scholarly journals PsiCLIP reveals dynamic RNA binding by DEAH-box helicases before and after exon ligation

2020 ◽  
Author(s):  
Lisa M. Strittmatter ◽  
Charlotte Capitanchik ◽  
Andrew J. Newman ◽  
Martina Hallegger ◽  
Christine M. Norman ◽  
...  
Keyword(s):  
Rna Binding ◽  
Positional Information ◽  
Mrna Splicing ◽  
Splicing Factors ◽  
Imaging Studies ◽  
Rna Helicases ◽  
Binding Profile ◽  
Exon Junction ◽  
Exon Ligation ◽  
Before And After

AbstractEight RNA helicases remodel the spliceosome to effect pre-mRNA splicing but their mechanism of action remains poorly understood. We have developed “purified spliceosome iCLIP” (psiCLIP) to define helicase-RNA contacts in specific spliceosomal states. psiCLIP reveals previously unappreciated dynamics of spliceosomal helicases. The binding profile of the helicase Prp16 is influenced by the distance between the branch-point and 3’ splice site, while Prp22 binds diffusely on the intron before exon ligation but switches to more narrow binding downstream of the exon junction after exon ligation. Notably, depletion of the exon-ligation factor Prp18 destabilizes Prp22 binding to the pre-mRNA, demonstrating that psiCLIP can be used to study the relationships between helicases and auxiliary splicing factors. Thus, psiCLIP is sensitive to spliceosome dynamics and complements the insights from structural and imaging studies by providing crucial positional information on helicase-RNA contacts during spliceosomal remodeling.

scholarly journals psiCLIP reveals dynamic RNA binding by DEAH-box helicases before and after exon ligation

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Lisa M. Strittmatter ◽  
Charlotte Capitanchik ◽  
Andrew J. Newman ◽  
Martina Hallegger ◽  
Christine M. Norman ◽  
...  
Keyword(s):  
Mechanism Of Action ◽  
Splice Site ◽  
Rna Binding ◽  
Mrna Splicing ◽  
Structural Studies ◽  
Rna Helicases ◽  
Exon Ligation ◽  
Before And After ◽  
The Stability

AbstractRNA helicases remodel the spliceosome to enable pre-mRNA splicing, but their binding and mechanism of action remain poorly understood. To define helicase-RNA contacts in specific spliceosomal states, we develop purified spliceosome iCLIP (psiCLIP), which reveals dynamic helicase-RNA contacts during splicing catalysis. The helicase Prp16 binds along the entire available single-stranded RNA region between the branchpoint and 3′-splice site, while Prp22 binds diffusely downstream of the branchpoint before exon ligation, but then switches to more narrow binding in the downstream exon after exon ligation, arguing against a mechanism of processive translocation. Depletion of the exon-ligation factor Prp18 destabilizes Prp22 binding to the pre-mRNA, suggesting that proofreading by Prp22 may sense the stability of the spliceosome during exon ligation. Thus, psiCLIP complements structural studies by providing key insights into the binding and proofreading activity of spliceosomal RNA helicases.

scholarly journals Structures of the Human Spliceosomes Before and After Release of the Ligated Exon

2018 ◽  
Author(s):  
Xiaofeng Zhang ◽  
Xiechao Zhan ◽  
Chuangye Yan ◽  
Wenyu Zhang ◽  
Dongliang Liu ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Splice Site ◽  
Mrna Splicing ◽  
Small Nuclear Rna ◽  
Exon Ligation ◽  
Cryo Electron Microscopy ◽  
Branch Point Sequence ◽  
Nuclear Rna ◽  
Before And After ◽  
Functional States

Pre-mRNA splicing is executed by the spliceosome, which has eight major functional states each with distinct composition. Five of these eight human spliceosomal complexes, all preceding exon ligation, have been structurally characterized. In this study, we report the cryo-electron microscopy structures of the human post-catalytic spliceosome (P complex) and intron lariat spliceosome (ILS) at average resolutions of 3.0 and 2.9 Å, respectively. In the P complex, the ligated exon remains anchored to loop I of U5 small nuclear RNA, and the 3'-splice site is recognized by the junction between the 5'-splice site and the branch point sequence. The ATPase/helicase Prp22, along with the ligated exon and eight other proteins, are dissociated in the P-to-ILS transition. Intriguingly, the ILS complex exists in two distinct conformations, one with the ATPase/helicase Prp43 and one without. Comparison of these three late-stage human spliceosomes reveals mechanistic insights into exon release and spliceosome disassembly.

scholarly journals miRNA-Based Regulation of Alternative RNA Splicing in Metazoans

2021 ◽  
Vol 22 (21) ◽  
pp. 11618
Author(s):  
Anna L. Schorr ◽  
Marco Mangone
Keyword(s):  
Neurological Disorders ◽  
Rna Splicing ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Mrna Splicing ◽  
Splicing Factors ◽  
Transcriptional Level ◽  
Dependent Manner ◽  
Non Coding Rnas ◽  
Specific Mrna

Alternative RNA splicing is an important regulatory process used by genes to increase their diversity. This process is mainly executed by specific classes of RNA binding proteins that act in a dosage-dependent manner to include or exclude selected exons in the final transcripts. While these processes are tightly regulated in cells and tissues, little is known on how the dosage of these factors is achieved and maintained. Several recent studies have suggested that alternative RNA splicing may be in part modulated by microRNAs (miRNAs), which are short, non-coding RNAs (~22 nt in length) that inhibit translation of specific mRNA transcripts. As evidenced in tissues and in diseases, such as cancer and neurological disorders, the dysregulation of miRNA pathways disrupts downstream alternative RNA splicing events by altering the dosage of splicing factors involved in RNA splicing. This attractive model suggests that miRNAs can not only influence the dosage of gene expression at the post-transcriptional level but also indirectly interfere in pre-mRNA splicing at the co-transcriptional level. The purpose of this review is to compile and analyze recent studies on miRNAs modulating alternative RNA splicing factors, and how these events contribute to transcript rearrangements in tissue development and disease.

scholarly journals Serine Phosphorylation of SR Proteins Is Required for Their Recruitment to Sites of Transcription In Vivo

1998 ◽  
Vol 143 (2) ◽  
pp. 297-307 ◽  
Author(s):  
Tom Misteli ◽  
Javier F. Cáceres ◽  
Jade Q. Clement ◽  
Adrian R. Krainer ◽  
Miles F. Wilkinson ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Transcriptional Activation ◽  
Active Sites ◽  
Rna Binding ◽  
Sr Proteins ◽  
Mrna Splicing ◽  
Serine Phosphorylation ◽  
Splicing Factors ◽  
Nuclear Speckles

Expression of most RNA polymerase II transcripts requires the coordinated execution of transcription, splicing, and 3′ processing. We have previously shown that upon transcriptional activation of a gene in vivo, pre-mRNA splicing factors are recruited from nuclear speckles, in which they are concentrated, to sites of transcription (Misteli, T., J.F. Cáceres, and D.L. Spector. 1997. Nature. 387:523–527). This recruitment process appears to spatially coordinate transcription and pre-mRNA splicing within the cell nucleus. Here we have investigated the molecular basis for recruitment by analyzing the recruitment properties of mutant splicing factors. We show that multiple protein domains are required for efficient recruitment of SR proteins from nuclear speckles to nascent RNA. The two types of modular domains found in the splicing factor SF2/ ASF exert distinct functions in this process. In living cells, the RS domain functions in the dissociation of the protein from speckles, and phosphorylation of serine residues in the RS domain is a prerequisite for this event. The RNA binding domains play a role in the association of splicing factors with the target RNA. These observations identify a novel in vivo role for the RS domain of SR proteins and suggest a model in which protein phosphorylation is instrumental for the recruitment of these proteins to active sites of transcription in vivo.

scholarly journals The RNA-binding profile of Acinus, a peripheral component of the exon junction complex, reveals its role in splicing regulation

RNA ◽  
2016 ◽  
Vol 22 (9) ◽  
pp. 1411-1426 ◽  
Author(s):  
Julie Rodor ◽  
Qun Pan ◽  
Benjamin J. Blencowe ◽  
Eduardo Eyras ◽  
Javier F. Cáceres
Keyword(s):  
Rna Binding ◽  
Exon Junction Complex ◽  
Splicing Regulation ◽  
Binding Profile ◽  
Exon Junction

scholarly journals Sip1, a Novel RS Domain-Containing Protein Essential for Pre-mRNA Splicing

1998 ◽  
Vol 18 (2) ◽  
pp. 676-684 ◽  
Author(s):  
Wan-Jiang Zhang ◽  
Jane Y. Wu
Keyword(s):  
Protein Interactions ◽  
Rna Binding ◽  
Sequence Similarity ◽  
Functional Characterization ◽  
Nuclear Extract ◽  
Mrna Splicing ◽  
Splicing Factor ◽  
Splicing Factors ◽  
Interacting Protein

ABSTRACT Previous studies have shown that protein-protein interactions among splicing factors may play an important role in pre-mRNA splicing. We report here identification and functional characterization of a new splicing factor, Sip1 (SC35-interacting protein 1). Sip1 was initially identified by virtue of its interaction with SC35, a splicing factor of the SR family. Sip1 interacts with not only several SR proteins but also with U1-70K and U2AF65, proteins associated with 5′ and 3′ splice sites, respectively. The predicted Sip1 sequence contains an arginine-serine-rich (RS) domain but does not have any known RNA-binding motifs, indicating that it is not a member of the SR family. Sip1 also contains a region with weak sequence similarity to the Drosophila splicing regulator suppressor of white apricot (SWAP). An essential role for Sip1 in pre-mRNA splicing was suggested by the observation that anti-Sip1 antibodies depleted splicing activity from HeLa nuclear extract. Purified recombinant Sip1 protein, but not other RS domain-containing proteins such as SC35, ASF/SF2, and U2AF65, restored the splicing activity of the Sip1-immunodepleted extract. Addition of U2AF65 protein further enhanced the splicing reconstitution by the Sip1 protein. Deficiency in the formation of both A and B splicing complexes in the Sip1-depleted nuclear extract indicates an important role of Sip1 in spliceosome assembly. Together, these results demonstrate that Sip1 is a novel RS domain-containing protein required for pre-mRNA splicing and that the functional role of Sip1 in splicing is distinct from those of known RS domain-containing splicing factors.

scholarly journals RBM20 phosphorylation on serine/arginine domain is crucial to regulate pre-mRNA splicing and protein shuttling in the heart

2020 ◽  
Author(s):  
Mingming Sun ◽  
Yutong Jin ◽  
Chaoqun Zhu ◽  
Yanghai Zhang ◽  
Martin Liss ◽  
...  
Keyword(s):  
Protein Trafficking ◽  
Rna Binding ◽  
Heart Function ◽  
Mrna Splicing ◽  
Splicing Factors ◽  
Binding Motif ◽  
Amino Acid Residues ◽  
Cellular Mechanisms ◽  
Sr Protein ◽  
Protein Protein Interaction

AbstractMolecular and cellular mechanisms of mutations of splicing factors in heart function are not well understood. The splicing of precursor mRNA is dependent on an essential group of splicing factors containing serine-arginine (SR) domain(s) that are critical for protein-RNA and protein-protein interaction in the spliceosome assembly. Phosphorylation of SR domains plays a key role in splicing control and the distribution of splicing factors in the cell. RNA binding motif 20 (RBM20) is a splicing factor predominantly expressed in muscle tissues with the highest expression level in the heart. However, its phosphorylation status is completely unknown up-to-date. In this study, we identified sixteen amino acid residues that are phosphorylated by middle-down mass spectrometry. Four of them are located in the SR domain, and two out of the four residues, S638 and S640, play an essential role in splicing control and facilitate RBM20 shuttling from the nucleus to the cytoplasm. Re-localization of RBM20 promotes protein aggregation in the cytoplasm. We have also verified that SR-protein kinases (SRPKs), cdc2-like kinases (CLKs) and protein kinase B (PKB or AKT) phosphorylate S638 and S640. Mutations of S638A and S640G reduce RBM20 phosphorylation and disrupt the splicing. Taken together, we determine the phosphorylation status of RBM20 and provide the first evidence that phosphorylation on SR domain is crucial for pre-mRNA splicing and protein trafficking. Our findings reveal a new role of RBM20 via protein shuttling in cardiac function.

scholarly journals Contribution of DEAH-box protein DHX16 in human pre-mRNA splicing

2010 ◽  
Vol 429 (1) ◽  
pp. 25-32 ◽  
Author(s):  
Marieta Gencheva ◽  
Mitsuo Kato ◽  
Alain N.S. Newo ◽  
Ren-Jang Lin
Keyword(s):  
Rna Binding ◽  
Rna Binding Proteins ◽  
Mrna Splicing ◽  
Dominant Negative ◽  
Helicase Domain ◽  
Rna Helicases ◽  
Mutant Proteins ◽  
Dead Box ◽  
Nuclear Extracts

Studies of mammalian splicing factors are often focused on small nuclear ribonucleoproteins or regulatory RNA-binding proteins, such as hnRNP (heterogeneous nuclear ribonucleoprotein) and SR proteins (serine/arginine-rich proteins); however, much less is known about the contribution of DExD/H-box proteins or RNA helicases in mammalian pre-mRNA splicing. The human DEAH-box protein DHX16 [also known as DBP2 (DEAD-box protein 2)], is homologous with Caenorhabditis elegans Mog-4, Schizosaccharomyces pombe Prp8 and Saccharomyces cerevisiae Prp2. In the present study, we show that DHX16 is required for pre-mRNA splicing after the formation of a pre-catalytic spliceosome. We found that anti-DHX16 antiserum inhibited the splicing reaction in vitro and the antibody immunoprecipitated pre-mRNA, splicing intermediates and spliceosomal small nuclear RNAs. Cells that expressed DHX16 that had a mutation in the helicase domain accumulated unspliced intron-containing minigene transcripts. Nuclear extracts isolated from the dominant-negative DHX16-G724N-expressing cells formed splicing complex B, but were impaired in splicing. Adding extracts containing DHX16-G724N or DHX16-S552L mutant proteins to HeLa cell nuclear extracts resulted in reduced splicing, indicating that the mutant protein directly inhibited splicing in vitro. Therefore our results show that DHX16 is needed for human pre-mRNA splicing at a step analogous to that mediated by the S. cerevisiae spliceosomal ATPase Prp2.

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