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ZRSR2 & ZRSR1 co-operate in splicing of U12 introns
AB
C
This was accompanied by significantly reduced MAPK9 and MAPK14 protein expression in human cells with silencing of ZRSR2 (Figure 5C). Amongst the two cell lines, K562 cells exhibited increased mis-splicing of both MAPK9 and MAPK14, which correlated with a more pro- found effect on their protein levels, compared to the TF1 cells (Figure 5A to C). These observations illustrate that mis-splicing of MAPK9 and MAPK14 results in lower lev- els of these proteins, potentially impacting their function in hematopoietic development.
Discussion
Mouse models have been effective in deciphering key splicing and hematopoietic defects caused by mutations of spliceosome genes. Hematopoietic cells from mice expressing either mutant SF3B1, SRSF2 or U2AF1 recapit- ulated the patterns of splicing changes observed in mutant MDS samples.6,12,15,16,18-20 Although the splice fac- tors are evolutionary conserved, intronic sequences are divergent between human and mouse. These differences in intronic splicing elements and regulatory motifs between the two species possibly lead to a divergent set of mis-spliced transcripts, which can contribute to pheno- typic differences in hematopoietic development. This presents a conundrum about the utility of mouse models to identify genes whose splicing is altered by spliceosome mutation in MDS. We have previously demonstrated that ZRSR2 mutations/deficiency impairs splicing of the U12-
Figure 5. Aberrant retention of U12-type introns in human MAPK9 and MAPK14 genes impacts their protein expression. (A and B) Normalized expression of U12-type introns of MAPK9 (A) and MAPK14 (B) in TF1 and K562 cells stably expressing short hairpin RNA (shRNA) targeting human ZRSR2. The expression of U12-type introns was measured relative to the expression of flanking exons using quantitative polymerase chain reaction (qPCR). Data are from five PCR experiments and represented as mean ± stan- dard error of the mean. *P<0.05, **P<0.01, ***P<0.001, ns: not significant. (C) Protein lev- els of human MAPK9 and MAPK14 in TF1 and K562 cells transduced with shRNA targeting ZRSR2.
type intron in human cells.8,17 U12-type introns are highly conserved during evolution, and the tissue-specific expression of transcripts harboring this class of introns is largely preserved between mouse and human.30,31 Therefore, we envisaged that a murine model would faithfully replicate the loss of ZRSR2 in leukemic human cells. Indeed, murine ZRSR2 KO myeloid cells exhibited aberrant retention of U12-type introns. Unexpectedly, splicing of the U12-type introns was unaffected in ZRSR2-deficient MEF, suggesting tissue-specific effects of ZRSR2-deficiency on splicing. A previous study also demonstrated a more pronounced mis-splicing of U12- type introns in blood cells compared to fibroblast and amniocytes in Taybi–Linder syndrome (TALS) cases har- boring germline mutations of a U12 spliceosome-specific component, RNU4ATAC.32
One limitation of our constitutive KO mouse model is that complete absence of ZRSR2 caused by germline loss of ZRSR2 might promote functional compensation during development. Therefore, findings from our KO mice may not reflect the consequences of acute loss of ZRSR2 in myeloid cells. Hematopoietic cell-specific inducible mod- els of ZRSR2 deficiency are needed to further elucidate the effect of conditional depletion of ZRSR2 during adult hematopoiesis, akin to what is observed in ZRSR2 mutant MDS.
Mice with constitutive ZRSR2 deletion developed nor- mally with no overt hematopoietic defects and function- ally competent hematopoietic stem cells. This is in con- trast with mice expressing mutant SF3B1, SRSF2 or
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