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Rakesh Chatrikhi, Wenhua Wang, Ankit Gupta, Sarah Loerch, Alexandre Maucuer, Clara L Kielkopf
Splicing factor 1 (SF1) recognizes 3' splice sites of the major class of introns as a ternary complex with U2AF(65) and U2AF(35) splicing factors. A conserved SPSP motif in a coiled-coil domain of SF1 is highly phosphorylated in proliferating human cells and is required for cell proliferation. The UHM kinase 1 (UHMK1), also called KIS, double-phosphorylates both serines of this SF1 motif. Here, we use isothermal titration calorimetry to demonstrate that UHMK1 phosphorylation of the SF1 SPSP motif slightly enhances specific binding of phospho-SF1 to its cognate U2AF(65) protein partner...
December 20, 2016: Biophysical Journal
Somsakul Pop Wongpalee, Ajay Vashisht, Shalini Sharma, Darryl Chui, James A Wohlschlegel, Douglas L Black
Polypyrimidine-tract binding protein PTBP1 can repress splicing during the exon definition phase of spliceosome assembly, but the assembly steps leading to an exon definition complex (EDC) and how PTBP1 might modulate them are not clear. We found that PTBP1 binding in the flanking introns allowed normal U2AF and U1 snRNP binding to the target exon splice sites but blocked U2 snRNP assembly in HeLa nuclear extract. Characterizing a purified PTBP1-repressed complex, as well as an active early complex and the final EDC by SILAC-MS, we identified extensive PTBP1-modulated changes in exon RNP composition...
November 24, 2016: ELife
Sarah Loerch, Clara L Kielkopf
U2AF homology motifs (UHM) that recognize U2AF ligand motifs (ULM) are an emerging family of protein-protein interaction modules. UHM-ULM interactions recur in pre-mRNA splicing factors including U2AF1 and SF3b1, which are frequently mutated in myelodysplastic syndromes. The core topology of the UHM resembles an RNA recognition motif and is often mistakenly classified within this large family. Here, we unmask the charade and review recent discoveries of UHM-ULM modules for protein-protein interactions. Diverse polypeptide extensions and selective phosphorylation of UHM and ULM family members offer new molecular mechanisms for the assembly of specific partners in the early-stage spliceosome...
December 2016: RNA
Lena Voith von Voithenberg, Carolina Sánchez-Rico, Hyun-Seo Kang, Tobias Madl, Katia Zanier, Anders Barth, Lisa R Warner, Michael Sattler, Don C Lamb
An essential early step in the assembly of human spliceosomes onto pre-mRNA involves the recognition of regulatory RNA cis elements in the 3' splice site by the U2 auxiliary factor (U2AF). The large (U2AF65) and small (U2AF35) subunits of the U2AF heterodimer contact the polypyrimidine tract (Py-tract) and the AG-dinucleotide, respectively. The tandem RNA recognition motif domains (RRM1,2) of U2AF65 adopt closed/inactive and open/active conformations in the free form and when bound to bona fide Py-tract RNA ligands...
November 15, 2016: Proceedings of the National Academy of Sciences of the United States of America
Pravin Kumar Ankush Jagtap, Divita Garg, Tobias G Kapp, Cindy L Will, Oliver Demmer, Reinhard Lührmann, Horst Kessler, Michael Sattler
U2AF homology motifs (UHMs) are atypical RNA recognition motif domains that mediate critical protein-protein interactions during the regulation of alternative pre-mRNA splicing and other processes. The recognition of UHM domains by UHM ligand motif (ULM) peptide sequences plays important roles during early steps of spliceosome assembly. Splicing factor 45 kDa (SPF45) is an alternative splicing factor implicated in breast and lung cancers, and splicing regulation of apoptosis-linked pre-mRNAs by SPF45 was shown to depend on interactions between its UHM domain and ULM motifs in constitutive splicing factors...
November 23, 2016: Journal of Medicinal Chemistry
Li Chen, Robert Weinmeister, Jana Kralovicova, Lucy P Eperon, Igor Vorechovsky, Andrew J Hudson, Ian C Eperon
The selection of 3' splice sites (3'ss) is an essential early step in mammalian RNA splicing reactions, but the processes involved are unknown. We have used single molecule methods to test whether the major components implicated in selection, the proteins U2AF35 and U2AF65 and the U2 snRNP, are able to recognize alternative candidate sites or are restricted to one pre-specified site. In the presence of adenosine triphosphate (ATP), all three components bind in a 1:1 stoichiometry with a 3'ss. Pre-mRNA molecules with two alternative 3'ss can be bound concurrently by two molecules of U2AF or two U2 snRNPs, so none of the components are restricted...
September 28, 2016: Nucleic Acids Research
Francesca Perez-Marques, Pippa Simpson, Ke Yan, Michael W Quasney, Nadine Halligan, Daniel Merchant, Mary K Dahmer
BACKGROUND: Previous work has demonstrated a strong association between lung injury in African American children with pneumonia and a polymorphic (TG)mTn region in cystic fibrosis transmembrane conductance (CFTR) involved in the generation of a nonfunctional CFTR protein lacking exon 9. A number of splicing factors that regulate the inclusion/exclusion of exon 9 have been identified. The objective of this study was to determine whether genetic variants in these splicing factors were associated with acute respiratory distress syndrome (ARDS) in children with pneumonia...
2016: Critical Care: the Official Journal of the Critical Care Forum
Jana Kralovicova, Igor Vorechovsky
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...
January 9, 2017: Nucleic Acids Research
Sharon Soucek, Yi Zeng, Deepti L Bellur, Megan Bergkessel, Kevin J Morris, Qiudong Deng, Duc Duong, Nicholas T Seyfried, Christine Guthrie, Jonathan P Staley, Milo B Fasken, Anita H Corbett
Numerous RNA binding proteins are deposited onto an mRNA transcript to modulate post-transcriptional processing events ensuring proper mRNA maturation. Defining the interplay between RNA binding proteins that couple mRNA biogenesis events is crucial for understanding how gene expression is regulated. To explore how RNA binding proteins control mRNA processing, we investigated a role for the evolutionarily conserved polyadenosine RNA binding protein, Nab2, in mRNA maturation within the nucleus. This work reveals that nab2 mutant cells accumulate intron-containing pre-mRNA in vivo We extend this analysis to identify genetic interactions between mutant alleles of nab2 and genes encoding the splicing factor, MUD2, and the RNA exosome, RRP6, with in vivo consequences of altered pre-mRNA splicing and poly(A) tail length control...
August 15, 2016: Molecular and Cellular Biology
Galina A Stepanyuk, Pedro Serrano, Eigen Peralta, Carol L Farr, Herbert L Axelrod, Michael Geralt, Debanu Das, Hsiu-Ju Chiu, Lukasz Jaroszewski, Ashley M Deacon, Scott A Lesley, Marc-André Elsliger, Adam Godzik, Ian A Wilson, Kurt Wüthrich, Daniel R Salomon, James R Williamson
RNA-binding protein 39 (RBM39) is a splicing factor and a transcriptional co-activator of estrogen receptors and Jun/AP-1, and its function has been associated with malignant progression in a number of cancers. The C-terminal RRM domain of RBM39 belongs to the U2AF homology motif family (UHM), which mediate protein-protein interactions through a short tryptophan-containing peptide known as the UHM-ligand motif (ULM). Here, crystal and solution NMR structures of the RBM39-UHM domain, and the crystal structure of its complex with U2AF65-ULM, are reported...
April 2016: Acta Crystallographica. Section D, Structural Biology
Anant A Agrawal, Enea Salsi, Rakesh Chatrikhi, Steven Henderson, Jermaine L Jenkins, Michael R Green, Dmitri N Ermolenko, Clara L Kielkopf
How the essential pre-mRNA splicing factor U2AF(65) recognizes the polypyrimidine (Py) signals of the major class of 3' splice sites in human gene transcripts remains incompletely understood. We determined four structures of an extended U2AF(65)-RNA-binding domain bound to Py-tract oligonucleotides at resolutions between 2.0 and 1.5 Å. These structures together with RNA binding and splicing assays reveal unforeseen roles for U2AF(65) inter-domain residues in recognizing a contiguous, nine-nucleotide Py tract...
March 8, 2016: Nature Communications
Yongcheng Wei, Qinghua Liu, Hongyu Dong, Zhichun Zhou, Yanping Hao, Xuelian Chen, Liuyi Xu
Pinus massoniaia Lamb has gained more and more attention as the most important tree species for timber and forestation in South China. Gene expression studies are of great importance to identify new and elite cultivars. Real-time quantitative PCR, a highly sensitive and specific method, is commonly used in the analysis of gene expression. The appropriate reference genes must be employed to normalize the calculation program for ascertaining repeatable and significant results. Herein, eleven housekeeping genes were evaluated during different stages of P...
2016: PloS One
Jana Kralovicova, Marcin Knut, Nicholas C P Cross, Igor Vorechovsky
ATM is an important cancer susceptibility gene that encodes a critical apical kinase of the DNA damage response (DDR) pathway. We show that a key nonsense-mediated RNA decay switch exon (NSE) in ATM is repressed by U2AF, PUF60 and hnRNPA1. The NSE activation was haplotype-specific and was most promoted by cytosine at rs609621 in the NSE 3' splice-site (3'ss), which is predominant in high cancer risk populations. NSE levels were deregulated in leukemias and were influenced by the identity of U2AF35 residue 34...
January 6, 2016: Scientific Reports
Sunghee Cho, Heegyum Moon, Tiing Jen Loh, Ha Na Jang, Yongchao Liu, Jianhua Zhou, Takbum Ohn, Xuexiu Zheng, Haihong Shen
U2 snRNP auxiliary factor 65 kDa (U2AF(65)) is a general splicing factor that contacts polypyrimidine (Py) tract and promotes prespliceosome assembly. In this report, we show that U2AF(65) stimulates alternative exon skipping in spinal muscular atrophy (SMA)-related survival motor neuron (SMN) pre-mRNA. A stronger 5' splice-site mutation of alternative exon abolishes the stimulatory effects of U2AF(65). U2AF(65) overexpression promotes its own binding only on the weaker, not the stronger, Py tract. We further demonstrate that U2AF(65) inhibits splicing of flanking introns of alternative exon in both three-exon and two-exon contexts...
August 11, 2015: Proceedings of the National Academy of Sciences of the United States of America
Hisashi Yoshida, Sam-Yong Park, Takashi Oda, Taeko Akiyoshi, Mamoru Sato, Mikako Shirouzu, Kengo Tsuda, Kanako Kuwasako, Satoru Unzai, Yutaka Muto, Takeshi Urano, Eiji Obayashi
The pre-mRNA splicing reaction of eukaryotic cells has to be carried out extremely accurately, as failure to recognize the splice sites correctly causes serious disease. The small subunit of the U2AF heterodimer is essential for the determination of 3' splice sites in pre-mRNA splicing, and several single-residue mutations of the U2AF small subunit cause severe disorders such as myelodysplastic syndromes. However, the mechanism of RNA recognition is poorly understood. Here we solved the crystal structure of the U2AF small subunit (U2AF23) from fission yeast, consisting of an RNA recognition motif (RRM) domain flanked by two conserved CCCH-type zinc fingers (ZFs)...
August 1, 2015: Genes & Development
Michael O Duff, Sara Olson, Xintao Wei, Sandra C Garrett, Ahmad Osman, Mohan Bolisetty, Alex Plocik, Susan E Celniker, Brenton R Graveley
Recursive splicing is a process in which large introns are removed in multiple steps by re-splicing at ratchet points--5' splice sites recreated after splicing. Recursive splicing was first identified in the Drosophila Ultrabithorax (Ubx) gene and only three additional Drosophila genes have since been experimentally shown to undergo recursive splicing. Here we identify 197 zero nucleotide exon ratchet points in 130 introns of 115 Drosophila genes from total RNA sequencing data generated from developmental time points, dissected tissues and cultured cells...
May 21, 2015: Nature
Ashish Misra, Jianhong Ou, Lihua J Zhu, Michael R Green
The mechanisms that regulate alternative precursor mRNA (pre-mRNA) splicing are largely unknown. Here, we perform an RNAi screen to identify factors required for alternative splicing regulation by RBFOX2, an RNA-binding protein that promotes either exon inclusion or exclusion. Unexpectedly, we find that two mRNA 3' end formation factors, cleavage and polyadenylation specificity factor (CPSF) and SYMPK, are RBFOX2 cofactors for both inclusion and exclusion of internal exons. RBFOX2 interacts with CPSF/SYMPK and recruits it to the pre-mRNA...
June 4, 2015: Molecular Cell
Tongbin Wu, Xiang-Dong Fu
The U2AF heterodimer is generally accepted to play a vital role in defining functional 3' splice sites in pre-mRNA splicing. Given prevalent mutations in U2AF, particularly in the U2AF1 gene (which encodes for the U2AF35 subunit) in blood disorders and other human cancers, there are renewed interests in these classic splicing factors to further understand their regulatory functions in RNA metabolism in both physiological and disease settings. We recently reported that U2AF has a maximal capacity to directly bind ˜88% of functional 3' splice sites in the human genome and that numerous U2AF binding events also occur in various exonic and intronic locations, thus providing additional mechanisms for the regulation of alternative splicing besides their traditional role in titrating weak splice sites in the cell...
2015: RNA Biology
Jana Kralovicova, Marcin Knut, Nicholas C P Cross, Igor Vorechovsky
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...
April 20, 2015: Nucleic Acids Research
Soraya Becerra, Marta Montes, Cristina Hernández-Munain, Carlos Suñé
The first stable complex formed during the assembly of spliceosomes onto pre-mRNA substrates in mammals includes U1 snRNP, which recognizes the 5' splice site, and the splicing factors SF1 and U2AF, which bind the branch point sequence, polypyrimidine tract, and 3' splice site. The 5' and 3' splice site complexes are thought to be joined together by protein-protein interactions mediated by factors that ensure the fidelity of the initial splice site recognition. In this study, we identified and characterized PRPF40B, a putative mammalian ortholog of the U1 snRNP-associated yeast splicing factor Prp40...
March 2015: RNA
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