Mutation in minor spliceosome components is linked to the developmental disorder microcephalic osteodysplastic primordial dwarfism type 1 (MOPD1). Here, we inactivated the minor spliceosome in the developing mouse cortex (pallium) by ablating , which encodes the crucial minor spliceosome small nuclear RNA (snRNA) U11. conditional knockout mice were born with microcephaly, which was caused by the death of self-amplifying radial glial cells (RGCs), while intermediate progenitor cells and neurons were produced. RNA sequencing suggested that this cell death was mediated by upregulation of p53 (Trp53 - Mouse Genome Informatics) and DNA damage, which were both observed specifically in U11-null RGCs. Moreover, U11 loss caused elevated minor intron retention in genes regulating the cell cycle, which was consistent with fewer RGCs in S-phase and cytokinesis, alongside prolonged metaphase in RGCs. In all, we found that self-amplifying RGCs are the cell type most sensitive to loss of minor splicing. Together, these findings provide a potential explanation of how disruption of minor splicing might cause microcephaly in MOPD1.

Minor introns constitute <0.5% of the introns in the human genome and have remained an enigma since their discovery. These introns are removed by a distinct splicing complex, the minor spliceosome. Both are ancient, tracing back to the last eukaryotic common ancestor (LECA), which is reflected by minor intron enrichment in specific gene families, such as the mitogen activated-protein kinase kinases, voltage-gated sodium and calcium ion channels, and E2F transcription factors. Most minor introns occur as single introns in genes with predominantly major introns. Due to this organization, minor intron-containing gene (MIG) expression requires the coordinated action of two spliceosomes, which increases the probability of missplicing. Thus, one would expect loss of minor introns via purifying selection. This has resulted in complete minor intron loss in at least nine eukaryotic lineages. However, minor introns are highly conserved in land plants and metazoans, where their importance is underscored by embryonic lethality when the minor spliceosome is inactivated. Conditional inactivation of the minor spliceosome has shown that rapidly dividing progenitor cells are highly sensitive to minor spliceosome loss. Indeed, we found that MIGs were significantly enriched in a screen for genes essential for survival in 341 cycling cell lines. Here, we propose that minor introns inserted randomly into genes in LECA or earlier and were subsequently conserved in genes crucial for cycling cell survival. We hypothesize that the essentiality of MIGs allowed minor introns to endure through the unicellularity of early eukaryotic evolution. Moreover, we identified 59 MIGs that emerged after LECA, and that many of these are essential for cycling cell survival, reinforcing our essentiality model for MIG conservation. This suggests that minor intron emergence is dynamic across eukaryotic evolution, and that minor introns should not be viewed as molecular fossils. We also posit that minor intron splicing was co-opted in multicellular evolution as a regulatory switch for en masse control of MIG expression and the biological processes they regulate. Specifically, this mode of regulation could control cell proliferation and thus body size, an idea supported by domestication syndrome, wherein MIGs are enriched in common candidate animal domestication genes.

Disruption of the minor spliceosome due to mutations in RNU4ATAC is linked to primordial dwarfism in microcephalic osteodysplastic primordial dwarfism type 1, Roifman syndrome, and Lowry-Wood syndrome. Similarly, primordial dwarfism in domesticated animals is linked to positive selection in minor spliceosome components. Despite being vital for limb development and size regulation, its role remains unexplored. Here, we disrupt minor spliceosome function in the developing mouse limb by ablating one of its essential components, U11 small nuclear RNA, which resulted in micromelia. Notably, earlier loss of U11 corresponded to increased severity. We find that limb size is reduced owing to elevated minor intron retention in minor intron-containing genes that regulate cell cycle. As a result, limb progenitor cells experience delayed prometaphase-to-metaphase transition and prolonged S-phase. Moreover, we observed death of rapidly dividing, distally located progenitors. Despite cell cycle defects and cell death, the spatial expression of key limb patterning genes was maintained. Overall, we show that the minor spliceosome is required for limb development via size control potentially shared in disease and domestication.

Minor spliceosome inhibition due to mutations in RNU4ATAC are linked to primary microcephaly. Ablation of Rnu11, a minor spliceosome snRNA, inhibits the minor spliceosome in the developing mouse pallium, causing microcephaly. There, cell cycle defects and p53-mediated apoptosis in response to DNA damage resulted in loss of radial glial cells (RGCs), underpinning microcephaly. Here, we ablated Trp53 to block cell death in the Rnu11 cKO mice. We report that Trp53 ablation failed to prevent microcephaly in these double knockout (dKO) mice. We show that the transcriptome of the dKO pallium was closer to the control compared to the Rnu11 cKO. We find aberrant minor intron splicing in MIGs involved in cell cycle regulation, resulting in more severely impaired mitotic progression and cell cycle lengthening of RGCs in the dKO that was detected earlier than the Rnu11 cKO. Furthermore, we discover a potential role of p53 in causing DNA damage in the developing pallium, as detection of γH2aX+ was delayed in the dKO. Thus, we postulate that microcephaly in minor spliceosome-related diseases is primarily caused by cell cycle defects.

Here we explored the role of minor spliceosome (MiS) function and minor intron-containing gene (MIG) expression in prostate cancer (PCa). We show MIGs are enriched as direct interactors of cancer-causing genes and their expression discriminates PCa progression. Increased expression of MiS U6atac snRNA, including others, and 6x more efficient minor intron splicing was observed in castration-resistant PCa (CRPC) versus primary PCa. Notably, androgen receptor signalling influenced MiS activity. Inhibition of MiS through siU6atac in PCa caused minor intron mis-splicing and aberrant expression of MIG transcripts and encoded proteins, which enriched for MAPK activity, DNA repair and cell cycle. Single cell-RNAseq confirmed cell cycle defects and lineage dependency on the MiS from primary to CRPC and neuroendocrine PCa. siU6atac was ~50% more efficient in lowering tumor burden of CRPC cells and organoids versus current state-of-the-art combination therapy. In all, MiS is a strong therapeutic target for lethal PCa and potentially other cancers.