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

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.

At the Intersection of Major and Minor Spliceosomes: Crosstalk Mechanisms and Their Impact on Gene Expression

“…In such cases, the outcome is typically increased minor intron retention combined with the activation of nearby cryptic U2-type splice sites. In more rare cases, splicing of the U12-type intron in question appears to be unperturbed and the splicing defect is observed only due to activation of U2-type cryptic splice sites such as those described for the SNRNPE , RCD8/EDC4 and SLC9A8 genes ( Figure 4 ; Turunen et al, 2008 ; de Wolf et al, 2021 ; Norppa and Frilander, 2021 ).…”

“…A similar case has been observed for the MAPK12 gene in a cell line carrying a U12 snRNA mutation linked to cerebellar ataxia. In that case minor spliceosome dysfunction induces an exon skipping event where the U2-type 5′ss of the upstream intron is used together with the 3′ss of the downstream U12-type intron ( Norppa and Frilander, 2021 ; Figure 4 ).…”

“…The consequences of competitive intron recognition by either minor and major spliceosome on cryptic splicing have thus far been studied in cases where the splicing of U12-type introns has been compromised ( Turunen et al, 2008 ; Cologne et al, 2019 ; de Wolf et al, 2021 ; Norppa and Frilander, 2021 ; Olthof et al, 2021 ). In such cases, the outcome is typically increased minor intron retention combined with the activation of nearby cryptic U2-type splice sites.…”

Human spliceosomal snRNA sequence variants generate variant spliceosomes

“…On the other hand, human U5 snRNAs require the 3 ′ stem-loop for proper expression and snRNP maturation (Hinz et al 1996). Similarly, single point mutations in the 3 ′ -terminal stem of U12 snRNA were found to destabilize the snRNA leading to the accumulation of truncated fragments in human cell lines (Norppa and Frilander 2021). Multiple U5 variants have a conserved Stem II, indicating a selective pressure to maintain a stable 3 ′ -terminal stem-loop (Fig.…”

Mutations in the non-codingRNU4ATACgene affect the homeostasis and function of the Integrator complex

“…It is thus plausible that U4atac108_126del and U4atac111G>A RNA species with aberrant 3′ ends are not protected by TOE1 and thus become substrates for degradation by the nuclear RNA exosome. A competition between TOE1 and the nuclear RNA exosome has indeed been observed in a recent study showing that TOE1 knockdown destabilizes a U12 snRNA mutant while knockdown of MTR4, a component of the nuclear exosome targeting (NEXT) complex, has an opposite effect ( 54 ).…”