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Interactions of aCPs with Cytosine-rich Polypyrimidine Tracts Enhance Splicing of Cassette Exons

ABSTRACT: Alternative splicing comprises a robust generator of mammalian transcriptome complexity. Splice site specification and activity are controlled by interactions of cis-acting determinants on a transcript with specific RNA binding proteins. A major subset of these interactions comprises interactions localized to the intronic U-rich polypyrimidine tract located immediately 5’ to the majority of splice acceptors. alphaCPs (also referred to as polyC-binding proteins (PCBPs) and hnRNP Es) comprise a subset of KH-domain proteins with high specificity and affinity for C-rich polypyrimidine motifs. Prior studies have revealed that binding of alphaCPs to C-rich motifs can modulate splicing and 3’ processing of the human alpha-globin mRNA transcript in the nucleus as well as stabilization of the halpha-globin mRNA in the cytoplasm. In the current report, we demonstrate that alphaCPs have a positive impact on the activity of splice acceptor sites in a defined subset of mammalian transcripts via binding to polypyrimidine tracts that are predominantly C-rich. These findings lead us to conclude that the alphaCPs play a global role in determining the splicing activity and levels of cassette exon inclusion within the mammalian transcriptome.

ORGANISM(S): Homo sapiens

PROVIDER: GSE71569 | GEO | 2016/03/01

SECONDARY ACCESSION(S): PRJNA291524

REPOSITORIES: GEO

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Interactions of aCPs with Cytosine-rich Polypyrimidine Tracts Enhance Splicing of Cassette Exons

Project description:Alternative splicing comprises a robust generator of mammalian transcriptome complexity. Splice site specification and activity are controlled by interactions of cis-acting determinants on a transcript with specific RNA binding proteins. A major subset of these interactions comprises interactions localized to the intronic U-rich polypyrimidine tract located immediately 5â to the majority of splice acceptors. alphaCPs (also referred to as polyC-binding proteins (PCBPs) and hnRNP Es) comprise a subset of KH-domain proteins with high specificity and affinity for C-rich polypyrimidine motifs. Prior studies have revealed that binding of alphaCPs to C-rich motifs can modulate splicing and 3â processing of the human alpha-globin mRNA transcript in the nucleus as well as stabilization of the halpha-globin mRNA in the cytoplasm. In the current report, we demonstrate that alphaCPs have a positive impact on the activity of splice acceptor sites in a defined subset of mammalian transcripts via binding to polypyrimidine tracts that are predominantly C-rich. These findings lead us to conclude that the alphaCPs play a global role in determining the splicing activity and levels of cassette exon inclusion within the mammalian transcriptome. To test the impact of aCP proteins on alternative splicing, aCP proteins were knockdown from K562 cells by siRNA. Since aCP1 and aCP2 have redundent function, we therefore designed siRNAs capable of knockdown both isoform at the same time. 3 aCP1/2 combined knockdown and 3 control siRNA knockdown were performed in K562 cells. RNA-seq were then performed to identify alternative splicing pattern mediated by aCP proteins.

2016-03-01 | E-GEOD-71569 | biostudies-arrayexpress

Transcriptome-wide analysis of U2AF2 RNA binding in a human hematopoietic cell line.

Project description:The essential pre-mRNA splicing factor U2AF2 (also called U2AF65) identifies polypyrimidine (Py) tract signals of nascent transcripts, despite length and sequence variations. Previous studies have shown that the U2AF2 RNA recognition motifs (RRM1 and RRM2) preferentially bind uridine-rich RNAs. Nonetheless, the specificity of the RRM1/RRM2 interface for the central Py tract nucleotide has yet to be investigated. Enhanced crosslinking and immunoprecipitation of endogenous U2AF2 in human erythroleukemia cells showed uridine-sensitive binding sites with lower sequence conservation at the central nucleotide positions of otherwise uridine-rich, U2AF2-bound splice sites. Altogether, these results highlight the importance of RNA flexibility for protein recognition and take a step towards relating splice site motifs to pre-mRNA splicing efficiencies. Keywords: splicing, RNA binding, U2AF2, U2AF65, 3'SS, 3' splice site, eCLIP, polypyrimidine tract, RNA recognition motif, RRM, U-rich, Py-tract

2022-06-01 | GSE195669 | GEO

PTB CLIP-seq

2009-12-24 | GSE19323 | GEO

PTB CLIP-seq

Project description:Recent transcriptome analysis indicates that >90% of human genes undergoes alternative splicing, underscoring the contribution of differential RNA processing to diverse proteomes in higher eukaryotic cells. The polypyrimidine tract binding protein PTB is a well-characterized splicing repressor, but PTB knockdown causes both exon inclusion and skipping. Genome-wide mapping of PTB-RNA interactions and construction of a functional RNA map now revealed that dominant PTB binding near a competing constitutive splice site generally induces exon inclusion whereas prevalent binding close to an alternative site often causes exon skipping. This positional effect was further demonstrated by disrupting or creating a PTB binding site on minigene constructs and testing their responses to PTB knockdown or overexpression. These findings suggest a mechanism for PTB to modulate splice site competition to produce opposite functional consequences, which may be generally applicable to RNA binding splicing factors to positively or negatively regulate alternative splicing in mammalian cells. Examination of PTB-RNA binding in Hela cells using CLIP-seq (Cross-Linking ImmunoPrecipitation coupled with high-throughput sequencing) method. Peaks: The four alignment files (linked as supplementary files on Sample records) were combined together for peak finding, as we found that most of the monomeric and dimeric tags are similarly distributed in the genome with high pearson correlation coefficient. The method to detect the peaks above gene-specific randomized background was similar to (Yeo et al., 2009) and described in the paper (Xue et al., 2009).

2009-12-24 | E-GEOD-19323 | biostudies-arrayexpress

Cis- and trans-regulation of alternative splicing by Adenosine Deaminase Acting on the double-stranded RNA (ADAR) proteins

Project description:RNA editing and splicing are the two major processes that dynamically regulate human transcriptome diversity. Despite growing evidence of crosstalk between RNA editing enzymes (mainly ADAR1) and splicing machineries, detailed mechanistic explanations and their biological importance in diseases, such as cancer are still lacking. Herein, we identify approximately a hundred high-confidence splicing events altered by ADAR1 and/or ADAR2, and ADAR1 or ADAR2 protein can regulate cassette exons in both directions. We unravel a binding tendency of ADARs to dsRNAs that involves GA-rich sequences for editing and splicing regulation. ADAR1 edits an intronic splicing silencer, leading to recruitment of SRSF7 and repression of exon inclusion. We also present a mechanism through which ADAR2 binds to dsRNA formed between GA-rich sequences and polypyrimidine (Py)-tract and precludes access of U2AF65 to 3′ splice site. Furthermore, we find these ADARs-regulated splicing changes per se influence tumorigenesis, not merely byproducts of ADARs editing and binding.

2019-12-22 | GSE131658 | GEO

mRNA profiling of wildtype, germline depleted, NMD mutant C. elegans whole worms and wildtype dissected gonads

Project description:Adjacent alternative 3’ splice sites, those separated by ≤18nt, provide a unique problem in the study of alternative splicing regulation; there is overlap of the cis-elements that define the adjacent sites. Identification of the intron's 3' end depends upon sequence elements that define the branchpoint, polypyrimidine tract and terminal AG dinucleotide. Starting with RNA-seq data from germline-enriched and somatic cell-enriched C. elegans samples, we identify hundreds of introns with adjacent alternative 3’ splice sites. We identify 203 events that undergo tissue-specific alternative splicing. For these, the regulation is mono-directional, with somatic cells preferring to splice at the distal 3' splice site and germline cells showing a distinct shift towards usage of the adjacent proximal 3' splice site. Splicing patterns in somatic cells follow consensus rules of 3’ splice site definition, using sites with a short stretch of pyrimidines and an AG dinucleotide. Splicing in germline cells occurs at proximal 3' splice sites that frequently lack a polypyrimidine tract or, occasionally, the AG dinucleotide. We provide evidence that use of germline-specific proximal 3' splice sites is conserved across Caenorhabditis species. We propose that divergent mechanisms exist between germline and somatic cells in determining an intron terminus at adjacent alternative 3’ splice sites. Examination of alternative splicing changes between germline- and somatic-cell enriched samples as well as nonsense-mediated decay mutants.

2015-04-22 | E-GEOD-64672 | biostudies-arrayexpress

A global regulatory mechanism for activating an exon network required for neurogenesis

Project description:The vertebrate and neural-specific SR-related protein nSR100/SRRM4 regulates an extensive program of alternative splicing with critical roles in nervous system development. However, the mechanism by which nSR100 controls its target exons is poorly understood. We demonstrate that nSR100-dependent neural exons are associated with a unique configuration of intronic cis-elements that promote rapid switch-like regulation during neurogenesis. A key feature of this configuration is the insertion of specialized intronic enhancers between polypyrimidine tracts and acceptor sites that bind nSR100 to potently activate exon inclusion in neural cells, while weakening 3' splice site recognition and contributing to exon skipping in non-neural cells. nSR100 further operates by forming multiple interactions with early spliceosome components bound proximal to 3' splice sites. These multifaceted interactions achieve dominance over neural exon silencing mediated by the splicing regulator PTBP1. The results thus illuminate a widespread mechanism by which a critical neural exon network is activated during neurogenesis. RNA-Seq was used to obtain mRNA profiles of various N2A and 293T cell lines from human and mouse, respectively, to investigate the roles of nSR100, Ptbp1 and U2af65 in alternative splicing regulation. PAR-iCLIP and iCLIP experiments followed by high throughput sequencing were conducted to obtain RNA binding profiles of nSR100, PTBP1 and U2af65.

2014-09-11 | E-GEOD-57278 | biostudies-arrayexpress

Homo sapiens

Project description:Interactions of aCPs with Cytosine-rich Polypyrimidine Tracts Enhance Splicing of Cassette Exons

| PRJNA291524 | ENA

mRNA profiling of wildtype, germline depleted, NMD mutant C. elegans whole worms and wildtype dissected gonads

2015-04-22 | GSE64672 | GEO

The exon junction complex controls transposable element activity by ensuring the faithful splicing of the piwi transcript

Project description:The exon junction complex (EJC) is a highly conserved ribonucleoprotein complex which binds RNAs at a late stage of the splicing reaction and remains associated following export to the cytoplasm. This complex is involved in several cellular post-transcriptional processes including mRNA localization, translation and degradation. The EJC plays an additional role in the splicing of a subset of genes in Drosophila and in human cells but the underlying mechanism remains to be elucidated. Here, we have found a novel function for the EJC and its splicing subunit RnpS1 in preventing transposon accumulation in both Drosophila germline and surrounding follicular cells. This function is mediated specifically through the control of the splicing of the piwi transcript. In absence of RnpS1 one of the piwi intron is retained. This intron contains a weak 5’ splice site as well as degenerate transposon fragments, reminiscent of heterochromatic introns. In addition, we identified a small A/T rich region, which alters its polypyrimidine tract (PPT) and confers the RnpS1’s dependency. Finally, we showed that the removal of this intron by RnpS1 requires the initial splicing of the flanking introns, suggesting a model in which the EJC facilitates the splicing of challenging introns following its initial deposition to adjacent exon junctions. In total there are 4 different conditions. Comparisons were made between piwi mutant vs control piwi and rnps1 KD vs controls RnpS1

2014-09-08 | E-GEOD-57710 | biostudies-arrayexpress

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