Splicing of intervening sequences from precursor-tRNA requires at least two functions, an endonuclease and a ligase. These enzymes catalyze excision of the introns and rejoining of the tRNA half-molecules through recognition of conserved features of tRNA structure. Despite the fact that tRNA splicing is readily amenable to genetic and biochemical analysis, little is known about the regulatory and structural genes involved, the potential interaction of proteins or other components that might form a splicing complex, or the possible relationship between splicing and transport of tRNA from nucleus to cytoplasm. Information on these aspects of tRNA splicing have not been forthcoming due to the intractable nature of the membrane-associated tRNA-splicing endonuclease. Previous attempts in several laboratories to study the endonuclease by either forward or reverse genetic techniques have not been successful. Recently, however, mutations in SEN (splicing endonuclease) genes were identified in this laboratory that cause partial deficiencies of endonuclease activity in the yeast Saccharomyces cerevisiae. One mutant also causes pre- tRNA steady-state accumulation and temperature-sensitive growth. By analyzing SEN genes and their products using genetic, biochemical, and immunological approaches the research in this proposal will provide critical information on the structure, organization, function, and intracellular location of tRNA splicing components. The most immediate question to be answered centers on which products are structural components of the endonuclease. Once this central question is answered it should be possible to address the more complex issues of how and where tRNA splicing occurs in intact cells.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM040310-01
Application #
3297722
Study Section
Genetics Study Section (GEN)
Project Start
1988-09-01
Project End
1993-08-31
Budget Start
1988-09-01
Budget End
1989-08-31
Support Year
1
Fiscal Year
1988
Total Cost
Indirect Cost
Name
University of Wisconsin Madison
Department
Type
Graduate Schools
DUNS #
161202122
City
Madison
State
WI
Country
United States
Zip Code
53715
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Ursic, D; Himmel, K L; Gurley, K A et al. (1997) The yeast SEN1 gene is required for the processing of diverse RNA classes. Nucleic Acids Res 25:4778-85
Rasmussen, T P; Culbertson, M R (1996) Analysis of yeast trimethylguanosine-capped RNAs by midwestern blotting. Gene 182:89-96
DeMarini, D J; Papa, F R; Swaminathan, S et al. (1995) The yeast SEN3 gene encodes a regulatory subunit of the 26S proteasome complex required for ubiquitin-dependent protein degradation in vivo. Mol Cell Biol 15:6311-21
Ursic, D; DeMarini, D J; Culbertson, M R (1995) Inactivation of the yeast Sen1 protein affects the localization of nucleolar proteins. Mol Gen Genet 249:571-84
Sturley, S L; Talmud, P J; Brasseur, R et al. (1994) Human apolipoprotein B signal sequence variants confer a secretion-defective phenotype when expressed in yeast. J Biol Chem 269:21670-5
Ursic, D; Sedbrook, J C; Himmel, K L et al. (1994) The essential yeast Tcp1 protein affects actin and microtubules. Mol Biol Cell 5:1065-80
DeMarini, D J; Winey, M; Ursic, D et al. (1992) SEN1, a positive effector of tRNA-splicing endonuclease in Saccharomyces cerevisiae. Mol Cell Biol 12:2154-64
Ursic, D; Culbertson, M R (1991) The yeast homolog to mouse Tcp-1 affects microtubule-mediated processes. Mol Cell Biol 11:2629-40
Sturley, S L; Culbertson, M R; Attie, A D (1991) Secretion and lipid association of human apolipoprotein E in Saccharomyces cerevisiae requires a host mutation in sterol esterification and uptake. J Biol Chem 266:16273-6

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