BIOSYNTHESIS OF RNAS

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Despite intensive study in numerous laboratories, our understanding of the molecular mechanisms which specify the accurate and efficient splicing of intron-containing mRNA nuclear transcripts has progressed only slightly beyond the phenomenological. The same is true for other steps in the maturation of messages, including the generation of 3' termini and polyadenylation. Attention has recently been drawn to a group of six small nuclear RNAs (snRNAs), whose extraordinary structural conservation, together with a variety of circumstantial evidence, suggest their participation in functionally conserved steps in eukaryotic gene expression. For example, U1 RNA is hypothesized to mediate intron removal via complementarity to conserved sequences at the splice junction(s). Because of the lack of tractable experimental systems among the higher eukaryotes, these hypotheses have yet to be critically tested. We propose to exploit the powerful genetic techniques uniquely available in yeast to achieve these ends. We are employing two complementary strategies. The first exploits our recent demonstration of the existence in Saccharomyces cerevisiae of snRNAs encoded by single copy genes; this allows us to perform definitive tests of these models by analyzing the consequences of null and conditional alleles in each of these genes. Moreover, the isolation of extragenic suppressors of these mutations will identify cellular components which interact with the snRNAs; these should include the snRNP proteins, as well as potential "target" RNAs: the pre-mRNA substrates. Our second approach takes advantage of our recent identification of a mutant 5' splice junction in a yeast mRNA intron. In this case, the ability to isolate extragenic suppressors should allow a direct test of the "Ul guide model" for RNA splicing