ZRSR2 Mutations Cause Dysregulated RNA Splicing in MDS

Recurrent somatic mutations have been uncovered in several components of the spliceosome in Myelodysplastic Syndrome (MDS). Recent high throughput sequencing of large cohorts of MDS has established RNA splicing as the pathway most frequently targeted by somatic mutations. These findings implicate dysregulated RNA splicing in the pathogenesis of MDS. However, the mechanism linking aberrant splicing to the development of MDS is unknown. ZRSR2, a frequently mutated spliceosome gene in MDS is located on the X chromosome. Somatic alterations of ZRSR2 are typically inactivating mutations (frameshift indels, nonsense point mutations or splice site mutations) which are observed predominantly in males. Mutations in ZRSR2are more prevalent in MDS subtypes without ring sideroblasts and chronic myelomonocytic leukemia (CMML) and are associated with elevated bone marrow blasts and higher rate of progression to AML. Although ZRSR2 has been suggested to interact with other splice proteins, U2AF2 and SRSF2, at the 3΄ splice sites during the pre-spliceosome assembly, its precise role in RNA splicing remains unexplored.

In this study, we demonstrate that deficiency of ZRSR2 leads to impaired splicing of U12-type introns which are dependent upon the minor spliceosome. RNA-Sequencing of MDS bone marrow harboring inactivating mutations of ZRSR2 revealed aberrant retention of U12-type introns as compared to bone marrow with wild-type ZRSR2. In addition, several U12-type introns displayed mis-splicing associated with recognition of cryptic splice-site. In contrast, the splicing of U2-type introns (dependent upon the major spliceosome) was largely unaffected. The mis-spliced introns were found in several key genes including PTEN, MAPK1, MAPK3, BRAF and E2F2, and the impaired splicing of several introns in ZRSR2 mutant bone marrow was validated experimentally. Further, short hairpin RNA (shRNA) mediated knockdown of ZRSR2 in MDS/AML TF1 cells led to impaired splicing specifically of U12-type introns. We also observe that ectopic expression of ZRSR2 in stable knockdown 293T cells resulted in increase in splicing efficiency of U12-type introns.

Further, the downregulation of ZRSR2 in leukemia cells results in reduced growth and clonogenic potential in leukemia cell lines. Moreover, knockdown of ZRSR2 in human CD34+ cells displayed altered differentiation potential towards erythroid and myeloid lineages in vitro.

Overall, the dysregulated RNA splicing of U12-type introns in ZRSR2 mutant samples affects several crucial genes involved in cell cycle, signaling, RNA binding and transport. These genes are potential mediators of MDS phenotype. Ours is a first study which demonstrates the functional consequences of ZRSR2 mutations in MDS and identifies a specific role of ZRSR2 in regulating RNA splicing. It underlines aberrant splicing of U12-type introns as a distinctive feature of ZRSR2 mutant MDS.