The integrity of the U12 snRNA 3′ stem–loop is necessary for its overall stability

Abstract

Abstract 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.