Minor spliceosome inhibition via ablation of its U11 snRNA gene results in multiple defects in murine spermatogenesis
Minor intron-containing genes (MIGs), which require the minor spliceosome (MiS) for their splicing, are highly represented among genes involved in mouse spermatogenesis. Leveraging gene- and intron-level ortholog data, we show that conservation of these genes as MIGs is particularly strong in chordates but not in other species groups, suggesting lineage-specific changes to their splicing regulation. A role for the MiS in splicing during spermatogenesis was reinforced by the cell-type specific expression patterns of MiS snRNAs and directly tested using Stra8-Cre mediated ablation of Rnu11, which encodes for the MiS U11 snRNA. Rnu11 mutant testes were smaller, and presented with multiple mitotic and meiotic defects, increased cell death, and a reduction in mature sperm at matched timepoints. RNA sequencing of P14 whole testes revealed minor intron retention and alternative splicing events in MIGs that are key regulators of genomic stability, cell cycle progression, and gene expression regulation, contributing to the variable cellular defects. RT-PCR of fractionated cell types of the testes confirmed splicing defects of key MIGs in cell-type enriched samples. The multifaceted molecular disruption resulted in defective meiotic recombination, telomere morphology, chromosome synapsis and XY-chromosome association. Overall, we highlight the molecular and cellular processes that are regulated by the MiS. Overall design: RNA-seq profiling of postnatal day 14 whole-testes dissected from control and mutant (Rnu11-cKO) animals (n=3). Libraries were prepared using the Illumina TruSeq Stranded Total RNA Library Sample Prep Kit with ribosomal RNA depletion by RiboZero, and sequenced on the Illumina NovaSeq 6000 platform.