Vertebrate genomes contain major (>99.5%) and minor (<0.5%) introns that are spliced by the major and minor spliceosomes, respectively. Major intron splicing follows the exon-definition model, whereby major spliceosome components first assemble across exons. However, since most genes with minor introns predominately consist of major introns, formation of exon-definition complexes in these genes would require interaction between the major and minor spliceosomes. Here, we report that minor spliceosome protein U11-59K binds to the major spliceosome U2AF complex, thereby supporting a model in which the minor spliceosome interacts with the major spliceosome across an exon to regulate the splicing of minor introns. Inhibition of minor spliceosome snRNAs and U11-59K disrupted exon-bridging interactions, leading to exon skipping by the major spliceosome. The resulting aberrant isoforms contained a premature stop codon, yet were not subjected to nonsense-mediated decay, but rather bound to polysomes. Importantly, we detected elevated levels of these alternatively spliced transcripts in individuals with minor spliceosome-related diseases such as Roifman syndrome, Lowry–Wood syndrome and early-onset cerebellar ataxia. In all, we report that the minor spliceosome informs splicing by the major spliceosome through exon-definition interactions and show that minor spliceosome inhibition results in aberrant alternative splicing in disease.

Splicing is a crucial regulatory node of gene expression that has been leveraged to expand the proteome from a limited number of genes. Indeed, the vast increase in intron number that accompanied vertebrate emergence might have aided the evolution of developmental and organismal complexity. Here, we review how animal models for core spliceosome components have provided insights into the role of splicing in vertebrate development, with a specific focus on neuronal, neural crest and skeletal development. To this end, we also discuss relevant spliceosomopathies, which are developmental disorders linked to mutations in spliceosome subunits. Finally, we discuss potential mechanisms that could underlie the tissue-specific phenotypes often observed upon spliceosome inhibition and identify gaps in our knowledge that, we hope, will inspire further research.

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.

Mutations in minor spliceosome components are linked to diseases such as Roifman syndrome, Lowry-Wood syndrome, and early-onset cerebellar ataxia (EOCA). Here we report that besides increased minor intron retention, Roifman syndrome and EOCA can also be characterized by elevated alternative splicing (AS) around minor introns. Consistent with the idea that the assembly/activity of the minor spliceosome informs AS in minor intron-containing genes (MIGs), inhibition of all minor spliceosome snRNAs led to upregulated AS. Notably, alternatively spliced MIG isoforms were bound to polysomes in the U11-null dorsal telencephalon, which suggested that aberrant MIG protein expression could contribute to disease pathogenesis. In agreement, expression of an aberrant isoform of the MIG Dctn3 by in utero electroporation, affected radial glial cell divisions. Finally, we show that AS around minor introns is executed by the major spliceosome and is regulated by U11-59K of the minor spliceosome, which forms exon-bridging interactions with proteins of the major spliceosome. Overall, we extend the exon-definition model to MIGs and postulate that disruptions of exon-bridging interactions might contribute to disease severity and pathogenesis.Keywords: Minor spliceosome/U11-59K/alternative splicing/exon-definition model/Lowry-Wood syndrome

The pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) revealed the world's unpreparedness to deal with the emergence of novel pathogenic viruses, pointing to the urgent need to identify targets for broad-spectrum antiviral strategies. Here, we report that proteins encoded by Minor Intron-containing Genes (MIGs) are significantly enriched in datasets of cellular proteins that are leveraged by SARS-CoV-2 and other viruses. Pointing to a general gateway for viruses to tap cellular machinery, MIG-encoded proteins (MIG-Ps) that react to the disruption of the minor spliceosome are most important points of viral attack, suggesting that MIG-Ps may pan-viral drug targets. While contemporary anti-viral drugs shun MIG-Ps, we surprisingly found that anti-cancer drugs that have been repurposed to combat SARS-CoV-2, indeed target MIG-Ps, suggesting that such genes can potentially be tapped to efficiently fight viruses.