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Series GSE71299 Query DataSets for GSE71299
Status Public on Aug 10, 2015
Title Disease-associated mutation in SRSF2 misregulates splicing by altering RNA binding affinities
Organism Homo sapiens
Experiment type Expression profiling by high throughput sequencing
Summary SRSF2 is an RNA binding protein that plays important roles in splicing of mRNA precursors. Mutations in SRSF2 are frequently found in patients with myelodysplastic syndromes and certain leukemias, but how they affect SRSF2 function has only begun to be examined. Here we used CRISPR/Cas9 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, 374 involve 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 less tightly to UGGAG sites. The pattern of exon inclusion or exclusion thus correlated in most cases 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 not only shed light on the mechanism of the disease-associated SRSF2 mutation in splicing regulation, but also reveal a group of mis-spliced mRNA isoforms for potential therapeutic targeting.
 
Overall design Examination of differentially spliced events in K562 CRISPR cell clones (with wild-type or mutant SRSF2) by RNA sequencing
 
Contributor(s) Manley JL, Mukherjee S
Citation(s) 26261309
Submission date Jul 24, 2015
Last update date May 15, 2019
Contact name James L. Manley
E-mail(s) jlm2@columbia.edu
Organization name Columbia University
Department Dept. of Biological Sciences
Lab 1119 Fairchild Center
Street address 1212 Amsterdam Ave
City New York
State/province New York
ZIP/Postal code 10027
Country USA
 
Platforms (1)
GPL11154 Illumina HiSeq 2000 (Homo sapiens)
Samples (9)
GSM1831901 K562 parental cells
GSM1831902 W19 (wild-type SRSF2)
GSM1831903 W33 (wild-type SRSF2)
Relations
BioProject PRJNA290859
SRA SRP061539

Download family Format
SOFT formatted family file(s) SOFTHelp
MINiML formatted family file(s) MINiMLHelp
Series Matrix File(s) TXTHelp

Supplementary file Size Download File type/resource
GSE71299_RAW.tar 1.9 Mb (http)(custom) TAR (of TXT)
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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