Proteomic study identifies Aurora-A mediated regulation of alternative splicing through multiple splicing factors
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PROVIDER: PRJNA1186114 | ENA |
REPOSITORIES: ENA
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Project description:The cell cycle regulator Aurora-A kinase presents an attractive target for cancer therapies, though its inhibition is also associated with toxic side effects. To gain a more nuanced understanding of Aurora-A function, we applied shotgun proteomics to identify 407 specific protein partners, including several splicing factors. Supporting a role in alternative splicing, we found that Aurora-A localizes to nuclear speckles, the storehouse of splicing proteins. Aurora-A interacts with and phosphorylates splicing factors both in vitro and in vivo, suggesting that it regulates alternative splicing by modulating the activity of these splicing factors. Consistently, Aurora-A inhibition significantly impacts the alternative splicing of 505 genes, with RNA motif analysis revealing an enrichment for Aurora-A interacting splicing factors. Additionally, we observed a significant positive correlation between the splicing events regulated by Aurora-A and those modulated by its interacting splicing factors. An interesting example is represented by CLK1 exon 4, which appears to be regulated by Aurora-A through SRSF3. Collectively, our findings highlight a broad role of Aurora-A in the regulation of alternative splicing.
2024-12-10 | GSE281881 | GEO
Project description:The goal of this study is to identify the effect of inhibition of Aurora-A kinase activity on gene expression and RNA splicing. The perturbation of Aurora-A is well known to affect cell cycle distribution. Therefore, we coupled the inhibition of Aurora-A with cell synchronization procedure in order to avoid the indirect effect of cell cycle perturbation on splicing changes. The mRNA -seq libraries were prepared and subjected to paired-end sequencing on Illumina HiSeq 2500 lanes. Differential gene expression and splicing analysis were carried using the edgeR tool and VAST-tools respectively. The RNA seq analysis identified that pharmacological inhibition of Aurora-A affects alternative splicing of 505 genes while having a marginal effect on gene expression. Overall our work identified Aurora-A as a novel splicing kinase and for the first time, describes a broad role of Aurora-A in regulating alternative splicing.
2024-11-21 | GSE160733 | GEO
Project description:Aurora-A phosphorylates splicing factors and regulates alternative splicing
| PRJNA674315 | ENA
Project description:In this study, we identified many new potential interactors of the Aurora-A kinase using a proteomic approach. A significant portion of Aurora-A interaction network is composed of proteins involved in pre-mRNA splicing, implying that Aurora-A signaling extends beyond its canonical function. Aurora-A directly interacts with many of the RRM domain-containing splicing factors such as SR proteins and hnRNP proteins and phosphorylates them in vitro. Aurora-A shows a subcellar distribution to nuclear speckles, the storehouse of splicing factors, consistent with its potential function in pre-mRNA splicing. Moreover, RNA-seq analysis of pharmacologically inhibited Aurora-A cells identified 261 genes whose RNA splicing is dependent on Aurora-A activity. These splicing affected genes are involved in various biological processes such as transcription, GTPase activity, ciliogenesis, DNA repair, RNA splicing and G2/M transition. Here, for the first time, we uncovered a relationship between Aurora-A activity and mRNA processing through a complex network of factors involved in RNA maturation.
2024-12-11 | PXD021093 | Pride
Project description:The Cell Division Cycle and Apoptosis Regulator (CCAR) protein family members have recently emerged as regulators of alternative splicing and transcription, as well as having other key physiological functions. For example, mammalian CCAR2/DBC1 forms a complex with the zinc factor protein ZNF326 to integrate alternative splicing with RNA polymerase II transcriptional elongation in AT-rich regions of the DNA. Additionally, Caenorhabditis elegans CCAR-1, a homolog to mammalian CCAR2, facilitates the alternative splicing of the perlecan unc-52 gene. However, much about the CCAR family's role in alternative splicing is unknown. We are interested in uncovering the role of the CCAR family in alternative splicing in vivo using Caenorhabditis elegans. We examined the role of CCAR-1 in genome-wide alternative splicing and identified new alternative splicing targets of CCAR-1 using RNA sequencing. Also, we found that CCAR-1 interacts with the spliceosome factors UAF-1 and UAF-2 using mass spectrometry, and that knockdown of ccar-1 affects alternative splicing patterns, motility, and proteostasis of UAF-1 mutant worms. Collectively, we demonstrate a role for CCAR-1 in the regulation of global alternative splicing in C. elegans and in conjunction with UAF-1
2024-02-26 | PXD043100 | Pride
Project description:Alternative pre-mRNA splicing increases proteomic diversity and provides a potential mechanism underlying both phenotypic diversity and susceptibility to genetic disorders in human populations. To investigate the variation in splicing among humans on a genomewide scale, we use a comprehensive exon-targeted microarray to examine alternative splicing in lymphoblasts derived from the CEPH HapMap population. We show the identification of transcripts containing annotated and novel sequence verified exon skipping, intron retention, and cryptic splice site usage that are specific between individuals. Using family-based linkage analysis, we demonstrate Mendelian inheritance and segregation of specific splice isoforms with regulatory haplotypes for three genes. Allelic association was further used to identify individual SNPs or regulatory haplotype blocks linked to the alternative splicing event, taking advantage of the high-resolution genotype information from the CEPH HapMap population. Keywords: Comparative genomic hybridiation within a 3 generation pedigree We used 14 individuals from the 3 generation CEPH/UTAH 1444 pedigree for our analysis of looking at heritability of differentially spliced exons within the family. 3 biological growths of individuals NA12739, NA12740, NA12750, and NA12751 were used and a single biological growth for the remaining 10 individuals.
2007-08-07 | E-GEOD-7952 | biostudies-arrayexpress
Project description:The goal of this study was to investigate the role of hnRNP L-like in alternative pre-mRNA splicing in human B-cells through an RNA-Seq approach. RNA-Seq was performed in DG75 cell line with over expression of hnRNP L-like or GFP as control.
2012-03-28 | E-GEOD-33013 | biostudies-arrayexpress
Project description:Glycogen Synthase Kinase-3 (GSK-3) is a constitutively active, ubiquitously expressed protein kinase that regulates multiple signaling pathways. Over 100 putative GSK-3 substrates have been reported in diverse cell types based on in vitro kinase assays or genetic and pharmacological manipulation of GSK-3. Many more have been predicted based on a recurrent GSK-3 consensus motif, but this prediction has not been tested by analyzing the GSK-3 phosphoproteome. We used stable isotope labeling of amino acids in culture (SILAC) and mass spectrometry to analyze the repertoire of GSK-3 dependent substrates in mouse embryonic stem cells (ESCs). A comparison of wild-type and Gsk3a;Gsk3b knockout (DKO) ESCs revealed prominent GSK-3-dependent phosphorylation of multiple splicing factors and regulators of RNA biosynthesis, as well as proteins that regulate transcription, translation, and cell division. We demonstrate direct, GSK-3-dependent phosphorylation of the splicing factors RBM8A and PSF as well as the nucleolar protein NPM1. RNA sequencing to compare the transcriptomes of wild-type and Gsk3 DKO ESCs identified more than 210 genes that are alternatively spliced in a GSK-3-dependent manner, supporting a broad role for GSK-3 in regulating alternative splicing. Overall, this study provides the first unbiased analysis of the GSK-3 phosphoproteome and strong evidence for GSK-3 as a regulator of alternative splicing.
2017-08-24 | GSE90921 | GEO
Project description:Alternative splicing (AS) of pre-mRNA is utilized by higher eukaryotes to achieve increased transcriptome and proteomic complexity. The serine/arginine (SR) splicing factors regulate tissue- or cell type-specific AS in a concentration and phosphorylation dependent manner. However, the mechanisms that modulate the cellular levels of active SR proteins remain to be elucidated. In the present study, we provide evidence for a role for the long nuclear-retained regulatory RNA (nrRNA), MALAT1 in AS regulation. MALAT1 interacts with SR proteins and influences the distribution of these and other splicing factors in nuclear speckle domains. Depletion of MALAT1 changes AS of endogenous pre-mRNAs, similar to what was observed upon overexpression of SR proteins. Furthermore, MALAT1 regulates cellular levels of phosphorylated forms of SR proteins. Taken together, our results suggest that MALAT1 regulates AS by modulating the levels of active SR proteins. Our results further highlight a novel role for a nrRNA in the regulation of gene expression. Malat1 Antisense and control knockdowns evaluated on a microarray platform to profile alternative splicing levels for 5782 cassette-type alternative exons.
2010-09-01 | E-GEOD-22963 | biostudies-arrayexpress
Project description:Alternative splicing is a key process underlying the evolution of increased proteomic and functional complexity and is especially prevalent in the mammalian nervous system. However, the factors and mechanisms governing nervous system-specific alternative splicing are not well understood. Through a genome-wide computational and expression profiling strategy, we have identified a tissue- and vertebrate-restricted Ser/Arg (SR)-repeat splicing factor, the neural-specific SR-related protein of 100 kDa (nSR100). We show that nSR100 regulates an extensive network of brain-specific alternative exons enriched in genes that function in neural cell differentiation. nSR100 acts by increasing the levels of the neural/brain-enriched polypyrimidine tract binding protein and by interacting with its target transcripts. Disruption of nSR100 prevents neural cell differentiation in cell culture and in the developing zebrafish. Our results thus reveal a critical neural-specific alternative splicing regulator, the evolution of which has contributed to increased complexity in the vertebrate nervous system. A microarray platform to profile alternative splicing levels for 8714 cassette-type alternative exons across a diverse spectrum of mouse tissues.
2010-05-15 | E-GEOD-16187 | biostudies-arrayexpress