SRSF2 is essential for hematopoiesis and its myelodysplastic syndromes-related mutations dysregulate alternative pre-mRNA splicing

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.

本研究在条件性基因敲除小鼠模型中解析了Srsf2在造血过程中的生物学功能。在造血细胞谱系中敲除Srsf2会导致胚胎致死，且Srsf2缺陷的胎肝细胞凋亡显著增强，造血干/祖细胞数量明显减少。经聚肌胞苷酸（polyinosinic:polycytidylic acid）诱导成年Mx1Cre/ Srsf2flox/flox小鼠的Srsf2条件性敲除后，其骨髓中lineage-/Sca+/cKit+细胞群体显著降低。为揭示SRSF2相关骨髓增生异常综合征（Myelodysplastic Syndromes, MDS）突变的功能影响，我们利用RASL-seq技术对MDS-L细胞系开展了全基因组可变剪接应答谱分析，发现P95H错义突变以及P95至R102区域的框内8个氨基酸缺失均可导致可变剪接发生显著改变。受影响的基因显著富集于癌症发生与细胞凋亡相关通路。上述研究结果表明，完整的Srsf2对于造血系统的功能完整性至关重要，其突变可能是骨髓增生异常综合征发生的关键驱动事件。本研究设置三组生物学重复的MDS-L细胞，分别仅转染srsf2短发夹RNA（short hairpin RNA, shRNA），或转染同时携带srsf2 shRNA与srsf2突变体互补DNA（complementary DNA, cDNA）的pTRIPZ载体（包含P95H突变体、P95 8氨基酸缺失突变体以及野生型构建体），随后加入多西环素（Doxycycline, Dox）诱导表达。提取总RNA后通过RASL-seq进行测序分析。