Date of Completion
minor spliceosome, U11 snRNA, alternative splicing, cortical development, ALS, disease, RNAseq
Rahul N Kanadia
Field of Study
Physiology and Neurobiology
Doctor of Philosophy
Expression of ~2% of eukaryotic genes requires the splicing of not just one, but two types of introns: major, as well as minor, introns. These minor introns contain divergent sequences at their splice sites which are recognized by the minor spliceosome (U11, U12, U4atac, U6atac and U5). Since splicing of both intron classes occurs co-transcriptionally, expression of minor intron-containing genes (MIGs) is thought to require the coordinated action of both the major and minor spliceosome. Proper MIG expression is especially important for nervous system development, as underscored by multiple diseases linked to pathogenic variants in minor spliceosome components. To understand this bias of nervous system involvement, I examined tissue-specific splicing of minor introns. To this end, I identified all minor introns in the mouse and human genome and developed new bioinformatics pipelines to study their splicing. Moreover, I identified a subset of MIGs whose aberrant splicing might underlie prevalent symptoms, like microcephaly, observed in minor spliceosome-related diseases. To determine how disrupted minor intron splicing can result in microcephaly we then ablated U11 snRNA in the dorsal telencephalon. U11 loss especially resulted in the death of self-amplifying radial glial cells, through cell cycle defects, DNA damage and p53-mediated apoptosis. This was due to retention and alternative splicing (AS) of minor introns found in genes with crucial roles in cell cycle and led me to explore the mechanism of regulating AS around minor introns. I found that AS is normally repressed through interactions between minor spliceosome component U11-59K and the major spliceosome. Inhibition of the minor spliceosome relieves this repression, resulting in elevated AS in individuals with pathogenic variants in U4atac and U12 snRNA. To test whether disrupted minor intron splicing also plays a role in amyotrophic lateral sclerosis (ALS), we then ubiquitously ablated U11 in juvenile mice and found that they recapitulated most of the pathological hallmarks associated with ALS. In all, I show how the major and minor spliceosome interact to regulate the proper splicing of minor introns in a tissue-specific manner. Moreover, we found that disrupted minor intron splicing affects survival of both neural progenitors and motor neurons.
Olthof, Anouk M., "The Minor Spliceosome Regulates Alternative Splicing of Minor Introns in Neuronal Development and Disease" (2020). Doctoral Dissertations. 2557.