Dataset Information

Disease-associated mutation in SRSF2 misregulates splicing by altering RNA-binding affinities.

ABSTRACT: Serine/arginine-rich splicing factor 2 (SRSF2) is an RNA-binding protein that plays important roles in splicing of mRNA precursors. SRSF2 mutations are frequently found in patients with myelodysplastic syndromes and certain leukemias, but how these mutations affect SRSF2 function has only begun to be examined. We used clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 nuclease to introduce the P95H mutation to SRSF2 in K562 leukemia cells, generating an isogenic model so that splicing alterations can be attributed solely to mutant SRSF2. We found that SRSF2 (P95H) misregulates 548 splicing events (<1% of total). Of these events, 374 involved the inclusion of cassette exons, and the inclusion was either increased (206) or decreased (168). We detected a specific motif (UCCA/UG) enriched in the more-included exons and a distinct motif (UGGA/UG) in the more-excluded exons. RNA gel shift assays showed that a mutant SRSF2 derivative bound more tightly than its wild-type counterpart to RNA sites containing UCCAG but bound less tightly to UGGAG sites. Thus in most cases the pattern of exon inclusion or exclusion correlated with stronger or weaker RNA binding, respectively. We further show that the P95H mutation does not affect other functions of SRSF2, i.e., protein-protein interactions with key splicing factors. Our results thus demonstrate that the P95H mutation positively or negatively alters the binding affinity of SRSF2 for cognate RNA sites in target transcripts, leading to misregulation of exon inclusion. Our findings shed light on the mechanism of the disease-associated SRSF2 mutation in splicing regulation and also reveal a group of misspliced mRNA isoforms for potential therapeutic targeting.

SUBMITTER: Zhang J

PROVIDER: S-EPMC4553800 | biostudies-literature | 2015 Aug

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Disease-associated mutation in SRSF2 misregulates splicing by altering RNA-binding affinities.

Zhang Jian J Lieu Yen K YK Ali Abdullah M AM Penson Alex A Reggio Kathryn S KS Rabadan Raul R Raza Azra A Mukherjee Siddhartha S Manley James L JL

Proceedings of the National Academy of Sciences of the United States of America 20150810 34

Serine/arginine-rich splicing factor 2 (SRSF2) is an RNA-binding protein that plays important roles in splicing of mRNA precursors. SRSF2 mutations are frequently found in patients with myelodysplastic syndromes and certain leukemias, but how these mutations affect SRSF2 function has only begun to be examined. We used clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 nuclease to introduce the P95H mutation to SRSF2 in K562 leukemia cells, generating a ...[more]

PMID: 26261309

Similar Datasets

Project description:BackgroundThe complicated cellular and biochemical changes that occur in brain during Alzheimer's disease are poorly understood. In a previous study we used an unbiased label-free quantitative mass spectrometry-based proteomic approach to analyze these changes at a systems level in post-mortem cortical tissue from patients with Alzheimer's disease (AD), asymptomatic Alzheimer's disease (AsymAD), and controls. We found modules of co-expressed proteins that correlated with AD phenotypes, some of which were enriched in proteins identified as risk factors for AD by genetic studies.MethodsThe amount of information that can be obtained from such systems-level proteomic analyses is critically dependent upon the number of proteins that can be quantified across a cohort. We report here a new proteomic systems-level analysis of AD brain based on 6,533 proteins measured across AD, AsymAD, and controls using an analysis pipeline consisting of isobaric tandem mass tag (TMT) mass spectrometry and offline prefractionation.ResultsOur new TMT pipeline allowed us to more than double the depth of brain proteome coverage. This increased depth of coverage greatly expanded the brain protein network to reveal new protein modules that correlated with disease and were unrelated to those identified in our previous network. Differential protein abundance analysis identified 350 proteins that had altered levels between AsymAD and AD not caused by changes in specific cell type abundance, potentially reflecting biochemical changes that are associated with cognitive decline in AD. RNA binding proteins emerged as a class of proteins altered between AsymAD and AD, and were enriched in network modules that correlated with AD pathology. We developed a proteogenomic approach to investigate RNA splicing events that may be altered by RNA binding protein changes in AD. The increased proteome depth afforded by our TMT pipeline allowed us to identify and quantify a large number of alternatively spliced protein isoforms in brain, including AD risk factors such as BIN1, PICALM, PTK2B, and FERMT2. Many of the new AD protein network modules were enriched in alternatively spliced proteins and correlated with molecular markers of AD pathology and cognition.ConclusionsFurther analysis of the AD brain proteome will continue to yield new insights into the biological basis of AD.

Project description:Recent developments in our understanding of the interactions between long non-coding RNAs (lncRNAs) and cellular components have improved treatment approaches for various human diseases including cancer, vascular diseases, and neurological diseases. Although investigation of specific lncRNAs revealed their role in the metabolism of cellular RNA, our understanding of their contribution to post-transcriptional regulation is relatively limited. In this study, we explore the role of lncRNAs in modulating alternative splicing and their impact on downstream protein-RNA interaction networks. Analysis of alternative splicing events across 39 lncRNA knockdown and wildtype RNA-sequencing datasets from three human cell lines-HeLa (cervical cancer), K562 (myeloid leukemia), and U87 (glioblastoma)-resulted in the high-confidence (false discovery rate (fdr) < 0.01) identification of 11,630 skipped exon events and 5895 retained intron events, implicating 759 genes to be impacted at the post-transcriptional level due to the loss of lncRNAs. We observed that a majority of the alternatively spliced genes in a lncRNA knockdown were specific to the cell type. In tandem, the functions annotated to the genes affected by alternative splicing across each lncRNA knockdown also displayed cell-type specificity. To understand the mechanism behind this cell-type-specific alternative splicing pattern, we analyzed RNA-binding protein (RBP)-RNA interaction profiles across the spliced regions in order to observe cell-type-specific alternative splice event RBP binding preference. Despite limited RBP binding data across cell lines, alternatively spliced events detected in lncRNA perturbation experiments were associated with RBPs binding in proximal intron-exon junctions in a cell-type-specific manner. The cellular functions affected by alternative splicing were also affected in a cell-type-specific manner. Based on the RBP binding profiles in HeLa and K562 cells, we hypothesize that several lncRNAs are likely to exhibit a sponge effect in disease contexts, resulting in the functional disruption of RBPs and their downstream functions. We propose that such lncRNA sponges can extensively rewire post-transcriptional gene regulatory networks by altering the protein-RNA interaction landscape in a cell-type-specific manner.

Dataset Information

Disease-associated mutation in SRSF2 misregulates splicing by altering RNA-binding affinities.

Publications

Disease-associated mutation in SRSF2 misregulates splicing by altering RNA-binding affinities.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets