Suppressors of a U4 snRNA mutation define a novel U6 snRNP protein with RNA-binding motifs

U4 and U6 small nuclear RNAs are associated by an extensive base-pairing interaction that must be disrupted and reformed with each round of splicing. U4 mutations within the U4/U6 interaction domain destabilize the complex in vitro and cause a cold-sensitive phenotype in vivo. Restabilization of the U4/U6 helix by dominant (gain-of-function), compensatory mutations in U6 results in wild-type growth. Cold-insensitive growth can also be restored by two classes of recessive (loss-of-function) suppressors: (1) mutations in PRP24, which we show to be a U6-specific binding protein of the RNP-consensus family; and (2) mutations in U6, which lie outside the interaction domain and identify putative PRP24-binding sites. Destabilization of the U4/U6 helix causes the accumulation of a PRP24/U4/U6 complex, which is undetectable in wild-type cells. The loss-of-function suppressor mutations inhibit the binding of PRP24 to U6, and thus presumably promote the release of PRP24 from the PRP24/U4/U6 complex and the reformation of the base-paired U4/U6 snRNP. We propose that the PRP24/U4/U6 complex is normally a highly transient intermediate in the spliceosome cycle and that PRP24 promotes the reannealing of U6 with U4.     

Structure of the Echinococcus multilocularis U1 snRNA gene repeat.

The gene encoding U1 snRNA in Echinococcus multilocularis has been cloned and sequenced. This gene is contained within a 1300-bp sequence which is tandemly repeated in the E. multilocularis genome. E. multilocularis U1 snRNA is 50-70% homologous to U1 snRNAs of other species. E. multilocularis U1 snRNA could assume a predicted secondary structure similar to that proposed for other U1 snRNAs, and appears shorter (157 bases) than the U1 snRNAs of higher eukaryotes (163-166 bases).     

Alternative 3′-end processing of U5 snRNA by RNase III

The cellular components required to form the 3′ ends of small nuclear RNAs are unknown. U5 snRNA from Saccharomyces cerevisiae is found in two forms that differ in length at their 3′ ends (U5L and U5S). When added to a yeast cell free extract, synthetic pre-U5 RNA bearing downstream genomic sequences is processed efficiently and accurately to generate both mature forms of U5. The two forms of U5 are produced in vitro by alternative 3′-end processing. A temperature-sensitive mutation in the RNT1 gene encoding RNase III blocks accumulation of U5L in vivo. In vitro, alternative cleavage of the U5 precursor by RNase III determines the choice between the two multistep pathways that lead to U5L and U5S, one of which (U5L) is strictly dependent on RNase III. These results identify RNase III as a trans-acting factor involved in 3′-end formation of snRNA and show how RNase III might regulate alternative RNA processing pathways.     

C16orf57, a gene mutated in poikiloderma with neutropenia, encodes a putative phosphodiesterase responsible for the U6 snRNA 3' end modification

C16orf57 encodes a human protein of unknown function, and mutations in the gene occur in poikiloderma with neutropenia (PN), which is a rare, autosomal recessive disease. Interestingly, mutations in C16orf57 were also observed among patients diagnosed with Rothmund-Thomson syndrome (RTS) and dyskeratosis congenita (DC), which are caused by mutations in genes involved in DNA repair and telomere maintenance. A genetic screen in Saccharomyces cerevisiae revealed that the yeast ortholog of C16orf57, USB1 (YLR132C), is essential for U6 small nuclear RNA (snRNA) biogenesis and cell viability. Usb1 depletion destabilized U6 snRNA, leading to splicing defects and cell growth defects, which was suppressed by the presence of multiple copies of the U6 snRNA gene SNR6. Moreover, Usb1 is essential for the generation of a unique feature of U6 snRNA; namely, the 3'-terminal phosphate. RNAi experiments in human cells followed by biochemical and functional analyses confirmed that, similar to yeast, C16orf57 encodes a protein involved in the 2',3'-cyclic phosphate formation at the 3' end of U6 snRNA. Advanced bioinformatics predicted that C16orf57 encodes a phosphodiesterase whose putative catalytic activity is essential for its function in vivo. Our results predict an unexpected molecular basis for PN, DC, and RTS and provide insight into U6 snRNA 3' end formation.