The central roles of snRNAs in spliceosome assembly and splicing have been appreciated for a number of years. A persistent mystery in splicing is how the many DExH/D proteins required for function are linked to specific snRNA rearrangements during assembly and activation of the spliceosome. A second major issue concerns how splicing regulators influence the basic process of spliceosome assembly to regulate splice site use. A third question concerns how splicing regulation is integrated into global gene expression programs with other regulatory modules. Finally, a fourth emerging set of questions concerns the integration of splicing with other steps in the gene expression pathway. We will address each of these major questions with a specific aim. We will test specific models for the RNA-RNA rearrangements that to lead to early spliceosome assembly and will learn how the newly discovered U2 branchpoint interaction stem loop (BSL) helps recognize the branchpoint, a key step in splicing. Our hypothesis is that control of RNA rearrangements is key to the progression and fidelity of splicing reactions. We will explore the function of the splicing factors Nam8p and Mer1p. Using splicing-sensitive microarrays we will dissect the global role of the Nam8p and Mer1p regulons in the vegetative and meiotic gene expression programs. Finally we will test mechanistic models for co-transcriptional control of splicing.
The specific aims are as follows: (1) We will determine roles and ordered requirements of DExD/H-box proteins Prp5p and Sub2p in promoting rearrangement of pre-mRNA, and U2 during spliceosome function. Spliceosome assembly and activation steps are incompletely mapped to the RNA- RNA rearrangements that accompany them. Starting with U2 and Prp5p, we will examine the mechanisms of RNA-RNA interaction transitions. (2) We will reveal mechanisms leading to Nam8p- dependent activation of splicing as well as Mer1p enhancer-dependent splicing activation by Nam8p and Mer1p. (3) We will explore the global integration of the Mer1p and Nam8p splicing regulons in the meiotic gene expression program to understand how eukaryotic regulatory networks are built. (4) We will determine mechanisms by which the process of transcript elongation impacts splicing. Our preliminary results provide a unique opportunity to investigate the parameters of co-transcriptional splicing, a process that plays a poorly understood role in gene expression, using the powerful yeast system. Our hypothesis is that splicing decisions can be influenced co-transcriptionally.
These aims encompass the major issues in the splicing field: How do snRNPs work? How is the basal splicing machinery controlled and influenced to regulate splicing? And how is the process of splicing integrated into genome function and evolution? The combined strengths of genetics, biochemistry, and genomics available in yeast and the conserved properties of the splicing machinery indicate that fundamental knowledge uncovered by these efforts will translate directly to the understanding of human gene expression.
Project Narrative - Splicing is a fundamental process of gene expression. Aberrant splicing leads to a multitude of human diseases ranging from myotonic dystrophy to nervous system dysfunctions to cancers. The baker's yeast Saccharomyces cerevisiae is a simple eukaryote which has proven to be a powerful model for studying underlying aspects of the splicing process and its regulation. Discoveries made using yeast are readily testable in more complex mammalian systems.
|Fu, Xiang-Dong; Ares Jr, Manuel (2014) Context-dependent control of alternative splicing by RNA-binding proteins. Nat Rev Genet 15:689-701|
|Turner, Rushia; Shefer, Kinneret; Ares Jr, Manuel (2013) Safer one-pot synthesis of the 'SHAPE' reagent 1-methyl-7-nitroisatoic anhydride (1m7). RNA 19:1857-63|
|Hall, Megan P; Nagel, Roland J; Fagg, W Samuel et al. (2013) Quaking and PTB control overlapping splicing regulatory networks during muscle cell differentiation. RNA 19:627-38|
|Munding, Elizabeth M; Shiue, Lily; Katzman, Sol et al. (2013) Competition between pre-mRNAs for the splicing machinery drives global regulation of splicing. Mol Cell 51:338-48|
|Effenberger, Kerstin A; Perriman, Rhonda J; Bray, Walter M et al. (2013) A high-throughput splicing assay identifies new classes of inhibitors of human and yeast spliceosomes. J Biomol Screen 18:1110-20|
|Mishra, Shravan Kumar; Ammon, Tim; Popowicz, Grzegorz M et al. (2011) Role of the ubiquitin-like protein Hub1 in splice-site usage and alternative splicing. Nature 474:173-8|
|Munding, Elizabeth M; Igel, A Haller; Shiue, Lily et al. (2010) Integration of a splicing regulatory network within the meiotic gene expression program of Saccharomyces cerevisiae. Genes Dev 24:2693-704|
|Perriman, Rhonda; Ares Jr, Manuel (2010) Invariant U2 snRNA nucleotides form a stem loop to recognize the intron early in splicing. Mol Cell 38:416-27|
|Du, Hongqing; Cline, Melissa S; Osborne, Robert J et al. (2010) Aberrant alternative splicing and extracellular matrix gene expression in mouse models of myotonic dystrophy. Nat Struct Mol Biol 17:187-93|
|Pohl, Andrew A; Sugnet, Charles W; Clark, Tyson A et al. (2009) Affy exon tissues: exon levels in normal tissues in human, mouse and rat. Bioinformatics 25:2442-3|
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