Abstract
Alternative pre-mRNA splicing is a key process of biological diversity and normal gene expression. More than 90% of human multi-exon genes undergo alternative pre-mRNA processing. Mis-regulation of normal splicing patterns may give rise to pathophysiological processes and has been associated with human diseases, such as cancer. In the past few years, numerous studies have reported mutations involving multiple components of the mRNA splicing machinery including SF3B1, SRSF2, U2AF1, ZRSR2, PRPF40B, U2AF65 and SF1 in patients with MDS. Furthermore, the most frequently mutated spliceosome component in MDS, SF3B1 (30% of cases), is mutated in 70-85% of refractory anemia with ringed sideroblasts (RARS) cases and is highly associated with the presence of ringed sideroblasts. However, the pathophysiological role of SF3B1 mutations in MDS has not yet been elucidated.
In this study, we analyzed the role of pre-mRNA-splicing factor Sf3b1 in hematopoiesis. While Sf3b1 homozygous knockout mice die during early embryonic development around the 16- to 32-cell stage, Sf3b1 heterozygous knockout mice (Sf3b1+/-) are born healthy. Sf3b1+/- maintained almost normal hematopoiesis by 70 weeks and did not develop any hematological malignancies during the observation period. Seventy-week-old mice, however, showed moderately enhanced apoptosis in LSK cells. In the noncompetitive BM repopulating assays, Sf3b1+/- BM cells showed no obvious defects in repopulating recipients’ hematopoiesis. In contrast, Sf3b1+/- BM cells showed modest but significant impairment in repopulating capacity in competitive settings, although they did not show obvious defects in differentiation.
We next knocked down Sf3b1 in Sf3b1+/- hematopoietic stem cells (HSCs) using lentiviruses expressing shRNAs against Sf3b1. We found that depletion of Sf3b1 to the levels around 1/4 of that in wild-type (WT) cells causes marked inhibition of cell growth in vitro. Even the control Sf3b1+/- HSCs showed mildly compromised proliferative capacity in culture. Furthermore, depletion of Sf3b1 abolished repopulating capacity of HSCs in vivo.
Microarray analysis revealed little difference in gene expression profiles between WT and Sf3b1+/- LSK cells. Of interest, however, the genes on the non-sense-mediated mRNA decay (NMD) pathway, which is reportedly activated in HeLa cells upon expression of an U2AF35 mutant from MDS patients, were not activated in Sf3b1+/- LSK cells in the GSEA analysis, suggesting heterozygosity for Sf3b1 is not enough to induce abnormal RNA splicing that induce the NMD activity for surveillance of abnormal transcripts.
Finally, we evaluated the frequencies of sideroblastic erythroblasts in erythroblasts from WT and Sf3b1+/- BM and those from recipients’ BM repopulated with WT and Sf3b1+/- BM cells at 11 months post-transplantation. Unexpectedly, however, Prussian blue iron staining revealed no significant changes in frequencies of sideroblasts by heterozygosity for Sf3b1.
In conclusion, our findings suggest that the level of Sf3b1 gene expression is critical in the maintenance of proliferative capacity of adult HSCs. Nonetheless, the heterozygisity for Sf3b1 alone does not induce apparent splicing abnormalities or MDS-like disease with sideroblasts. Evaluation of more severe Sf3b1 hypomorphic mice or mice expressing SF3B1 mutations will be needed to precisely understand the pathological role of SF3B1 mutations in MDS.
Disclosures:
No relevant conflicts of interest to declare.
Spliceosome activation by PRP2 ATPase prior to the first transesterification reaction of pre-mRNA splicing.
