Serine-arginine rich splicing factor 3 (SRSF3) is an RNA binding protein previously implicated in controlling somatic reprogramming, but the underlying molecular mechanisms remain elusive. Consistent with the central role of SRSF3 in reprogramming, SRSF3 mRNA expression significantly increases during reprogramming. In order to understand the mechanisms through which SRSF3 regulates reprogramming efficiency and the maintenance of pluripotency, we generated a reprogrammable Srsf3 knockout mouse model with a tamoxifen inducible Cre-lox system to knockout Srsf3 and doxycycline inducible expression of Oct4-Klf4-Sox2-Myc (OKSM) transcription factors. Mouse embryonic fibroblasts (MEFs) reprogrammed with a significantly lower efficiency following SRSF3 depletion. Consistent with studies in cancer cells, loss of SRSF3 led to reduced cell proliferation and cell cycle arrest at G1-S transition during the early stages of reprogramming. On the other hand, the overexpression of SRSF3 in reprogrammable MEFs increased cell proliferation and reprogramming efficiency.

Crosslinking and immunoprecipitation (iCLIP) assay performed in pluripotent cells resulted in an increased fraction of SRSF3 crosslink sites within noncoding RNAs, including pronounced binding to microRNAs (miRNAs). Many miRNAs have been shown to play a critical role in the regulation of gene expression both during reprogramming and in pluripotent cells. SRSF3 crosslink sites were found downstream of pri-miRNA hairpins of ES cell specific miRNA clusters miR290 and miR-17-92, which produce more than 70% of all mature miRNAs in pluripotent cells. Knockdown of SRSF3 in iPS cells did not affect the levels of pri-miRNAs, but the level of mature miRNAs were significantly reduced, providing evidence for the in vivo significance of SRSF3 binding to miRNA precursor to mediate their processing in pluripotent cells. One of the key miR290 and miR-17-92 family targets in pluripotent cells is Cdkn1a (encoding P21) which did not have SRSF3 crosslink sites. SRSF3 depleted iPS cells showed increased expression of Cdkn1a, a known inhibitor of the G1-S transition of the cell cycle. Further, SRSF3 depleted iPS cells had decreased level of c-Myc, a promoter of the G1-S transition which is also known to be regulated by ES cell specific miRNA. We conclude that SRSF3 enhances cell proliferation and maintains the cell cycle in pluripotent cells through the iPS cell specific regulation of Cdkn1a, c-Myc and potentially other miR290 and miR-17-92 targets.