Pre-mRNA splicing is an essential step in eukaryotic gene regulation and a central process for encoding genetic complexity in higher organisms. Splicing is carried out by a MegaDalton complex of RNA and proteins called the spliceosome. Critical to the splicing process is the correct choice of the splice sites (locations of chemistry) in the pre-mRNA in order to preserve the reading frame of the transcript and produce the proper mRNA isoform by alternative splicing. The focus of this ROO application is to use single molecule fluorescence methods to elucidate the mechanisms of 5'splice site and branchsite recognition during spliceosome assembly in yeast. These mechanisms will serve as a paradigm for understanding splice site selection and alternative splicing in humans and human disease. Single molecule fluorescence methods developed during the K99 phase (see Hoskins et al., Science, 2011) allow complex reaction schemes to be dissected by following splicing pathways on individual pre-mRNAs from start to finish. These methods can be directly applied to analysis of splice site selection during the ROO phase. The 5'splice site is initially recognized by the spliceosomal U1 snRNP. The U1 snRNP engages in a number of RNA:RNA, RNA:protein, and protein:protein interactions with the pre-mRNA that all collaborate to confer affinity and fidelity. Using single molecule fluorescence, the various contributions these interactions make to the stability of the U1/5'splice site interaction will be quantified (Specific Aim 1). Auxiliary proteins often contribute to promote spliceosome assembly (e.g. splicing regulatory proteins in humans). Yeast also contain factors that can promote spliceosome assembly, and the mechanisms by which cap binding proteins promote splicing of meiotically regulated pre-mRNAs will be elucidated with single molecule methods (Specific Aim 2). Finally, correct choice ofthe branchsite by the U2 snfRNP requires ATP hydrolysis by the DEAD-box ATPase, Prp5. Single molecufe-^methods will be used to elucidate Prp5/U2/pre-mRNA interactions that promote branchsite fidelity by kinetic proofreading (Specific Aim 3).

Public Health Relevance

Pre-mRNA splicing is fundamental to human biology, and errors in splicing are implicated in a wide range of diseases including cancer, cystic fibrosis, and retinitis pigmentosa. Many of these errors stem from the choice of incorrect splice sites in precursor to messenger (pre-mRNA) transcripts. This proposal seeks to elucidate the mechanisms of splice site choice in order to improve human health.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Transition Award (R00)
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Special Emphasis Panel (NSS)
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Lewis, Catherine D
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University of Wisconsin Madison
Schools of Earth Sciences/Natur
United States
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Hoskins, Aaron A; Rodgers, Margaret L; Friedman, Larry J et al. (2016) Single molecule analysis reveals reversible and irreversible steps during spliceosome activation. Elife 5:
Rodgers, Margaret L; Tretbar, U Sandy; Dehaven, Alexander et al. (2016) Conformational dynamics of stem II of the U2 snRNA. RNA 22:225-36
Cornilescu, Gabriel; Didychuk, Allison L; Rodgers, Margaret L et al. (2016) Structural Analysis of Multi-Helical RNAs by NMR-SAXS/WAXS: Application to the U4/U6 di-snRNA. J Mol Biol 428:777-89
DeHaven, Alexander C; Norden, Ian S; Hoskins, Aaron A (2016) Lights, camera, action! Capturing the spliceosome and pre-mRNA splicing with single-molecule fluorescence microscopy. Wiley Interdiscip Rev RNA 7:683-701
Rodgers, Margaret L; Paulson, Joshua; Hoskins, Aaron A (2015) Rapid isolation and single-molecule analysis of ribonucleoproteins from cell lysate by SNAP-SiMPull. RNA 21:1031-41
Larson, Joshua; Kirk, Matt; Drier, Eric A et al. (2014) Design and construction of a multiwavelength, micromirror total internal reflectance fluorescence microscope. Nat Protoc 9:2317-28
Wang, Tracy Y; Friedman, Larry J; Gelles, Jeff et al. (2014) The covalent trimethoprim chemical tag facilitates single molecule imaging with organic fluorophores. Biophys J 106:272-8
Anderson, Eric G; Hoskins, Aaron A (2014) Single molecule approaches for studying spliceosome assembly and catalysis. Methods Mol Biol 1126:217-41
Carrocci, Tucker J; Hoskins, Aaron A (2014) Imaging of RNAs in live cells with spectrally diverse small molecule fluorophores. Analyst 139:44-7
Larson, Joshua D; Rodgers, Margaret L; Hoskins, Aaron A (2014) Visualizing cellular machines with colocalization single molecule microscopy. Chem Soc Rev 43:1189-200

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