Disease-associated mutation in SRSF2 misregulates splicing by altering RNA-binding affinities.
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.
Department of Biological Sciences, Columbia University, New York, NY 10027;
Jian
J
Zhang
Irving Cancer Research Center, Columbia University, New York, NY 10032;
Yen K
YK
Lieu
Irving Cancer Research Center, Columbia University, New York, NY 10032;
Abdullah M
AM
Ali
Department of Systems Biology, Columbia University, New York, NY 10032;
Alex
A
Penson
Department of Biological Sciences, Columbia University, New York, NY 10027;
Kathryn S
KS
Reggio
Department of Systems Biology, Columbia University, New York, NY 10032; Department of Biomedical Informatics, Columbia University, New York, NY 10032;
Raul
R
Rabadan
Irving Cancer Research Center, Columbia University, New York, NY 10032; Division of Hematology and Oncology, Department of Medicine, Columbia University, New York, NY 10032.
Azra
A
Raza
Irving Cancer Research Center, Columbia University, New York, NY 10032; Division of Hematology and Oncology, Department of Medicine, Columbia University, New York, NY 10032 sm3252@cumc.columbia.edu jlm2@columbia.edu.
Siddhartha
S
Mukherjee
Department of Biological Sciences, Columbia University, New York, NY 10027; sm3252@cumc.columbia.edu jlm2@columbia.edu.
James L
JL
Manley
D001665:Binding Sites; D064130:CRISPR-Associated Proteins / Q000235:genetics; D005091:Exons; D006801:Humans; D009154:Mutation; D009687:Nuclear Proteins / Q000235:genetics* / Q000502:physiology; D012313:RNA / Q000378:metabolism*; D012326:RNA Splicing; D012261:Ribonucleoproteins / Q000235:genetics* / Q000502:physiology; D000068103:Serine-Arginine Splicing Factors
D064130:CRISPR-Associated Proteins; D009687:Nuclear Proteins; D012261:Ribonucleoproteins; C073288:SRSF2 protein, human; D000068103:Serine-Arginine Splicing Factors; D012313:RNA
leukemia; myelodysplastic syndromes; pre-mRNA splicing; serine/arginine-rich proteins; spliceosome