Direct regulation of alternative splicing by SMAD3 through interaction with PCBP1 is essential for the tumor-promoting role of TGF-beta
ABSTRACT: To assess the global impact of TGF-beta on alternative splicing, we conducted RNA-seq of total RNAs isolated from various lines of HeLa cells that were generated for this study and treated without or with TGF-beta and EGF. Using rMAT tool from the RNA-Seq data and annotation of transcripts in GTF format, we identified differential alternative splicing events between untreated and treated cell lines. Our study shows that the TGF-beta-mediated alternative splicing affects the protein products of a large number of genes enriched in pathways critical for EMT, cykeskeleton organization, and adherens junction signaling. PCBP1 was required for the most, if not all, alternative splicing events detected. SRA accession number: SRP061634, BioProject ID PRJNA290799. RNAs from Hela cells were treated with TGF-β and EGF, in triplicates and sequenced on HiSeq2000 with 2 samples pooled into one lane with Illumina TruSeq v3 chemistry.
Project description:Alternative polyadenylation has been implicated as an important regulator of gene expression. In some cases, alternative polyadenylation is known to couple with alternative splicing to influence last intron removal. However, it is unknown whether alternative polyadenylation events influence alternative splicing decisions at upstream exons. Knockdown of the polyadenylation factors CFIm25 or CstF64 was used as an approach in identifying alternative polyadenylation and alternative splicing events on a genome-wide scale. Although hundreds of alternative splicing events were found to be differentially spliced in the knockdown of CstF64, genes associated with alternative polyadenylation did not exhibit an increased incidence of alternative splicing. These results demonstrate that the coupling between alternative polyadenylation and alternative splicing is usually limited to defining the last exon. The striking influence of CstF64 knockdown on alternative splicing can be explained through its effects on UTR selection of known splicing regulators such as hnRNP A2/B1, thereby indirectly influencing splice site selection. We conclude that changes in the expression of the polyadenylation factor CstF64 influences alternative splicing through indirect effects. HeLa cell line was stably transfected with shRNA plasmids targeting CstF64. Total RNA was isolated from CstF64 KD cells and wild-type control cells using Trizol according to manufacturer’s protocols. Samples were deep sequenced in duplicate using the Illumina GAIIx system.
Project description:Analysis of the alternative pre-mRNA procesing after SF3b155 siRNA knock-down in HeLa cells employing a custom microarray platform sensitive to splicing. HeLa cells were transfected with either a scrambled RNA or one of the two different SF3b155 siRNA oligonucleotides (oligo 3: 5’-GACAGCAGAUUUGCUGGAUACGUGA-3; oligo 5: 5’-CCCUGUGGCAUUGCUUAAUGAUAU-3’). Total RNA samples from three biological replicates were labeled in direct and dye-swap hybridizations. Labeled samples were hybridized into our custom splicing-sensitive agilent platform which contains 1804 splicing events from 482 genes.
Project description:We performed EGF treatment and hnRNP A1 knockdown in HeLa cells and analyzed alternative splicing patterns by high-throughput RNA sequencing. Overall design: The mRNA profiles of control/EGF-treated/hnRNP A1-knockdown HeLa cells were generated by deep sequencing using Illumina HiSeq2000.
Project description:Abstract: Alternative splicing (AS) plays a major role in the generation of proteomic diversity and in gene regulation. However, the role of the basal splicing machinery in regulating AS remains poorly understood. Here we show that the core snRNP protein SmB/B’ self-regulates its expression by promoting the inclusion of a highly-conserved alternative exon in its own pre-mRNA that targets the spliced transcript for nonsense-mediated mRNA decay (NMD). Depletion of SmB/B’ in human cells results in reduced levels of snRNPs and in a striking reduction in the inclusion levels of hundreds of alternative exons, with comparatively few effects on constitutive exon splicing levels. The affected alternative exons are enriched in genes encoding RNA processing and other RNA binding factors, and a subset of these exons also regulate gene expression by activating NMD. Our results thus demonstrate a role for the core spliceosomal machinery in controlling an exon network that appears to modulate the levels of many RNA processing factors. HeLa cells were transfected with a control non-targeting siRNA pool (siNT), or with siRNA pools designed to knockdown SmB/B' or SRSF1 (also known as SF2/ASF/SFRS1). Sequence reads were aligned to exon-exon junction sequences in a database of EST/cDNA-mined cassette-type alternative splicing events. Processed data files (.bed and .txt) provided as supplementary files on the Series record. Processed data file build information: hg18.
Project description:Alternative splicing profiling of apopotosis related genes in human HeLa cells (cervical cancer cell line) transfected with a plasmid expressing shRNAs targetting p68 helicase (DDX5, DEAD (Asp-Glu-Ala-Asp) box polypeptide 5) cloned into the pSuper expression vector compared to empty vector. Keywords: treated vs. untreated comparison; alternative splicing Two-condition experiment, where the p68 DDX5 gene product levels was inhibited by siRNA transfection and compared to transfection with the negative control (scrambled siRNA). Biological replicates: 2, all independently grown and harvested. A dye swapping technical duplicate was performed for each biological replicate. Samples were hybridized onto a 44290 feature array designed by ExonHit to detect splicing events in apopotosis related genes and manufactured by Agilent using in situ synthesis of oligonucleotides by SurePrint technology.
Project description:This SuperSeries is composed of the following subset Series: GSE23513: Position-dependent alternative splicing activity revealed by global profiling of alternative splicing events regulated by PTB (HJAY) GSE23514: Position-dependent alternative splicing activity revealed by global profiling of alternative splicing events regulated by PTB (Exon array) Refer to individual Series
Project description:Mediator complex is an integrative hub for transcriptional regulation. Here we show that Mediator regulates alternative mRNA processing via its Med23 subunit. Combining tandem affinity purification and mass spectrometry, we identified a number of mRNA processing factors that bind to a soluble recombinant Mediator subunit MED23 but not to several other Mediator components. One of these factors, hnRNP L, specifically interacts with MED23 in vitro and in vivo. Consistently, Mediator partially colocalizes with hnRNP L and the splicing machinery in the cell. Functionally Med23 regulates a subset of hnRNP L-targeted alternative splicing (AS) and alternative cleavage and polyadenylation (APA) events as shown by minigene reporters and exon array analysis. ChIP-seq analysis revealed that Med23 can regulate hnRNP L occupancy at their co-regulated genes. Taken together, these results demonstrate a crosstalk between Mediator and the splicing machinery, suggesting a novel mechanism for coupling mRNA processing to transcription. We performed an exon array experiment using HeLa cells expressing Med23, hnRNP L or control siRNAs which were established by a virus-mediated siRNA technology. Each sample was done in three biological replicates. Total RNA of these cell lines was processed and hybridized to the Affymetrix human exon array.
Project description:Mediator complex is an integrative hub for transcriptional regulation. Here we show that Mediator regulates alternative mRNA processing via its Med23 subunit. Combining tandem affinity purification and mass spectrometry, we identified a number of mRNA processing factors that bind to a soluble recombinant Mediator subunit MED23 but not to several other Mediator components. One of these factors, hnRNP L, specifically interacts with MED23 in vitro and in vivo. Consistently, Mediator partially colocalizes with hnRNP L and the splicing machinery in the cell. Functionally Med23 regulates a subset of hnRNP L-targeted alternative splicing (AS) and alternative cleavage and polyadenylation (APA) events as shown by minigene reporters and exon array analysis. ChIP-seq analysis revealed that Med23 can regulate hnRNP L occupancy at their co-regulated genes. Taken together, these results demonstrate a crosstalk between Mediator and the splicing machinery, suggesting a novel mechanism for coupling mRNA processing to transcription. Examination of hnRNP L and H3K36me3 enrichment in sictrl and si23 Hela cells
Project description:The Ewing's Sarcoma Protein EWS belongs to the TET family (FUS/TLS, EWS, TAF15) of RNA and DNA binding proteins, implicated in DNA transcription, pre-mRNA splicing and maintenance of genome integrity. To identify in vivo RNA targets of EWS we performed a combinatorial approach of CLIP-Seq In this dataset, we include the expression data obtained from three biological replicates of HeLa cells transfected with either scrambled or siEWS oligonucleotides. These data allowed us to identify 805 genes affected at the expression level by EWS knockdown, of which 395 upregulated and 410 downregulated. Moreover, HJAY identified 206 differentially regulated alternative splicing events in 114 genes. Three biological replicates were labeled and hybridized onto Affymetrix HJAY