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V. Madan et al.
U2AF1, which displayed a range of hematopoietic pheno- types.6,12,15,16,18-20,33 Isolated loss-of-function mutations of ZRSR2 have been associated with macrocytic anaemia in patients with myeloid diseases.34 However, our Zrsr2 KO mice did not display any significant changes in size and counts of blood cells. Moreover, while ZRSR2 deficiency affected splicing of U12-type introns in murine hematopoietic cells, magnitude of splicing defects was modest compared to either MDS BM cells or human leukemia cell lines. This led us to investigate whether ZRSR1 can functionally compensate for the lack of ZRSR2.
ZRSR1 is a retrotransposed copy of ZRSR2, which orig- inated via independent transposition events in rodents and primates. Murine Zrsr1 is an imprinted gene and expressed from the unmethylated, paternal allele.35-40 Unlike the human counterpart, which is designated as a pseudogene, the murine gene is expressed in hematopoi- etic cells, albeit at lower levels compared with Zrsr2. Our analyses revealed open chromatin and enrichment of epi- genetic marks associated with transcriptional activation only at the murine Zrsr1 locus, supporting evidence for transcription of the murine retrogene.
In our study, silencing of Zrsr1 had no evident effect on the splicing of U12-type introns in Zrsr2 WT myeloid cells. In contrast, deficiency of ZRSR1 clearly exacerbated the mis-splicing of U12-type introns in cells lacking ZRSR2, thereby underlining that both ZRSR1 and ZRSR2 collectively contribute to splicing of U12-type introns in mouse hematopoietic cells. While RNA splicing was not studied in Zrsr1 KO mice,41 recent studies have shown that expression of truncated Zrsr1 impacts splicing of the U12-type introns in testis and hypothalamus.42,43 Interestingly, Zrsr1-mutant mice exhibit defects in ery- throcyte maturation and fewer peripheral red blood cells, with apparent morphological abnormalities.42 The differ- ences observed in impact of ZRSR1 deficiency on splicing of U12-type introns in our study (myeloid precursors) ver- sus studies investigating mutant Zrsr1-expressing sperma- tocytes/hypothalamus could possibly arise because of rel- ative levels of ZRSR2 and ZRSR1 in different cell types. Another possibility is that the truncated ZRSR1 can impair recruitment of ZRSR2 to U12-type intron splice site, thereby perturbing its splicing function.
Signal transduction mediated by MAP kinase pathway plays a vital role in numerous biological processes via phosphorylation of several downstream substrates.44 Genes encoding several members of MAP kinase family harbor U12-type introns, hence their splicing is depend- ent on ZRSR2 activity. We identified two candidates, MAPK9 and MAPK14, where aberrant splicing resulted in decreased protein levels both in human and murine cells. The effect of ZRSR2/ZRSR1 double deficiency on MAPK9 and MAPK14 protein levels in murine cells was less pronounced compared to human cells lacking ZRSR2, which corresponds with milder effect on splicing of U12- type introns in murine cells. While MAPK9 regulates T- cell apoptosis and proliferation, and MAPK14 is indispen- sable for definitive erythropoiesis in mice,45,46 these pro- teins have not been directly implicated in pathogenesis of MDS. Although we experimentally validated mis-splicing of just two of the MAPK members, a broader effect on splicing of multiple components can be envisaged. Moreover, in murine myeloid precursors, we also validat- ed aberrant retention of U12-type intron of MCTS1,
which modulates MAPK pathway by promoting phos- phorylation of MAPK1 and MAPK3.47 Given an essential role of MAPK proteins in regulating hematopoiesis,29 col- lective decrease in their protein levels can potentially be detrimental to myeloid/erythroid differentiation and expansion, thereby contributing to the disease phenotype in MDS.
Taken together, unlike human ZRSR1 pseudogene, Zrsr1 in mice is a functional autosomal copy of Zrsr2 and contributes to splicing of U12-type introns. This is also supported by a recent study which demonstrated that expression of at least one copy of either maternal Zrsr2 or paternal Zrsr1 is necessary for viability of murine embryos.48 Additionally, our study highlights that splicing of U12-type introns in murine cells depends conceivably on the balance between expression levels of ZRSR2 and ZRSR1. Hence, 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. Notably, germline expression of truncated Zrsr1 and Zrsr2 alleles showed that double mutant mice are non-viable, with Zrsr1/Zrsr2 double mutant embryos exhibiting defects in early preimplantation development.48 Hence, conditional KO alleles of both Zrsr1 and Zrsr2 are required to investigate their combined loss in adult mice.
Disclosures
No conflicts of interest to disclose.
Contributions
VM conceived the study, designed and performed research, analysed data and wrote the manuscript; ZC designed and per- formed research, analysed data and wrote the manuscript; WWT, LH, PS and MJ performed research and analysed data; PD performed bioinformatics and statistical analyses and wrote the manuscript; SZ, JL and HY performed and supervised bioin- formatics and statistical analyses; SJ, YS and MZH performed blastocyst injections to generate chimeras from targeted ES cells; WJC supervised the study and wrote the manuscript; HPK con- ceived and supervised the study, interpreted the data and wrote the manuscript. All authors reviewed and approved the manu- script.
Acknowledgements
We thank the staff of Comparative Medicine, NUS for their support in maintaining mouse colonies. We also acknowledge expert help and support from the FACS facility at CSI, Singapore.
Funding
This work was funded by the Leukemia and Lymphoma Society, the Singapore Ministry of Health’s National Medical Research Council (NMRC) under its Singapore Translational Research (STaR) Investigator Award to HPK (NMRC/STaR/0021/2014), the NMRC Center Grant award- ed to the National University Cancer Institute of Singapore (NMRC/CG/012/2013) and the National Research Foundation Singapore and the Singapore Ministry of Education under its Research Centers of Excellence initiatives. This research is also supported by the RNA Biology Center at the Cancer Science Institute of Singapore, NUS, as part of funding under the Singapore Ministry of Education’s Tier 3 grants, grant number MOE2014-T3-1-006. We thank the Melamed Family for their generous support.
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haematologica | 2022; 107(3)