Genetic Analysis of Yeast snRNAs
We have established that Saccharomyces cerevisiae contains several dozen snRNAs; in the eight cases tested to date, these are encoded by single copy genes (designated SNR). Preliminary mapping analyses indicate that these genes are distributed over at least four chromosomes.
By performing gene replacement experiments, we have demonstrated that at least five SNR genes are completely dispensable for wild-type growth; moreover, the phenotype of the quintulpe mutant is also indistinguishable from wild-type. A sixth snRNA, snR10, plays some important biological role, in that snr10::LEU2 haploid cells display cold-sensitive and osmotic-sensitive growth. The phenotype of the sextuple mutant, however, is no more impaired than is the single snr10 mutant. Preliminary experiments suggest the possibility that snR10 plays some role in ribosome biosynthesis.
At least three SNR genes (SNR7, SNR14, andSNR20) are essential for viability. The respective snRNAs have binding sites for the Sm antigen and are constituents of the spliceosome. These RNAs contain domains that share regions of structural homology with U5, U4, and U2, respectively. In the case of snR20 and snR7, the yeast RNAs are considerably longer than their metazoan counterparts; this difference is most exaggerated between snR20 (1175 nucleotides) and U2 (196 nucleotides). We have also recently identified a small RNA (snR6) with high sequence homology to U6.
We have established the conditional synthesis of snR7 by fusing the coding sequences of SNR7 to the GAL1 promoter. Haploid cells in which this construction provides the only source of snR7 grow normally on galactose and die after a shift to glucose. By analyzing RNA populations in a window of still exponential growth five generations after carbon source shift, blocks in splicing have been identified in four different transcripts tested. We conclude that a U54ike snRNA is required for efficient splicing of (probably all) yeast introns. Interestingly, this is despite the fact that, in at least several cases, these introns lack a counterpart of the polypyrimidine stretch which is a binding site for the U5 snRNP in mammalian introns.
By the construction of compensatory mutations, we have demonstrated a base-pairing interaction between a highly conserved region within the U2-like RNA, snR20, and the so-called TACTAAC box. We believe that this interaction is one of the earliest in spliceosome assembly, and that it plays an important role in identifying the subsequent site of branch formation, which occurs at the 3’-most A of this invariant sequence.
By the isolation of extragenic suppressors of intron mutations (within a genetically manipulable splicing substrate), we have identified a dominant, allele- specific suppressor that allows more efficient recognition of a C residue at the site of branch formation. The properties of this suppressor suggest that it encodes an essential component of the splicing machinery. This strategy, as well as the related approach of generating unlinked suppressors to point mutations within SNR genes, should ultimately allow the genetic identification of a network of macromolecules involved in splicing. Minimally, we expect these to include the snRNP proteins themselves.
KeywordsGene Disruption Biochemical Phenotype Intron Mutation Branch Formation Small Nuclear RNAs
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