The nuclear functions of the m7G cap, a signature feature of RNA polymerase II transcripts, are mediated by a heterodimeric nuclear cap binding complex (CBC). CBC engages the m7G caps of nascent transcripts and facilitates co- and post-transcriptional RNA processing, including splicing. Trimethylguanosine (TMG) caps are characteristic of a subset of RNA polymerase II transcripts, including the U1, U2, U4 and U5 snRNAs that direct pre-mRNA splicing. TMG is formed by the enzyme Tgs1, which catalyzes two successive methyl additions to the N2 atom of the m7G cap. Whereas m7G caps are essential for viability of eukarya, TMG caps are dispensable for vegetative growth of eukaryal cells. The key discoveries underlying the current proposal are our findings that: (1) TMG capping is essential for yeast sporulation via a TMG requirement for splicing of specific meiotic mRNAs, and (2) whereas the effects of weakening CBC-m7G cap interactions (by altering the cap binding pocket of the Cbc2 subunit) are buffered by other actors in the splicing pathway during yeast mitotic growth, an intact cap binding pocket is essential for meiotic development. Based on these findings, and the work of others (and us) regarding Mer1/Nam8-dependent meiotic splicing, we hypothesize that meiotic pre- mRNAs with non-consensus splice sites (and other unusual RNA features) are especially sensitive to control by general splicing factors and RNA caps. In support of this idea, we've delineated meiotic splicing regulons - encompassing distinct sets of pre-mRNAs whose splicing is dependent on vegetatively optional splicing factors (e.g. Nam8, required for splicing of SPO22, MER2, MER3, AMA1 and PCH2 pre-mRNAs), snRNA modifications (TMG caps; needed for SAE3 and PCH2 splicing) or pre-mRNA m7G cap binding by Cbc2 (required for MER3 and SAE3 splicing). Our long-term goal is to elucidate the full spectrum of meiotic splicing controls and the biochemical mechanisms involved. Here, we propose to: (1) dissect the steps in the splicing pathway that are sensitive to meiotic controls (esp. the TMG caps, and Nam8/Mer1 proteins) using in vitro splicing assays and site-specific crosslinking approaches; (2) investigate how nuclear CBC governs splicing of specific meiotic pre-mRNAs; and (3) interrogate the existence and target spectra of additional meiotic splicing controls, focusing first on Mud2 and the branchpoint binding protein Msl5. We expect to gain new and general insights to the question of how splice site choice can be modulated by components of the basal splicing machinery in response to distinct features in the pre-mRNA.
This project addresses pre-mRNA splicing, a fundamental step in gene expression. Defects or imbalances in pre-mRNA splicing underlie a broad spectrum of human diseases, either via mutations of core splicing machinery or splicing regulatory factors (such lesions are linked to retinitis pigmentosa and Prader- Willi syndrome), or, mutations of RNA signals that direct or regulate splice site utilization. Such lesions are associated with spinal muscular atrophy, cystic fibrosis, familial dysautonomia, tauopathies, myotonic dystrophy, neurofibromatosis, and breast cancer.
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