Intervening sequences are spliced from eukaryotic precursor-tRNAs through the action of three enzymes, a heterotrimeric endonuclease, a multi-functional but monomeric ligase, and an NAD-linked phosphatase. The endonuclease and ligase form a physical complex that is localized in the nuclear periphery in the vicinity of nuclear pores, suggesting the potential for functional coupling of splicing and nuclear export. Two genes, SEN1 and SEN2, were identified previously in the yeast Saccharomyces cerevisiae that are required for endonuclease activity. SEN1 codes for a nuclear-localized, positive effector of endonuclease activity that appears not to be a catalytic subunit of the enzyme. SEN2 codes for the 42 Kd catalytic subunit of the enzyme. We have isolated mutations in six new genes that affect SEN1 function. Mutations in two of the genes, SEN3 and SEN4, affect the expression of SEN1 at a post-translational level. One temperature-sensitive mutation, sen3-l, also causes excess accumulation of tRNA splicing intermediates in vivo. In addition, four genes have been identified that suppress the temperature-sensitive senl-l mutation when present in multiple copies and may encode products that interact with the SEN1 protein. By continuing with our analysis of SEN1 and SEN2 function and by initiating analyses of SEN3, SEN4, and the high-copy suppressor genes, we hope to provide critical information on the structure, organization, function, and intracellular location of the splicing complex. It is also possible that we may learn something about the import of splicing enzymes into the nucleus and the export of spliced tRNA products from the nucleus.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM040310-09
Application #
2180250
Study Section
Molecular Cytology Study Section (CTY)
Project Start
1988-09-01
Project End
1999-08-31
Budget Start
1996-09-01
Budget End
1999-08-31
Support Year
9
Fiscal Year
1996
Total Cost
Indirect Cost
Name
University of Wisconsin Madison
Department
Biochemistry
Type
Other Domestic Higher Education
DUNS #
161202122
City
Madison
State
WI
Country
United States
Zip Code
53715
Rasmussen, T P; Culbertson, M R (1998) The putative nucleic acid helicase Sen1p is required for formation and stability of termini and for maximal rates of synthesis and levels of accumulation of small nucleolar RNAs in Saccharomyces cerevisiae. Mol Cell Biol 18:6885-96
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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