The focus of this application is to establish the relationship between structure and function of the pre-mRNA branch site, the RNA formation that contains the attacking nucleophile for the first of pre-mRNA splicing. The long-range objective of our work is to understand how RNA and protein components of the eukaryotic splicing apparatus assemble, define splice sites, and catalyze the removal of noncoding sequences (introns) from pre-mRNA molecules. Splicing is an integral step in the maturation of eukaryotic RNAs, and alternative splice site selection is a source of developmental stage- and tissue-specific protein diversity. Since errors in splicing are the basis of certain cancers and neurodegenerative diseases, an understanding of the splicing process is of biomedical importance. The spliceosome of eukaryotic nuclei is a dynamic assembly of five recyclable small nuclear (sn)RNAs and numerous proteins, whereas the Group II intron, found in certain prokaryotes and eukaryotic organelles, is a single RNA molecule comprising six secondary structural domains. The two splicing systems remove introns via the identical chemical mechanism and stereochemistry, and share certain sequence similarities, but only the Group II intron can carry out the splicing reaction in vitro in the absence of proteins. It is therefore likely that the two systems share evolutionary ancestry and that RNA also plays a major catalytic role in the spliceosomal reaction. Gaps in structural information have limited comparison.
The Specific Aims are: 1) determine the contributions of individual RNA-RNA interactions to the spliceosomal branch site structure and their implications on splicing activity; and 2) probe the structure of the pre-mRNA branch site of the Group II self-splicing intron in order to compare it with its spliceosomal counterpart. Structural studies in solution will be carried out by homonuclear and heteronuclear NMR; the effect of various mutations and modifications on splicing activity will be assessed by perturbations to product formation of in vitro splicing assays. A number of innovative NMR techniques will be exploited to assist in determination of high resolution solution structural models and orientation of RNA domains. The proposed experiments will provide new insights into the ancestry and the molecular basis of splicing activity in eukaryotic cells.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM054008-04A2
Application #
6687521
Study Section
Molecular and Cellular Biophysics Study Section (BBCA)
Program Officer
Lewis, Catherine D
Project Start
1998-05-01
Project End
2007-07-31
Budget Start
2003-08-05
Budget End
2004-07-31
Support Year
4
Fiscal Year
2003
Total Cost
$216,947
Indirect Cost
Name
Florida State University
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
790877419
City
Tallahassee
State
FL
Country
United States
Zip Code
32306
Popovi?, Milena; Nelson, Joycelynn D; Schroeder, Kersten T et al. (2012) Impact of base pair identity 5' to the spliceosomal branch site adenosine on branch site conformation. RNA 18:2093-103
Schlatterer, Jorg C; Greenbaum, Nancy L (2008) Specificity of Mg2+ binding at the Group II intron branch site. Biophys Chem 136:96-100
Yuan, Faqing; Griffin, Laura; Phelps, LauraJane et al. (2007) Use of a novel Forster resonance energy transfer method to identify locations of site-bound metal ions in the U2-U6 snRNA complex. Nucleic Acids Res 35:2833-45
Schlatterer, Jorg C; Crayton, Samuel H; Greenbaum, Nancy L (2006) Conformation of the Group II intron branch site in solution. J Am Chem Soc 128:3866-7
Jennings, T L; Schlatterer, J C; Singh, M P et al. (2006) NSET molecular beacon analysis of hammerhead RNA substrate binding and catalysis. Nano Lett 6:1318-24
Breiner, Boris; Schlatterer, Jorg C; Kovalenko, Serguei V et al. (2006) Protected 32P-labels in deoxyribonucleotides: investigation of sequence selectivity of DNA photocleavage by enediyne-, fulvene-, and acetylene-lysine conjugates. Angew Chem Int Ed Engl 45:3666-70
Xu, Darui; Greenbaum, Nancy L; Fenley, Marcia O (2005) Recognition of the spliceosomal branch site RNA helix on the basis of surface and electrostatic features. Nucleic Acids Res 33:1154-61
Newby, Meredith I; Greenbaum, Nancy L (2002) Investigation of Overhauser effects between pseudouridine and water protons in RNA helices. Proc Natl Acad Sci U S A 99:12697-702