The spliceosome is an intricate biological nanomachine that is unique to the eukaryotic lineage of life. It performs the essential process of removing introns from the primary transcripts of eukaryotic genes, thus creating functional messenger RNAs for protein production. Failure of the spliceosome to accurately and efficiently remove introns, either due to mutations in individual introns or in spliceosome components, results in a range of human diseases including autosomal dominant retinitis pigmentosa (adRP), which causes blindness. We propose to continue our ongoing investigation into the molecular mechanisms of wild-type and mutant spliceosome function. Five small nuclear RNAs, U1, U2, U4, U5 and U6, are central to the splicing process. Each RNA associates with proteins to form a snRNP, and the five snRNPs assemble on an intron to reconstitute a spliceosome. After assembly, a complex activation process ensues that results in expulsion of the U1 and U4 snRNPs, and alignment of the intron splice sites on a U2/U6/U5 scaffold. It is thought that the U2/U6 complex then carries out the two catalytic steps of splicing in conjunction with key protein factors. Our studies will follow the progression of U6 RNA through the splicing cycle. We will determine the mechanism of assembly of the U4/U6 di-snRNP, which is thought to keep U6 in an inactive state during the spliceosome assembly process. In particular, we will test our hypothesis that U2 RNA is actively involved in this process, as well as the proteins Prp24 and Slt11. During the subsequent transition from U4/U6 complex to U2/U6 complex, U6 RNA changes structure to form a remarkably conserved internal stem-loop (ISL), which is thought to be required for catalysis of splicing. Engineered mutations that further stabilize the ISL result i a cold-sensitive block to splicing, providing a genetic and biochemical tool for studying the interconversion of U4/U6 and U2/U6. We will exploit one such mutation to probe the function of the essential U6 RNA-binding protein Cwc2 in spliceosome activation. We will determine the structures of key sub-complexes identified in our studies. The results of these studies will be a deeper understanding of an ancient molecular machine essential to all eukaryotic cells, and possible diagnostic and therapeutic interventions for devastating human diseases.

Public Health Relevance

The spliceosome is an intricate cellular machine composed of 5 RNAs and more than 75 proteins. The spliceosome is so vital to the cell that even a subtle defect in just one of its components results in the disease retinitis pigmentosa, which causes blindness. Our work will result in a better understanding of the mechanism by which this remarkable molecular machine processes messenger RNA so that proteins can be made correctly by cells.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM065166-11
Application #
8598884
Study Section
Macromolecular Structure and Function B Study Section (MSFB)
Program Officer
Preusch, Peter C
Project Start
2002-04-01
Project End
2016-11-30
Budget Start
2013-12-01
Budget End
2014-11-30
Support Year
11
Fiscal Year
2014
Total Cost
$292,654
Indirect Cost
$94,811
Name
University of Wisconsin Madison
Department
Biochemistry
Type
Schools of Earth Sciences/Natur
DUNS #
161202122
City
Madison
State
WI
Country
United States
Zip Code
53715
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Burke, Jordan E; Sashital, Dipali G; Zuo, Xiaobing et al. (2012) Structure of the yeast U2/U6 snRNA complex. RNA 18:673-83
Martin-Tumasz, Stephen; Richie, Ashley C; Clos 2nd, Lawrence J et al. (2011) A novel occluded RNA recognition motif in Prp24 unwinds the U6 RNA internal stem loop. Nucleic Acids Res 39:7837-47
Martin-Tumasz, Stephen; Reiter, Nicholas J; Brow, David A et al. (2010) Structure and functional implications of a complex containing a segment of U6 RNA bound by a domain of Prp24. RNA 16:792-804
Venditti, Vincenzo; Clos 2nd, Lawrence; Niccolai, Neri et al. (2009) Minimum-energy path for a u6 RNA conformational change involving protonation, base-pair rearrangement and base flipping. J Mol Biol 391:894-905
Butcher, Samuel E (2009) The spliceosome as ribozyme hypothesis takes a second step. Proc Natl Acad Sci U S A 106:12211-2
Martin-Tumasz, Stephen; Butcher, Samuel E (2009) (1)H, (13)C and (15)N resonance assignments of a ribonucleoprotein complex consisting of Prp24-RRM2 bound to a fragment of U6 RNA. Biomol NMR Assign 3:227-30
Imker, Heidi J; Singh, Jaya; Warlick, Benjamin P et al. (2008) Mechanistic diversity in the RuBisCO superfamily: a novel isomerization reaction catalyzed by the RuBisCO-like protein from Rhodospirillum rubrum. Biochemistry 47:11171-3
Venditti, Vincenzo; Niccolai, Neri; Butcher, Samuel E (2008) Measuring the dynamic surface accessibility of RNA with the small paramagnetic molecule TEMPOL. Nucleic Acids Res 36:e20
Bae, Euiyoung; Reiter, Nicholas J; Bingman, Craig A et al. (2007) Structure and interactions of the first three RNA recognition motifs of splicing factor prp24. J Mol Biol 367:1447-58

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