Bulk RNAseq of megakaryocytes and Lin- cells of mice with Srsf2-P95H mutation, Jak2-V617F mutation, both, or none
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ABSTRACT:
PROVIDER: PRJEB54749 | ENA |
REPOSITORIES: ENA
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Project description:We performed bulk RNAseq of sorted megakaryocytes and subsequent Lin- cells to study the impact of the combination of Jak2 V617F mutations and Srsf2 P95H mutations
2023-07-01 | E-MTAB-11960 | biostudies-arrayexpress
Project description: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. Examination of differentially spliced events in K562 CRISPR cell clones (with wild-type or mutant SRSF2) by RNA sequencing
2015-08-10 | E-GEOD-71299 | biostudies-arrayexpress
Project description: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.
2015-08-10 | GSE71299 | GEO
Project description:Causal mutations in multiple classes of genes have been identified in MDS patients with some patients harboring more than one mutation. Interestingly, double mutations tend to occur in different classes, rather than the same classes of genes, as exemplified by frequent co-occurring mutations in the transcription factor RUNX1 and the splicing factor SRSF2. Here we report a mouse model in which Runx1 knockout was combined with the Srsf2 P95H mutation to cause multi-lineage hematopoietic defects. Besides their additive and synergistic effects, we also unexpectedly noted a degree of antagonizing activity of single mutations in specific hematopoietic progenitors. To uncover the mechanism, we further developed a cellular model using human K562 cells and performed parallel gene expression and splicing analyses in both human and murine contexts. We performed RNA-seq on sorted Lineage- c-Kit+ (LK) hematopoietic progenitors from bone marrow of mice from WT, runx1 KO , Srsf2 P95H knock in and runx1 Srsf2 P95H double mutants. We also performed RNA-seq on set of isogenic K562 cell lines: wild type (WT), SRSF2 P95H knock-in (SRSF2P95H/+/+), RUNX1 knockdown (RUNX1 KD), and SRSF2 P95H knock-in with RUNX1 knockdown (Double). Strikingly, while RUNX1 deficiency was responsible for altered transcription in both single and double mutants, it also induced dramatic changes in global splicing, as seen with mutant SRSF2, and only their combination induced mis-splicing of genes selectively enriched in the DNA damage response and cell cycle checkpoint pathways. Collectively, these data reveal the convergent impact of a prototypic MDS-associated double mutant on RNA processing and suggest that aberrant DNA damage repair and cell cycle regulation critically contribute to MDS development.
2022-09-27 | GSE200446 | GEO
Project description:We report the biological function of Srsf2 in hematopoiesis in conditional knockout mouse models. Ablation of Srsf2 in the hematopoietic lineage caused embryonic lethality, and Srsf2-deficient fetal liver cells showed significantly enhanced apoptosis and decreased hematopoietic stem/progenitor cells. Induced ablation of Srsf2 in adult Mx1Cre/ Srsf2flox/flox mice upon polyinosinic:polycytidylic acid injection demonstrated a significant decrease in lineage-/Sca+/cKit+ cells in bone marrow. To reveal the functional impact of MDS-associated mutations in SRSF2, we profiled global splicing responses on an MDS-L cell line using RASL-seq, and found that the P95H missense mutation and P95 to R102 in-frame 8 amino-acid deletion caused significant changes in alternative splicing. The affected genes were enriched in cancer development and apoptosis. These findings suggest that intact Srsf2 is essential for the functional integrity of the hematopoietic system, and its mutations are likely key driver events to MDS. MDS-L cells (in triplicate) were transfected by srsf2 shRNA only, or pTRIPZ vectors containing both srsf2 shRNA and srsf2 mutants cDNA including P95H and P95 8 amino acid deletion as well as wild-type construct, followed by Dox induction. Total RNAs were extracted and been analyzed by RASL-seq.
2015-03-01 | E-GEOD-61052 | biostudies-arrayexpress
Project description:Recurrent Glycogen synthase kinase-3 (GSK-3) phosphorylates multiple splicing factors, including SRSF2, and regulates the splicing of a broad range of mRNAs in human cells. Inhibition of GSK-3 disrupts splicing and promotes cell death in hematopoietic cells with heterozygous mutations in SRSF2 and not in cells with wild-type splicing factors. To characterize how GSK-3 inhibition alters the cellular proteome in SRSF2-P95H/+ cells, we have performed quantitative mass spectrometry (qMS) on K562 cells with SRSF2P95H/+ knocked into the endogenous locus (PMID 30799057) and parental K562 cells, treated with the GSK-3 inhibitor CHIR99021. We focused on the mitochondrial proteome using human proteome (UniprotKB) and mitochondrial (MitoCarta 3.0) databases for protein identification, identifying 163 mitochondrial proteins whose levels are affected by SRSF2 mutation.
2024-06-22 | PXD043213 | Pride
Project description:Transcriptome analysis of Hoxb8 imortalised GM-CSF cell lines with SRSF2 P95H mutation [RNA-seq]
| PRJNA694744 | ENA
Project description:Mutations in the splicing factor Serine Arginine Rich Splicing Factor 2 (SRSF2) occur in over 30% of patients with myelodysplasia (MDS) and a subset of patients with acute myeloid leukemia (AML) and portend a poor prognosis. SRSF2 is ubiquitously expressed and directs exon inclusion or exclusion in a sequence and context dependent manner. SRSF2 mutations almost exclusively affect the proline at position 95 in the C-terminus of the RNA binding domain. We here show how a mutation in SRSF2 (P95H) affects its normal function in vivo on a transcriptome-wide basis via RNA crosslinking and immunoprecipitation (HITS-CLIP) and parallel quantification of transcript levels (RNA-seq), leading to the identification of differentially bound and alternatively spliced SRSF2 targets. Keywords: Splicing, myeloid neoplasia, SRSF2, HITS-CLIP
2018-06-18 | GSE111900 | GEO
Project description:Current strategies to target RNA splicing mutant myeloid cancers propose disruption of normal splicing activity by targeting the splicing apparatus therapeutically. This approach is only modestly sensitizing and is also toxic to non-mutant bearing wild-type cells. To explore potentially exploitable genetic interactions with spliceosome mutations, we combined data mining and functional screening for synthetic lethal interactions with an Srsf2P95H/+ mutation. Analysis of mis-splicing events in a series of both human and murine SRSF2P95H mutant samples across multiple myeloid diseases (AML, MDS, CMML) was first performed to identify conserved mis-splicing events. From this analysis, we identified that the DNA repair and cell cycle pathways were overrepresented within the conserved mis-spliced transcript sets. In parallel, to functionally define pathways essential for survival and proliferation Srsf2P95H/+ cells, we performed a genome-wide pooled CRISPR loss of function screen using Hoxb8 immortalised R26-CreERki/+ Srsf2P95H/+ and R26-CreERki/+ Srsf2+/+ cell lines. We assessed for loss of sgRNA representation at three timepoints: immediately after Srsf2P95H/+ activation, and at one week and two weeks post Srsf2P95H/+ mutation. Pathway analysis demonstrated that the DNA damage response and cell cycle pathways were amongst the top synthetic lethal pathways with Srsf2P95H/+ mutation., Based on the loss of guide RNAs targeting Cdk6, we identified that Palbocilib, a CDK6 inhibitor, showed preferential sensitivity in Srsf2P95H/+ cell lines and in primary non-immortalised lin-cKIT+Sca-1+ cells compared to wild type controls. Our data strongly suggest that cell cycle and DNA damage response pathways are required for Srsf2P95H/+ cell survival, and that Palbociclib could be an alternative therapeutic option for targeting SRSF2 mutant myeloid cancers.
2021-09-15 | GSE165504 | GEO
Project description:We report the biological function of Srsf2 in hematopoiesis in conditional knockout mouse models. Ablation of Srsf2 in the hematopoietic lineage caused embryonic lethality, and Srsf2-deficient fetal liver cells showed significantly enhanced apoptosis and decreased hematopoietic stem/progenitor cells. Induced ablation of Srsf2 in adult Mx1Cre/ Srsf2flox/flox mice upon polyinosinic:polycytidylic acid injection demonstrated a significant decrease in lineage-/Sca+/cKit+ cells in bone marrow. To reveal the functional impact of MDS-associated mutations in SRSF2, we profiled global splicing responses on an MDS-L cell line using RASL-seq, and found that the P95H missense mutation and P95 to R102 in-frame 8 amino-acid deletion caused significant changes in alternative splicing. The affected genes were enriched in cancer development and apoptosis. These findings suggest that intact Srsf2 is essential for the functional integrity of the hematopoietic system, and its mutations are likely key driver events to MDS.
2015-03-01 | GSE61052 | GEO