ZRSR1 Cooperates with ZRSR2 in Regulating Splicing of U12-Type Introns in Murine Hematopoietic Cells
Authors: Cao Zeya , Madan Vikas , Teoh Weoi Woon , Dakle Pushkar , Han Lin , Shyamsunder Pavithra , Zhou Siqin , Li Jia , Nordin Hazimah Binte Mohd , Jizhong Shi , Shuizhou Yu , Yang Henry , Hossain Md Zakir , Chng Wee-Joo , Koeffler H. Phillip 
Published: November 2020

Mutations in genes encoding components of the RNA splicing machinery constitute the leading class of genetic alterations in myelodysplastic syndromes (MDS). Somatic inactivating alterations of splicing factor, ZRSR2, are observed in ~10% of MDS patients. Mutations/loss of ZRSR2 in human myeloid cells causes aberrant splicing, primarily impairing splicing of the U12-type introns. However, the precise role of ZRSR2 in splicing and hematopoietic development needs further exploration.
To understand the function of ZRSR2 in hematopoietic development and splicing, we generated Zrsr2 knockout mice. RNA-Seq of sorted myeloid precursor populations (common myeloid progenitors, granulocyte monocyte progenitors and megakaryocyte erythrocyte progenitors) revealed aberrant retention of U12-type introns in ZRSR2-deficient murine cells, similar to our previous observation in ZRSR2 mutant MDS bone marrow cells (Madan et al, Nat Commun 2015). However, despite complete loss of ZRSR2 in our mouse model, the effect on splicing of U12-type introns was modest compared to the ZRSR2 mutant MDS bone marrow and ZRSR2 knockdown human AML cells (K562 and TF1). Moreover, our comprehensive analyses of hematopoietic compartment in ZRSR2-deficient (Zrsr2Δ/Y) compared to WT (Zrsr2+/Y) male mice, indicated that ZRSR2 was dispensable for hematopoietic differentiation. We did not observe any significant difference in peripheral blood counts, bone marrow cellularity, proportion of hematopoietic stem cells (HSCs) and myeloid precursors (CMP, GMP, MEP) in the bone marrow of both young (7-10 weeks) and old (&gt;1 year) mice of either genotypes. Competitive and non-competitive reconstitution assays also demonstrated that loss of ZRSR2 does not affect repopulation potential of HSCs. Collectively, our data demonstrate that ZRSR2 is not essential for hematopoietic development in mice.
Further, to understand the underlying determinant for our unexpected observation that deletion of Zrsr2 in mice did not impact hematopoietic development and affected modestly splicing of U12-type introns, we investigated the role of a closely-related homolog, ZRSR1, in splicing of U12-type introns. We utilized shRNA-mediated silencing of ZRSR1 in murine myeloid precursors (Lin−Kit+ bone marrow cells), and assessed splicing using RNA-Sequencing. Wildtype myeloid cells expressing Zrsr1 shRNA did not display significant mis-splicing of the U12-type introns. However, knockdown of Zrsr1 in ZRSR2-deficient myeloid cells exacerbated mis-splicing of the U12-introns. This suggested that ZRSR1 contributes to regulation of U12-spliceosome in murine hematopoietic cells. Increased mis-splicing of U12-type introns in ZRSR1/ZRSR2-deficient cells was validated using quantitative RT-PCR. Further to verify the compensatory role of murine ZRSR1 in splicing of U12-type introns, we used CRISPR/Cas9 technology to generate 32D cells lacking either one or both ZRSR proteins. Quantitative RT-PCR analysis showed notable aberrant retention of U12-type introns in Zrsr1/Zrsr2 double-deficient 32D cells compared to either WT or single KO cells. We also demonstrated that aberrant retention of U12-type introns of MAPK9 and MAPK14 caused by deficiency of ZRSR proteins led to their reduced protein expression in both human and murine myeloid cells.
Taken together, our study highlights functional role of murine ZRSR1 in splicing of U12-type introns in murine hematopoietic cells where it can partially compensate for the deficiency of ZRSR2. Therefore, deficiency of ZRSR2 alone is insufficient to impact extensively RNA splicing in mice, and further studies with concurrent deficiency of ZRSR1 and ZRSR2 are warranted to replicate complete loss of ZRSR activity.

Disclosures
Chng: Novartis: Honoraria; Abbvie: Honoraria; Amgen: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Janssen: Honoraria, Research Funding.