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ZRSR2 & ZRSR1 co-operate in splicing of U12 introns
SF3B1, SRSF2 and U2AF1, alterations of ZRSR2 are trun- cating mutations spread throughout the transcript. ZRSR2 is located on human chromosome X and somatic muta- tions are primarily observed in males, suggesting its loss- of-function in MDS.1 It is involved in 3’ splice site recogni- tion and interacts with the U2AF2/U2AF1 heterodimer and SRSF2 during pre-spliceosome assembly.21 ZRSR2 is recruited in an ATP-dependent fashion to the U12-type intron splice site, and is required for the formation of the spliceosome complex.22 We have previously illustrated that either truncating mutations of ZRSR2 in MDS or its silenc- ing in acute myeloid leukemia (AML) cells impair predom- inantly splicing of U12-type introns, excision of which is mediated by minor spliceosome assembly.8 However, ani- mal models of ZRSR2 deficiency have not been reported and an in-depth understanding of its function is lacking.
In this study, we have generated the first mouse model of ZRSR2 deficiency to further uncover its function in splicing and to evaluate the effect of its loss on normal and malignant hematopoiesis. Although deletion of Zrsr2 induces aberrant splicing of U12-type introns in mouse myeloid precursors, myeloid differentiation is largely unaffected in young, as well as ≥1-year old Zrsr2 knock- out (KO) male mice. ZRSR2-deficient hematopoietic stem cells (HSC) retain multilineage reconstitution ability, sug- gesting limited function of ZRSR2 in mouse hematopoiesis. We further investigated its closely related homolog, ZRSR1, and uncovered that it compensates for the loss of ZRSR2 in mouse hematopoietic cells, and con- current deficiency of both proteins leads to more pro- found defects in splicing of U12-type introns. Hence, murine models lacking both ZRSR2 and ZRSR1 are nec- essary to replicate faithfully mis-splicing caused by loss- of-function mutations of ZRSR2 in MDS.
Methods
Generation of Zrsr2 knockout mice
C57BL/6N embryonic stem (ES) cells (Zrsr2tm1(KOMP)Vlcg) with tar-
geted deletion of mouse Zrsr2 were obtained from UC Davis KOMP Repository. Mice with a constitutive deletion of Zrsr2 were generated at the transgenic facility of CSI Singapore. Briefly, ES cells were microinjected into BALB/cJInv blastocysts and resulting chimera were mated with C57BL/6 mice. Black offspring were genotyped, and mice carrying the Zrsr2 null allele were used to establish a colony of Zrsr2 knockout (KO) mice. Subsequently, mice were crossed with CMV-Cre strain to remove the neomycin selection cassette. In this study, Zrsr2 KO mice refer to those before or after Cre-mediated excision, as they were phenotypical- ly identical in all experiments. Primers used for genotyping are listed in the Online Supplementary Table S1.
All animal experiments were approved by the Institutional Animal Care and Use Committee of the National University of Singapore, Singapore.
Flow cytometric analysis and fluorescence-activated cell sorting
Single cell suspensions from bone marrow (BM), spleen and thymus were stained with fluorochrome-conjugated antibodies for 30 minutes. The lineage cocktail was comprised of antibod- ies targeting murine CD19, CD3ε, CD11b, Gr1 and TER119. Cells were washed with 2% fetal bovine serum in phosphate buffered saline and resuspended in SYTOX Blue Dead Cell Stain (ThermoFisher Scientific). Cells were acquired on FACS LSR II
(BD Biosciences) and data were analyzed using FACSDIVA soft- ware (BD Biosciences). Cells were sorted on FACSAria cell sorter (BD Biosciences).
RNA sequencing
Total RNA was extracted from sorted myeloid precursors (common myeloid precursors [CMP], granulocyte monocyte precursors [GMP] and megakaryocyte erythroid precursors [MEP]), murine embryonic fibroblasts [MEF] and ex vivo cultured Lin−Kit+ BM cells using either RNeasy Micro or Mini Kits (Qiagen). Libraries of polyA-selected RNA were prepared using either TruSeq sample preparation kit (CMP, GMP, MEP and MEF) or NEBNext Ultra RNA Library Prep Kit (Lin−Kit+ BM cells). Libraries were sequenced on HiSeq 4000, and paired-end reads were mapped to either mouse reference transcriptome (GRCm38/mm10; Ensemble version 84) or human hg38 refer- ence genome using the STAR aligner23 with params ‘-- outSAMstrandField intronMotif --alignSJDBoverhangMin 6 -- alignIntronMax 299999 --outFilterMultimapNmax 4 -- scoreGapATAC -4’.
Differential splicing analysis
A list of valid introns was extracted from the Gencode24 gene transfer format file post removing transcripts with biotype ‘retained_intron’. Introns overlapping with an exon at their junc- tions were removed using pybedtools25,26 and gffutils (https://github.com/daler/gffutils). They were subsequently classified as either U2-type or U12-type based on position weight matrices from splicerack using gimmemotifs.27,28
MSI (mis-splicing index) values were calculated for each intron as described before.8 Only those introns with a coverage of at least one read at each of their junctions and with total cov- erage of the two junctions higher than four were considered. Also, introns which did not have a coverage of at least one for 95% of their length were filtered out. In order to identify differ- entially spliced introns, differences in MSI values (DMSI) were calculated as DMSI=MSIknockout−MSIwildtype. Significance of this dif- ference was computed using a Fisher’s test, and the obtained P- values were corrected for multiple testing.
The python and R scripts used for analyses are available at https://github.com/pd321/intron-retention-scripts.
Accession codes
RNA sequencing data were deposited in Gene Expression Omnibus database repository under accession numbers GSE151470 and GSE152432.
Results
Aberrant retention of U12-type introns in ZRSR2-deficient murine hematopoietic cells
Mice lacking the entire Zrsr2 coding sequence were obtained by germline transmission of the targeted allele (Figure 1A). Following this, neomycin selection cassette was removed through mating with CMV-Cre transgenic mice, in effect replacing the Zrsr2 coding sequence with β- galactosidase gene (Figure 1A). Complete lack of Zrsr2 tran- scripts was evident in quantitative polymerase chain reac- tion (qPCR) analysis of BM, spleen and thymus cells, as well as RNA sequencing of targeted ES cells (Online Supplementary Figure S1A and B). Loss of Zrsr2 did not alter expression of its homolog, Zrsr1, in hematopoietic cells (Online Supplementary Figure S1B).
In order to assess the effect of ZRSR2 deficiency on
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