RNA splicing-the removal of introns and ligation of exons-is an essential step in eukaryotic gene expression and must occur precisely. Precision depends on accurate recognition of the splice sites within RNAs by a macromolecular machine called the spliceosome. Spliceosomes are assembled at particular locations in transcripts from protein and small nuclear ribonucleoprotein (snRNP) components. In humans, most RNAs are alternatively spliced meaning that the spliceosome can incorporate multiple regulatory signals to control the splicing fate of a given transcript. Many of the key steps in regulating alternative splicing and splicing efficiency occur in the earliest stages of spliceosome assembly. During these steps in yeast, the 5' splice site (SS) and the branchsite (BS) are first recognized by the U1 snRNP and the BBP/Mud2 protein heterodimer, respectively. This forms the so-called commitment complex (CC) that then recruits U2 to form the pre-spliceosome. Pre-spliceosome formation is believed to determine the alternative splicing fate of many transcripts and defects in human CC and pre-spliceosome components are linked to genetic diseases including myelodysplastic syndrome (MDS). The ultimate goals of this project are to understand the pathways by which spliceosomes assemble on RNAs. While the identities of the players in these processes are known, their mechanisms of action remain unclear. Investigation of these events will lead to a better understanding of this fundamental process as well as provide new insights into diseases linked to splicing. Here, we focus on formation of the spliceosomal CC and its transition into the pre-spliceosome. In these experiments, we exploit the unique capabilities of single molecule fluorescence as our primary tool.
In Aim 1, we will purify the components of CC and reconstitute its assembly in vitro. A key outcome of Aim 1 is a purified, biochemically characterized system for studying CC formation. This is a necessary step in our long-term objective of biochemically reconstituting spliceosome assembly.
In Aim 2, we study the disassembly of single molecules of CC and the formation of pre-spliceosomes using a novel combination of purified components and yeast cell extracts.
In Aim 3, we use a variety of approaches to study the binding and conformational dynamics of the Prp5 ATPase during pre- spliceosome formation. Together these experiments will provide much needed new insights into spliceosome assembly and the ways in which it can be regulated.

Public Health Relevance

RNA splicing is an essential step in humans for the transmission of genetic information and defects in splicing can lead to a number of diseases including cancers, blindness, or muscular atrophies. This project uses innovative technologies to address several issues of fundamental importance to the splicing reaction including how the splicing machinery can find particular sequences of RNA in a substrate, how RNAs and proteins collaborate to tether the splicing machinery to substrates, and how cells can regulate which splicing pathways particular RNAs employ. The outcomes of these studies will reveal the molecular details of how the spliceosome interacts with target RNAs and this information may lead to more effective treatments for a variety of genetic disorders related to defects in the earliest stages of spliceosome assembly.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM112735-04
Application #
9416170
Study Section
Molecular Genetics A Study Section (MGA)
Program Officer
Bender, Michael T
Project Start
2015-02-01
Project End
2020-01-31
Budget Start
2018-02-01
Budget End
2019-01-31
Support Year
4
Fiscal Year
2018
Total Cost
Indirect Cost
Name
University of Wisconsin Madison
Department
Biochemistry
Type
Earth Sciences/Resources
DUNS #
161202122
City
Madison
State
WI
Country
United States
Zip Code
53715
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van der Feltz, Clarisse; DeHaven, Alexander C; Hoskins, Aaron A (2017) Stress-induced Pseudouridylation Alters the Structural Equilibrium of Yeast U2 snRNA Stem II. J Mol Biol :
van der Feltz, Clarisse; Hoskins, Aaron A (2017) Methodologies for studying the spliceosome's RNA dynamics with single-molecule FRET. Methods 125:45-54
Carrocci, Tucker J; Zoerner, Douglas M; Paulson, Joshua C et al. (2017) SF3b1 mutations associated with myelodysplastic syndromes alter the fidelity of branchsite selection in yeast. Nucleic Acids Res 45:4837-4852
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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

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