Impaired Spermatogenesis, Muscle, and Erythrocyte Function in U12 Intron Splicing-Defective Zrsr1 Mutant Mice

Abstract

The U2AF35-like ZRSR1 has been implicated in the recognition of 3' splice site during spliceosome assembly, but ZRSR1 knockout mice do not show abnormal phenotypes. To analyze ZRSR1 function and its precise role in RNA splicing, we generated ZRSR1 mutant mice containing truncating mutations within its RNA-recognition motif. Homozygous mutant mice exhibited severe defects in erythrocytes, muscle stretch, and spermatogenesis, along with germ cell sloughing and apoptosis, ultimately leading to azoospermia and male sterility. Testis RNA sequencing (RNA-seq) analyses revealed increased intron retention of both U2- and U12-type introns, including U12-type intron events in genes with key functions in spermatogenesis and spermatid development. Affected U2 introns were commonly found flanking U12 introns, suggesting functional cross-talk between the two spliceosomes. The splicing and tissue defects observed in mutant mice attributed to ZRSR1 loss of function suggest a physiological role for this factor in U12 intron splicing.

RNA Binding Motif Protein 48 is required for U12 splicing and maize endosperm differentiation

“…Defects in MIG splicing have very similar patterns in both rbm48 and rgh3 with adjacent U2-type introns being mis-spliced in a subset of MIGs. Mis-splicing of neighboring U2-type introns in U12 splicing mutants has been observed in multiple species (Madan et al, 2015;Horiuchi et al, 2018). These data indicate that maize RBM48 is a U12 splicing factor.…”

“…The protein-protein interactions we found between RBM48 with U2AF, RGH3, and ARMC7 may help explain the impact on U2-type introns in a subset of U12 splicing mutants (Verma et al, 2017). A low frequency of U2 splicing defects have been observed for mutations in ZRSR2 homologs and RNPC3 (Madan et al, 2015;Gault et al, 2017;Horiuchi et al, 2018;Argente et al, 2014). RGH3/ZRSR2 and RNPC3 are required for U12-type intron recognition (Shen et al, 2010;Frilander and Steitz, 1999).…”

“…A subset of human myelodysplasia patients have mutations in ZRSR2 that result in aberrant U12 splicing and reduced terminal differentiation of myeloid blood cell types (Madan et al, 2015). Mutation of the mouse ZRSR1 paralog of ZRSR2 leads to blood and sperm differentiation defects (Horiuchi et al, 2018). Patients with Roifman syndrome have mutations in the human U4atac snRNA along with defects in both myeloid and lymphoid blood cell differentiation (Heremans et al, 2018).…”

“…Our study also illustrates the importance of genetic analysis of RNA splicing factors to determine RNA processing functions. Recently, mutant models for several splicing factors including ZRSR2, ZRSR1, SMN, and FUS showed a predominant in vivo function in minor intron splicing even though the proteins are thought to have broader roles based on in vitro splicing assays or protein-protein interactions (Madan et al, 2015;Gault et al, 2017;Horiuchi et al, 2018;Reber et al, 2016;Doktor et al, 2017;Jangi et al, 2017). RNA-seq analysis of mutant transcriptomes were able to resolve the predominant defects as impacting U12-type introns.…”

“…The rgh3 mutation of the maize ZRSR2 ortholog has a conserved U12 splicing function and inhibits differentiation of endosperm cells (Fouquet et al, 2011). Other U12 splicing mutants in humans and mouse models demonstrate roles in hematopoiesis, muscle strength, as well as bone and neural development (Edery et al, 2011;He et al, 2011;Horiuchi et al, 2018). A mutation in zebrafish indicates U12 splicing functions in intestinal epithelium and endodermal organ development (Markmiller et al, 2014).…”