The long-term goal of this project is to elucidate the detailed molecular mechanisms by which intervening sequences or introns are removed from nascent RNA transcripts through the process of pre-mRNA splicing. Alterations in this essential step in eukaryotic gene expression are known to underly many human diseases, so detailed understanding of the mechanisms involved is important for the betterment of human health. Pre- mRNA splicing is carried out by the spliceosome, a ~3 MDa macromolecular complex consisting of 5 small nuclear RNAs (snRNAs: U1, U2, U4, U5 and U6) and >100 polypeptides. While much progress has been made in defining these component parts, much remains to be learned about how these myriad pieces function together to mediate precise and timely intron removal. This proposal describes development of methodologies for application of single molecule total internal reflection fluorescence (SM-TIRF) microscopy to complex macromolecular systems such as the spliceosome. A novel SM-TIRF microscope with multi-wavelength capabilities designed and implemented in Jeff Gelles'laboratory at Brandeis University allows for simultaneous monitoring of multiple fluorophores interacting with single fluorescent pre-mRNA molecules tethered to a glass coverslip. Using this Colocalization Single Molecule Spectroscopy (CoSMoS) approach, one can directly observe the detailed kinetics of snRNP and splicing factor association/dissociation with pre-mRNA over the full course of spliceosome assembly and splicing. As there currently exists no fully-reconstituted system that faithfully replicates all stages of the splicing pathway, all experiments are carried out in crude cell lysates using fluorescently-tagged endogenous proteins. This approach will be used to: (1) Establish the first complete kinetic framework for spliceosome assembly on a model pre-mRNA;(2) Determine to what extent spliceosome assembly proceeds via the same or different dynamic pathway(s) on other transcripts;(3) Investigate the nature of spliceosomal discard pathways;and (4) Investigate the dynamics of DExH/D-box proteins with the spliceosome. Because CoSMoS analysis does not require purified components and can be carried out in highly complex mixtures, this technology should be broadly applicable to the study of many other macromolecular machines that, like the spliceosome, cannot be reassembled from component parts in vitro. )

Public Health Relevance

This project seeks to elucidate the molecular mechanisms by which sequences interrupting genes (introns) are removed during the process of gene expression. Such intron splicing is an essential process in all multicellular organisms, and missplicing is a major contributor to many human diseases. Only by gaining a better understanding the cellular machinery mediating this process will we ultimately be able to treat such splicing-related diseases. )

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM053007-17
Application #
8399727
Study Section
Molecular Genetics A Study Section (MGA)
Program Officer
Bender, Michael T
Project Start
1995-08-01
Project End
2014-12-31
Budget Start
2013-01-01
Budget End
2013-12-31
Support Year
17
Fiscal Year
2013
Total Cost
$375,083
Indirect Cost
$147,069
Name
University of Massachusetts Medical School Worcester
Department
Biochemistry
Type
Schools of Medicine
DUNS #
603847393
City
Worcester
State
MA
Country
United States
Zip Code
01655
Chen, Weijun; Shulha, Hennady P; Ashar-Patel, Ami et al. (2014) Endogenous U2·U5·U6 snRNA complexes in S. pombe are intron lariat spliceosomes. RNA 20:308-20
Anderson, Eric G; Hoskins, Aaron A (2014) Single molecule approaches for studying spliceosome assembly and catalysis. Methods Mol Biol 1126:217-41
Singh, Guramrit; Ricci, Emiliano P; Moore, Melissa J (2014) RIPiT-Seq: a high-throughput approach for footprinting RNA:protein complexes. Methods 65:320-32
Chen, Weijun; Moore, Melissa J (2014) The spliceosome: disorder and dynamics defined. Curr Opin Struct Biol 24:141-9
Kucukural, Alper; Ozadam, Hakan; Singh, Guramrit et al. (2013) ASPeak: an abundance sensitive peak detection algorithm for RIP-Seq. Bioinformatics 29:2485-6
Shcherbakova, Inna; Hoskins, Aaron A; Friedman, Larry J et al. (2013) Alternative spliceosome assembly pathways revealed by single-molecule fluorescence microscopy. Cell Rep 5:151-65
Crawford, Daniel J; Hoskins, Aaron A; Friedman, Larry J et al. (2013) Single-molecule colocalization FRET evidence that spliceosome activation precedes stable approach of 5' splice site and branch site. Proc Natl Acad Sci U S A 110:6783-8
Hoskins, Aaron A; Moore, Melissa J (2012) The spliceosome: a flexible, reversible macromolecular machine. Trends Biochem Sci 37:179-88
Hoskins, Aaron A; Friedman, Larry J; Gallagher, Sarah S et al. (2011) Ordered and dynamic assembly of single spliceosomes. Science 331:1289-95
Hoskins, Aaron A; Gelles, Jeff; Moore, Melissa J (2011) New insights into the spliceosome by single molecule fluorescence microscopy. Curr Opin Chem Biol 15:864-70

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