW7
• FIR (PUF60)
• alternative splicing (AS)
• cyclin E
• esophageal squamous cell carcinoma (ESCC)

• Anti-PUF60 antibodies
• Clinically amyopathic dermatomyositis
• Disease activity
• Idiopathic inflammatory myopathies

Here we identified PUF60, a splicing factor and a U2 small nuclear ribonucleoprotein auxiliary factor, as a versatile regulator of transcriptional and post-transcriptional steps in expression of hepatitis B virus (HBV) 3.5 kb, precore plus pregenomic RNA. We demonstrate that PUF60 is involved in: 1) up-regulation of core promoter activity through its interaction with transcription factor TCF7L2, 2) promotion of 3.5 kb RNA degradation and 3) suppression of 3.5 kb RNA splicing. When the 1.24-fold HBV genome was introduced into cells with the PUF60-expression plasmid, the 3.5 kb RNA level was higher at days 1–2 post-transfection but declined thereafter in PUF60-expressing cells compared to viral replication control cells. Deletion analyses showed that the second and first RNA recognition motifs (RRMs) within PUF60 are responsible for core promoter activation and RNA degradation, respectively. Expression of PUF60 mutant deleting the first RRM led to higher HBV production. To our knowledge, this is the first to identify a host factor involved in not only positively regulating viral gene expression but also negative regulation of the same viral life cycle. Functional linkage between transcriptional and post-transcriptional controls during viral replication might be involved in mechanisms for intracellular antiviral defense and viral persistence.

The switch of pyruvate kinase (PK) M1 to PKM2 is pivotal for glucose metabolism in cancers. The PKM1/M2 shift is controlled by the alternative splicing of two mutually exclusive exons in the PKM gene. PKM1 is expressed in differentiated tissues, whereas PKM2 is expressed in cancer tissues. This study revealed that the haplodeficiency of FUSE-binding protein (FBP)-interacting repressor (FIR), a transcriptional repressor of the c-myc gene, contributed to the splicing of PKM1 to PKM2 in mice thymic lymphoma and/or T-cell type acute lymphoblastic leukemia (T-ALL) using six-plex tandem mass tag (TMT) quantitative proteomic analysis. TMT revealed 648 proteins that were up- or downregulated in mice thymic lymphoma tissues compared with wild type mouse. These proteins included transcription factors and proteins involved in DNA damage repair, DNA replication, T-cell activation/proliferation, apoptosis, etc. Among them, PKM2 protein, but not PKM1, was upregulated in the thymic lymphoma as well as T-ALL. Using qRT-PCR, we revealed that the activation of PKM2 mRNA was higher in thymic lymphoma cells of FIR^+/−TP53^−/− mice than that in control lymphocytes of FIR^+/+TP53^−/− sorted by flow cytometry. FIR knockdown by siRNA suppressed hnRNPA1 expression in HeLa cells. These results indicated that FIR haplodeficiency contributes the alternative splicing of PKM1 to PKM2 by partly inhibiting hnRNPA1 expression in the thymic lymphoma cells prior to T-ALL. Taken together, our findings suggest that FIR and its related spliceosomes are potential therapeutic targets for cancers, including T-ALL.

• FUSE-binding protein-interacting repressor
• LC-MS/MS
• pyruvate kinase M2
• six-plex tandem mass tags labeling
• thymic lymphoma

Proteins and RNA are often found in ribonucleoprotein particles (RNPs), where they function in cellular processes to synthesize proteins (the ribosome), chemically modify RNAs (small nucleolar RNPs), splice pre-mRNAs (the spliceosome), and, on a larger scale, sequester RNAs, degrade them, or process them (P bodies, Cajal bodies, and nucleoli). Each RNA–protein interaction is a story in itself, as both molecules can change conformation, compete for binding sites, and regulate cellular functions. Recent studies of Xist long non-coding RNP, the U4/5/6 tri-small nuclear RNP complex, and an activated state of a spliceosome reveal new features of RNA interactions with proteins, and, although their stories are incomplete, they are already fascinating.

• RNA-protein interaction
• RNPs
• Ribonucleoprotein Particles
• U4/5/6 tri-snRNP complex
• Xist
• lncRNA

• RRM
• SF3B1
• U2AF1
• UHM
• protein–protein interaction

• Humans
• Ligands
• Protein Binding
• Proteins/chemistry
• Ribonucleoproteins/metabolism

• P30 AI078498 NIAID NIH HHS
• R01 GM070503 NIGMS NIH HHS
• R01 GM117005 NIGMS NIH HHS

The auxiliary factor of U2 small nuclear ribonucleoprotein (U2AF) facilitates branch point (BP) recognition and formation of lariat introns. The gene for the 35-kD subunit of U2AF gives rise to two protein isoforms (termed U2AF35a and U2AF35b) that are encoded by alternatively spliced exons 3 and Ab, respectively. The splicing recognition sequences of exon 3 are less favorable than exon Ab, yet U2AF35a expression is higher than U2AF35b across tissues. We show that U2AF35b repression is facilitated by weak, closely spaced BPs next to a long polypyrimidine tract of exon Ab. Each BP lacked canonical uridines at position -2 relative to the BP adenines, with efficient U2 base-pairing interactions predicted only for shifted registers reminiscent of programmed ribosomal frameshifting. The BP cluster was compensated by interactions involving unpaired cytosines in an upstream, EvoFold-predicted stem loop (termed ESL) that binds FUBP1/2. Exon Ab inclusion correlated with predicted free energies of mutant ESLs, suggesting that the ESL operates as a conserved rheostat between long inverted repeats upstream of each exon. The isoform-specific U2AF35 expression was U2AF65-dependent, required interactions between the U2AF-homology motif (UHM) and the α6 helix of U2AF35, and was fine-tuned by exon Ab/3 variants. Finally, we identify tandem homologous exons regulated by U2AF and show that their preferential responses to U2AF65-related proteins and SRSF3 are associated with unpaired pre-mRNA segments upstream of U2AF-repressed 3′ss. These results provide new insights into tissue-specific subfunctionalization of duplicated exons in vertebrate evolution and expand the repertoire of exon repression mechanisms that control alternative splicing.

• CAPERα
• HCC1
• RBM39
• RNA-binding protein 39
• RNA-binding proteins
• U2AF homology motif
• U2AF5
• UHM
• ULM
• alternative splicing

• Crystallography, X-Ray
• Humans
• Jurkat Cells
• Multiprotein Complexes/chemistry
• Nuclear Magnetic Resonance, Biomolecular
• Nuclear Proteins/chemistry
• Protein Domains
• Protein Structure, Quaternary
• RNA-Binding Proteins/chemistry
• Splicing Factor U2AF/chemistry

• P41 GM103393 NIGMS NIH HHS
• U01 GM094653 NIGMS NIH HHS
• U54 GM094586 NIGMS NIH HHS
• P41GM103393 NIGMS NIH HHS

Splicing factor 1 (SF1) recognizes the branch point sequence (BPS) at the 3′ splice site during the formation of early complex E, thereby pre-bulging the BPS adenosine, thought to facilitate subsequent base-pairing of the U2 snRNA with the BPS. The 65-kDa subunit of U2 snRNP auxiliary factor (U2AF65) interacts with SF1 and was shown to recruit the U2 snRNP to the spliceosome. Co-immunoprecipitation experiments of SF1-interacting proteins from HeLa cell extracts shown here are consistent with the presence of SF1 in early splicing complexes. Surprisingly almost all U2 snRNP proteins were found associated with SF1. Yeast two-hybrid screens identified two SURP domain-containing U2 snRNP proteins as partners of SF1. A short, evolutionarily conserved region of SF1 interacts with the SURP domains, stressing their role in protein–protein interactions. A reduction of A complex formation in SF1-depleted extracts could be rescued with recombinant SF1 containing the SURP-interaction domain, but only partial rescue was observed with SF1 lacking this sequence. Thus, SF1 can initially recruit the U2 snRNP to the spliceosome during E complex formation, whereas U2AF65 may stabilize the association of the U2 snRNP with the spliceosome at later times. In addition, these findings may have implications for alternative splicing decisions.

• AREs, Adenylate and uridylate Rich Elements
• AU, Analytical Ultracentrifugation
• CARM1, Coactivator associated Arginine Methyltransferase 1
• CD, Circular Dichroism
• Cdk1, Cyclin-dependent kinase 1
• Chk2, Checkpoint kinase 2
• ELAV1, Embryonic Lethal Abnormal Vision system human homolog 1
• EMSA, Electrophoretic Mobility Shift Assay
• FIR, FBP-Interacting Repressor
• FL, Full-Length, HNS, HuR Nucleocytoplasmic Shuttling Sequence
• HSQC, Heteronuclear Single-Quantum Correlation
• HuR, Human antigen R
• Human antigen R (HuR)
• MD, Molecular Dynamics
• NMR, Nuclear Magnetic Resonance
• NOE, Nuclear Overhauser Effect
• Nuclear Magnetic Resonance (NMR)
• PCA, Principal Component Analysis
• PKCα, Protein Kinase C α
• PKCδ, Protein Kinase C δ
• PMSF, PhenylMethylSulfonyl Fluoride
• PTB, Polypyrimidine Tract Binding protein
• RBPs, RNA Binding Proteins
• RNA binding
• RNA binding protein (RBP)
• RNA recognition motif (RRM)
• RRMs, RNA Recognition Motifs
• SPR, Surface Plasmon Resonance
• Serine Phosphorylation
• WT, Wild-Type
• dimerization
• hnRNP1, heterogeneous nuclear RiboNucleoprotein C protein

The auxiliary factor of U2 small nuclear RNA (U2AF) is a heterodimer consisting of 65- and 35-kD proteins that bind the polypyrimidine tract (PPT) and AG dinucleotides at the 3′ splice site (3′ss). The gene encoding U2AF35 (U2AF1) is alternatively spliced, giving rise to two isoforms U2AF35a and U2AF35b. Here, we knocked down U2AF35 and each isoform and characterized transcriptomes of HEK293 cells with varying U2AF35/U2AF65 and U2AF35a/b ratios. Depletion of both isoforms preferentially modified alternative RNA processing events without widespread failure to recognize 3′ss or constitutive exons. Over a third of differentially used exons were terminal, resulting largely from the use of known alternative polyadenylation (APA) sites. Intronic APA sites activated in depleted cultures were mostly proximal whereas tandem 3′UTR APA was biased toward distal sites. Exons upregulated in depleted cells were preceded by longer AG exclusion zones and PPTs than downregulated or control exons and were largely activated by PUF60 and repressed by CAPERα. The U2AF(35) repression and activation was associated with a significant interchange in the average probabilities to form single-stranded RNA in the optimal PPT and branch site locations and sequences further upstream. Although most differentially used exons were responsive to both U2AF subunits and their inclusion correlated with U2AF levels, a small number of transcripts exhibited distinct responses to U2AF35a and U2AF35b, supporting the existence of isoform-specific interactions. These results provide new insights into function of U2AF and U2AF35 in alternative RNA processing.

• FBP interacting repressor (FIR)
• P53
• T-ALL
• haplo-insufficiency
• leukemia
• splicing variant

• Adult
• Alternative Splicing
• Animals
• Disease Progression
• Female
• Haploinsufficiency
• Humans
• Male
• Mice
• Mice, Inbred C57BL
• Mice, Knockout
• Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/genetics
• Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/metabolism
• Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/pathology
• Proto-Oncogene Proteins c-myc/genetics
• Proto-Oncogene Proteins c-myc/metabolism
• RNA Splicing Factors
• RNA-Binding Proteins/genetics
• RNA-Binding Proteins/metabolism
• Receptor, Notch1/genetics
• Receptor, Notch1/metabolism
• Repressor Proteins/genetics
• Repressor Proteins/metabolism
• Tumor Suppressor Protein p53/genetics
• Tumor Suppressor Protein p53/metabolism

The retention and splicing (RES) complex is a conserved spliceosome-associated module that was shown to enhance splicing of a subset of transcripts and promote the nuclear retention of unspliced pre-mRNAs in yeast. The heterotrimeric RES complex is organized around the Snu17p protein that binds to both the Bud13p and Pml1p subunits. Snu17p exhibits an RRM domain that resembles a U2AF homology motif (UHM) and Bud13p harbors a Trp residue reminiscent of an UHM-ligand motif (ULM). It has therefore been proposed that the interaction between Snu17p and Bud13p resembles canonical UHM-ULM complexes. Here, we have used biochemical and NMR structural analysis to characterize the structure of the yeast Snu17p-Bud13p complex. Unlike known UHMs that sequester the Trp residue of the ULM ligand in a hydrophobic pocket, Snu17p and Bud13p utilize a large interaction surface formed around the two helices of the Snu17p domain. In total 18 residues of the Bud13p ligand wrap around the Snu17p helical surface in an U-turn-like arrangement. The invariant Trp²³² in Bud13p is located in the center of the turn, and contacts surface residues of Snu17p. The structural data are supported by mutational analysis and indicate that Snu17p provides an extended binding surface with Bud13p that is notably distinct from canonical UHM-ULM interactions. Our data highlight structural diversity in RRM-protein interactions, analogous to the one seen for nucleic acid interactions.

• Gene Regulation
• Protein Complex
• Protein Structure
• Protein-protein Interaction
• RNA-binding Protein
• Spliceosome
• U2AF Homology Motif

• Crystal Structure
• Gene Regulation
• Protein Domain
• Protein Structure and Folding
• Protein-Protein Interaction
• RNA Splicing

AIM: To investigate a novel therapeutic strategy to target and suppress c-myc in human cancers using far up stream element (FUSE)-binding protein-interacting repressor (FIR).

METHODS: Endogenous c-Myc suppression and apoptosis induction by a transient FIR-expressing vector was examined in vivo via a HA-tagged FIR (HA-FIR) expression vector. A fusion gene-deficient, non-transmissible, Sendai virus (SeV) vector encoding FIR cDNA, SeV/dF/FIR, was prepared. SeV/dF/FIR was examined for its gene transduction efficiency, viral dose dependency of antitumor effect and apoptosis induction in HeLa (cervical squamous cell carcinoma) cells and SW480 (colon adenocarcinoma) cells. Antitumor efficacy in a mouse xenograft model was also examined. The molecular mechanism of the anti-tumor effect and c-Myc suppression by SeV/dF/FIR was examined using Spliceostatin A (SSA), a SAP155 inhibitor, or SAP155 siRNA which induce c-Myc by increasing FIR∆exon2 in HeLa cells.

RESULTS: FIR was found to repress c-myc transcription and in turn the overexpression of FIR drove apoptosis through c-myc suppression. Thus, FIR expressing vectors are potentially applicable for cancer therapy. FIR is alternatively spliced by SAP155 in cancer cells lacking the transcriptional repression domain within exon 2 (FIR∆exon2), counteracting FIR for c-Myc protein expression. Furthermore, FIR forms a complex with SAP155 and inhibits mutual well-established functions. Thus, both the valuable effects and side effects of exogenous FIR stimuli should be tested for future clinical application. SeV/dF/FIR, a cytoplasmic RNA virus, was successfully prepared and showed highly efficient gene transduction in in vivo experiments. Furthermore, in nude mouse tumor xenograft models, SeV/dF/FIR displayed high antitumor efficiency against human cancer cells. SeV/dF/FIR suppressed SSA-activated c-Myc. SAP155 siRNA, potentially produces FIR∆exon2, and led to c-Myc overexpression with phosphorylation at Ser62. HA-FIR suppressed endogenous c-Myc expression and induced apoptosis in HeLa and SW480 cells. A c-myc transcriptional suppressor FIR expressing SeV/dF/FIR showed high gene transduction efficiency with significant antitumor effects and apoptosis induction in HeLa and SW480 cells.

CONCLUSION: SeV/dF/FIR showed strong tumor growth suppression with no significant side effects in an animal xenograft model, thus SeV/dF/FIR is potentially applicable for future clinical cancer treatment.

• Cancer gene therapy
• c-myc suppressor
• Far up stream element-binding protein-interacting repressor
• Sendai virus vector

• Animals
• Apoptosis
• Cell Line, Tumor
• Cell Survival
• Colonic Neoplasms/metabolism
• Colonic Neoplasms/therapy
• DNA, Complementary/metabolism
• Genes, myc
• Genetic Vectors
• Green Fluorescent Proteins/metabolism
• Guanine Nucleotide Exchange Factors/genetics
• Guanine Nucleotide Exchange Factors/metabolism
• HeLa Cells
• Humans
• Male
• Mice
• Mice, Inbred BALB C
• Neoplasm Transplantation
• Phosphorylation
• Plasmids/metabolism
• Pyrans/chemistry
• RNA Splicing Factors
• RNA-Binding Proteins/genetics
• RNA-Binding Proteins/metabolism
• Repressor Proteins/genetics
• Repressor Proteins/metabolism
• Sendai virus/genetics
• Spiro Compounds/chemistry

• Alternative Splicing
• Animals
• Cell Line
• Disks Large Homolog 4 Protein
• Exons
• Gene Expression Regulation
• Gene Library
• Green Fluorescent Proteins/genetics
• Guanylate Kinases/genetics
• High-Throughput Screening Assays/methods
• Humans
• Luminescent Proteins/genetics
• Membrane Proteins/genetics
• Mice
• RNA Interference
• Reproducibility of Results
• Red Fluorescent Protein

• R01 GM097230 NIGMS NIH HHS
• R01 GM049662 NIGMS NIH HHS
• Howard Hughes Medical Institute
• K99 MH096807 NIMH NIH HHS
• R01GM49662 NIGMS NIH HHS
• R01GM097230 NIGMS NIH HHS

• Amino Acid Sequence
• Animals
• Base Sequence
• Cell Proliferation
• Crystallography, X-Ray
• DNA-Binding Proteins/chemistry
• DNA-Binding Proteins/physiology
• HEK293 Cells
• HeLa Cells
• Humans
• Hydrogen Bonding
• Mice
• Models, Molecular
• Molecular Sequence Data
• NIH 3T3 Cells
• Nuclear Proteins/chemistry
• Nuclear Proteins/physiology
• Phosphorylation
• Protein Binding
• Protein Interaction Domains and Motifs
• Protein Processing, Post-Translational
• Protein Structure, Quaternary
• Protein Structure, Secondary
• RNA Splice Sites
• RNA Splicing Factors
• Ribonucleoproteins/chemistry
• Ribonucleoproteins/physiology
• Splicing Factor U2AF
• Transcription Factors/chemistry
• Transcription Factors/physiology

• CA92584 NCI NIH HHS
• R01 GM035490 NIGMS NIH HHS
• P41RR001209 NCRR NIH HHS
• RR01646 NCRR NIH HHS
• S10 RR026501 NCRR NIH HHS
• P41 RR001209 NCRR NIH HHS
• P01 CA092584 NCI NIH HHS
• R01 GM070503 NIGMS NIH HHS
• P41 RR001646 NCRR NIH HHS