OF FUNDED AWARD NIH GM112735, Mechanisms of Spliceosome Assembly and Splice Site Selection 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. This administrative supplement will allow us to construct a powerful single molecule fluorescence microscope (CoSMoS 2.0) for furthering this research. It will greatly increase our temporal resolution, providing novel insights into CC interactions in Aims 1 and 2. CoSMoS 2.0 will also enhance Aim 3 by letting us probe ATP hydrolysis by Prp5 in real-time. These experiments will ultimately be critical for connecting ATP hydrolysis to discrete steps in splicing. Together our experiments will provide much needed new insights into spliceosome assembly and the ways in which it can be regulated.
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 goal of this administrative supplement is to allow my laboratory to construct the state-of-the-art CoSMoS 2.0 microscope, which will be used to further the goals of the awarded grant by allowing us to carry out novel experiments with increased temporal resolution, greater throughput, and with higher concentrations of fluorescent molecules.
| Kaur, Harpreet; Jamalidinan, Fatemehsadat; Condon, Samson G F et al. (2018) Analysis of spliceosome dynamics by maximum likelihood fitting of dwell time distributions. Methods : |
| van der Feltz, Clarisse; DeHaven, Alexander C; Hoskins, Aaron A (2018) Stress-induced Pseudouridylation Alters the Structural Equilibrium of Yeast U2 snRNA Stem II. J Mol Biol 430:524-536 |
| Carrocci, Tucker J; Paulson, Joshua C; Hoskins, Aaron A (2018) Functional analysis of Hsh155/SF3b1 interactions with the U2 snRNA/branch site duplex. RNA 24:1028-1040 |
| van der Feltz, Clarisse; Hoskins, Aaron A (2017) Methodologies for studying the spliceosome's RNA dynamics with single-molecule FRET. Methods 125:45-54 |
| Larson, Joshua Donald; Hoskins, Aaron A (2017) Dynamics and consequences of spliceosome E complex formation. Elife 6: |
| Panchapakesan, Shanker Shyam S; Ferguson, Matthew L; Hayden, Eric J et al. (2017) Ribonucleoprotein purification and characterization using RNA Mango. RNA 23:1592-1599 |
| 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 |
| Rodgers, Margaret L; Didychuk, Allison L; Butcher, Samuel E et al. (2016) A multi-step model for facilitated unwinding of the yeast U4/U6 RNA duplex. Nucleic Acids Res 44:10912-10928 |
| Hansen, S R; Rodgers, M L; Hoskins, A A (2016) Fluorescent Labeling of Proteins in Whole Cell Extracts for Single-Molecule Imaging. Methods Enzymol 581:83-104 |
| Rodgers, Margaret L; Tretbar, U Sandy; Dehaven, Alexander et al. (2016) Conformational dynamics of stem II of the U2 snRNA. RNA 22:225-36 |
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