The integrity of the U12 snRNA 3 ' stem-loop is necessary for its overall stability

Disruption of minor spliceosome functions underlies several genetic diseases with mutations in the minor spliceosome-specific small nuclear RNAs (snRNAs) and proteins. Here, we define the molecular outcome of the U12 snRNA mutation (84C>U) resulting in an early-onset form of cerebellar ataxia. To understand the molecular consequences of the U12 snRNA mutation, we created cell lines harboring the 84C>T mutation in the U12 snRNA gene (RNU12). We show that the 84C>U mutation leads to accelerated decay of the snRNA, resulting in significantly reduced steady-state U12 snRNA levels. Additionally, the mutation leads to accumulation of 3 '-truncated forms of U12 snRNA, which have undergone the cytoplasmic steps of snRNP biogenesis. Our data suggests that the 84C>U-mutant snRNA is targeted for decay following reimport into the nucleus, and that the U12 snRNA fragments are decay intermediates that result from the stalling of a 3 '-to-5 ' exonuclease. Finally, we show that several other single-nucleotide variants in the 3 ' stem-loop of U12 snRNA that are segregating in the human population are also highly destabilizing. This suggests that the 3 ' stem-loop is important for the overall stability of the U12 snRNA and that additional disease-causing mutations are likely to exist in this region.Peer reviewe

Mutations in the U11/U12-65K protein associated with isolated growth hormone deficiency lead to structural destabilization and impaired binding of U12 snRNA

Mutations in the components of the minor spliceosome underlie several human diseases. A subset of patients with isolated growth hormone deficiency (IGHD) harbors mutations in the RNPC3 gene, which encodes the minor spliceosome-specific U11/U12-65K protein. Although a previous study showed that IGHD patient cells have defects in U12-type intron recognition, the biochemical effects of these mutations on the 65K protein have not been characterized. Here, we show that a proline-to-threonine missense mutation (P474T) and a nonsense mutation (R502X) in the C-terminal RNA recognition motif (C-RRM) of the 65K protein impair the binding of 65K to U12 and U6atac snRNAs. We further show that the nonsense allele is targeted to the nonsense-mediated decay (NMD) pathway, but in an isoform-specific manner, with the nuclear-retained 65K long-3'UTR isoform escaping the NMD pathway. In contrast, the missense P474T mutation leads, in addition to the RNA-binding defect, to a partial defect in the folding of the C-RRM and reduced stability of the full-length protein, thus reducing the formation of U11/U12 di-snRNP complexes. We propose that both the C-RRM folding defect and NMD-mediated decrease in the levels of the U11/U12-65K protein reduce formation of the U12-type intron recognition complex and missplicing of a subset of minor introns leading to pituitary hypoplasia and a subsequent defect in growth hormone secretion.Peer reviewe