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ZRSR2 & ZRSR1 co-operate in splicing of U12 introns
RNA splicing, we performed RNA sequencing on sorted wild-type (WT) and KO myeloid precursor populations from BM: CMP (Lin−Kit+Sca1−CD34+FcγRII/IIIlo), GMP (Lin−Kit+Sca1−CD34+FcγRII/IIIhi), MEP (Lin−Kit+Sca1−CD34−FcγRII/III−), as well as MEF from male WT and Zrsr2 KO embryos. Aberrant retention of U12- type introns was observed in all three ZRSR2-deficient myeloid progenitors, but was not evident in MEF (Figure 1B and C; Online Supplementary Table S2). Despite a trend towards mis-splicing of U12-type introns in murine hematopoietic cells, we noted that the effect of ZRSR2 deficiency on splicing (as indicated by DMSI values) was significantly lower than that previously observed by us in ZRSR2 mutant human MDS BM and ZRSR2-deficient human AML cell lines, TF1 and K562 (Figure 1B and E; Online Supplementary Table S3).8 The number of aberrantly retained U12-type introns in Zrsr2 KO murine myeloid pre- cursors was notably lower than ZRSR2 knockdown K562 and TF1 cells (Figure 1F). Only a modest effect on splicing was unexpected, especially given a complete loss of ZRSR2 expression in our mouse model. Moreover, U12-type introns are highly conserved between human and mouse genomes (Online Supplementary Figure S2), so a similar effect of ZRSR2 deficiency would be expected in the two species. Overall, our findings indicated reduced dependence of the U12-spliceosome on ZRSR2 in murine myeloid cells.
Normal myeloid development in mice lacking ZRSR2
We analyzed hematopoietic compartment in ZRSR2- deficient (Zrsr2D/Y) compared to WT (Zrsr2+/Y) male mice. No significant difference was observed in peripheral blood counts of young mice of both genotypes; and age-depen- dent defects were also not apparent (Figure 2A; Online Supplementary Figure S3). Initial analysis of young males (7- 10 weeks old) showed that deficiency of ZRSR2 neither affected the BM cellularity nor the frequency of Lin−Sca1+Kit+ (LSK) cells (Figure 2B and C). Proportion of HSC (CD34–Flt3– LSK cells) and progenitors also remained unaltered in young male mice (Figure 2D). MDS is prima- rily a disease of elderly, and in order to understand if loss of ZRSR2 manifested its effect with aging, BM of ≥1-year old male mice were examined. Surprisingly, old ZRSR2- deficient males also exhibited normal BM cellularity and frequencies of HSC and multipotent progenitors (Figure 2B to D). Moreover, proportion of myeloid precursors, CMP, GMP and MEP, and stages of erythroid development were also largely unaltered in the BM of young and old ZRSR2- deficient male mice (Figure 2E; Online Supplementary Figure S4A). Normal myeloid differentiation was also evident by unchanged frequencies of granulocytes in spleen and BM of Zrsr2 KO mice (Online Supplementary Figure S4B).
In order to evaluate the repopulation ability of Zrsr2 KO HSC, competitive repopulation assays were per- formed. ZRSR2-deficient HSC reconstituted both myeloid and lymphoid lineages in recipient mice as effi- ciently as WT cells (Figure 2F), suggesting that repopula- tion potential of HSC is maintained in the absence of ZRSR2. Further, in non-competitive repopulation assays, loss of ZRSR2 did not affect the peripheral blood cell counts in recipient mice even 1 year after transplantation (Online Supplementary Figure S5). Collectively, our com- prehensive analyses of hematopoietic development in Zrsr2 KO mice and reconstitution ability of ZRSR2-defi- cient HSC demonstrated that ZRSR2 is not essential for hematopoietic development in mice.
Murine Zrsr1 is a putative functional copy of Zrsr2 Given our unexpected observations that deletion of Zrsr2 in mice did not impact hematopoietic development and modestly affected splicing of U12-type introns, we postulated that other spliceosome protein(s) might com- pensate for its absence. ZRSR1, a closely-related homolog, is a single exon gene formed by retrotransposi- tion of ZRSR2 cDNA sequence. This autosomal copy of X-linked ZRSR2 gene is highly similar to the parent gene in coding sequence (95% and 77% identical in human and mouse, respectively) and amino acid sequences (92% and 75% identical in human and mouse, respectively) with a conserved open reading frame. While ZRSR2 is ubiquitously expressed, human ZRSR1 is designated as a pseudogene with negligible transcript levels in human tis- sues (https://gtexportal.org/home/), including the hematopoietic cells (Figure 3A). In contrast, the mouse Zrsr1 gene is expressed in hematopoietic cells, albeit at levels lower than Zrsr2 (Figure 3B). This difference in tran- scriptional regulation of murine and human ZRSR1 genes is possibly caused by their location in distinct, non- orthologous genomic loci (Figure 3C). Human ZRSR1 is located in the intron of the REEP5 gene on chromosome 5q while the mouse gene localizes to the first intron of the Commd1 gene on chromosome 11 (Figure 3C), a genomic locus not syntenic to human chromosome 5. This demonstrates that retrotransposition of ZRSR2 gene occurred independently in rodents and primates, after the evolutionary divergence between the two mammalian
orders.
Furthermore, we inspected the chromatin structure and
presence of histone marks associated with transcriptional activation at the ZRSR1 locus in human and murine cells, using available DNase-seq/ATAC-seq and ChIP-Seq data, respectively. While no ZRSR1 locus-specific signal was observed for histone marks associated with gene activa- tion (H3K4me3, H3K4me1 and H3K27ac) in human com- mon myeloid progenitors, significant ChIP-Seq peaks for all three histone modifications were detected in murine hematopoietic cells (Figure 3D). Correspondingly, accessi- ble chromatin was evident the murine but not human ZRSR1 locus (Figure 3D). Based on epigenetic profiles and expression levels, we hypothesized that murine ZRSR1 encodes for a functional protein that could possibly regu- late splicing of the U12-type introns.
Loss of ZRSR1 impairs splicing of U12-type introns in the absence of ZRSR2
In order to investigate if ZRSR1 functionally compen- sates for deficiency of ZRSR2 in splicing of U12-type introns, we silenced expression of Zrsr1 in murine myeloid precursors using short hairpin RNA (shRNA) (Online Supplementary Figure S6A). Stable knockdown of Zrsr1 in WT Lin−Kit+ BM cells did not notably alter splic- ing of the U12-type introns, although it resulted in a reduced number of myeloid colonies in the methylcellu- lose medium (Figure 4A; Online Supplementary Figures S6B and S7A; Online Supplementary Table S4). Zrsr2 KO myeloid cells exhibited sizable retention of the U12-type introns (Figure 4A and B; Online Supplementary Figure S7A; Online Supplementary Table S4), similar to that previously described for sorted CMP, GMP and MEP populations (Figure 1B and C). Notably, knockdown of Zrsr1 in ZRSR2-deficient myeloid cells further exacerbated mis- splicing of the U12-type introns (Figure 4A and B; Online
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