Structural basis for the regulation of splicing of a yeast messenger RNA

Abstract

In Saccaromyces cerevisiae, ribosomal protein L32 regulates its own production by inhibiting the splicing of its own transcript. To locate sequences within the transcript responsible for this regulation, two chimeric genes were constructed between RPL32 and another ribosomal protein gene, RPS10A; each was fused at the middle of its intron to the proper half of the other gene. Genetic analyses demonstrated that both hybrid genes are functional. Splicing of the transcript from the hybrid gene bearing the 5{dollar}\sp\prime{dollar} portion of RPL32 can be regulated by L32 protein while that of the gene bearing the 3{dollar}\sp\prime{dollar} portion cannot. These chimeric genes were subsequently utilized in in vivo assays to further identify sequences responsible for regulation. Deletion analysis localized the site of regulation to within the 61 nucleotide 5{dollar}\sp\prime{dollar} exon and the first 14 nucleotides of the intron. The 5{dollar}\sp\prime{dollar} splice site of RPL32 is G/GUCAGU and is unusual in yeast which normally utilize G/GUAUGU. Conversion to a consensus 5{dollar}\sp\prime{dollar} splice site or insertion of either an AAG or ACG codon just upsteam of the 5{dollar}\sp\prime{dollar} splice site abolished regulation. Substitution of the first seven nucleotides at the 5{dollar}\sp\prime{dollar} end of the transcript with 5{dollar}\sp\prime{dollar} sequences of CYC1, as well as a number of point mutations also within this 5{dollar}\sp\prime{dollar} end region abolished regulation. Thus, two regions of the transcript are involved in the regulation. An RNA secondary structure predicted by an RNA folding program was evaluated by mutational analysis; this structure was refuted. To take advantage of phylogenetic comparison of RNA structure, RPL32 was cloned and sequenced from the closely related yeast species, Kluveromyces lactis. The splicing of its transcript was determined to be similarly regulated. Comparison of the 5{dollar}\sp\prime{dollar} exon and intron from the two organisms revealed only two short regions of near identity: 13 nucleotides near the 5{dollar}\sp\prime{dollar} end of the transcript and 22 nucleotides that include the 5{dollar}\sp\prime{dollar} splice site. An RNA secondary structure model is proposed whereby the 5{dollar}\sp\prime{dollar} end of the transcript can interact with the 5{dollar}\sp\prime{dollar} splice site that would thus preclude its interaction with spliceosomal U1 snRNA. The phenotypes of numerous point mutations that disrupt intramolecular base pairs of the predicted structure disrupt regulation, in contrast to those that do not. Compensatory double mutations predicted to restore this structure restore regulation. This secondary structure that forms within the 5{dollar}\sp\prime{dollar} portion of the RPL32 transcript is predicted to be weak. We infer that it is stabilized in the presence of L32 protein, thereby leading to the autogenous regulation of splicing.