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In eukaryotic gene expression, the removal of introns from pre-mRNA is an essential function carried out by spliceosomes. Human cells have two distinct spliceosomes: U2-dependent and U12-dependent. U2- dependent spliceosomes, or major spliceosomes, remove over 99% of introns, whereas U-12 dependent spliceosomes remove less than 0.5% of introns. Mutations in spliceosome machinery are a notable cause of human disease. In particular, mutations to components unique to the minor spliceosome demonstrate that it plays a vital role in human development. Mutations in the gene encoding the minor spliceosomal small nuclear RNA (snRNA) U12, RNU12, are associated with two rare developmental disorders: 1) CDAGS syndrome (craniosynostosis and clavicular hypoplasia; delayed closure of the fontanelles, cranial defects, and, in some patients, deafness; anal anomalies; genitourinary malformations; and skin eruption) and 2) early onset cerebellar ataxia. Recent work identified rare biallelic variants in RNU12 as the likely cause of CDAGS syndrome, while a single homozygous mutation was identified as the likely cause of early onset cerebellar ataxia. Mutations associated with these diseases are clustered in or near the stem- loop III of U12 snRNA, with three of the mutations located in the Sm protein binding site. Further investigation of the mutation associated with early onset cerebellar ataxia suggests that mutations in RNU12 disrupt U12 snRNA function through the destabilization of the 3’ stem-loop, which precedes overall destabilization of the U12 snRNA. Using our in vivo orthogonal splicing assay, we quantified the effects of pathogenic RNU12 mutations on U12-dependent splicing. Splicing activity varies depending on the location of the mutation. Splicing was significantly reduced among three U12 variants located in the Sm protein binding site. Sm proteins are responsible for proper assembly of snRNPs (small nuclear ribonucleoproteins) prior to pre-mRNA splicing. Similarly, splicing activity was substantially impaired in the mutation located three nucleotides downstream of the U12 snRNA sequence. While the impact of this variant on the minor spliceosome complex is unclear, one possibility is that this mutation could affect 3’ end processing of U12 snRNA. The effects of the two variants within stem-loop III of U12 snRNA differ. Whereas splicing activity was considerably diminished with the 86G>A mutation, activity in cells with the 84C>T mutation was near wild-type levels. It is presumed that these mutations affect the secondary structure of the snRNA due to the fact that they are located at the base of stem-loop III, farther away from the U12-65K protein binding site. Further work remains to fully define the mechanisms of splicing impairment that are the result of disease-associated mutations in U12 snRNA.

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Disruption of Minor Intron Splicing by Disease-associated Mutations in U12 snRNA

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