The proposed work is the continuation of a multifaceted project whose long range goal is an understanding of the mechanisms that regulate RNA stability as a key element of genetic control. The studies proposed for the next project period are intended to provide fundamental information about certain of the genes, enzymes, and substrate properties that govern RNA degradation. They will utilize a combination of genetic and biochemical approaches and will employ RNAI, an antisense repressor of the replication of ColEl-type plasmids, as a model system.
The specific aims of the proposed investigations are to: 1) Isolate mutant RNAI substrates having sequence changes that affect the rate of ams/rne-dependent decay of RNAI in vivo. Identify sequence motifs and other structural features that affect the rate and/or site of cleavage in vivo and determine the effects of these changes on RNAseE-mediated cleavage of RNAI in vitro, 2) Isolate and characterize mutations in the ams/rne gene that alter plasmid copy number by increasing the rate of cleavage of RNAI in vivo; determine the effects of these mutations on other ams/rne-controlled ribonucleolytic activities. Isolate RNAseE from mutated cells and characterize its activity in vitro, 3) Characterize the RNAI cleaving activity from ams/rne ts mutant cells complemented by cloned yeast and mammalian cDNA and elucidate the basis for complementation. Determine the nature of the gene product encoded by the complementing clones, and 4) Isolate E. coli mutants that affect the activity of the growth-rate controlled enzyme that degrades RNAI variants lacking an RNAseE cleavage site; elucidate the basis of the growth rate control. Collectively, these experiments will provide answers to questions that are of fundamental importance to an understanding of the regulatory role of RNA in key biological processes crucial to current efforts to develop approaches that employ antisense and RNA molecules and ribozymes intracellularly in the treatment of disease.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM027241-11A2
Application #
3274631
Study Section
Biochemistry Study Section (BIO)
Project Start
1979-12-01
Project End
1997-06-30
Budget Start
1993-07-01
Budget End
1994-06-30
Support Year
11
Fiscal Year
1993
Total Cost
Indirect Cost
Name
Stanford University
Department
Type
Schools of Medicine
DUNS #
800771545
City
Stanford
State
CA
Country
United States
Zip Code
94305
Claverie-Martin, F; Wang, M; Cohen, S N (1997) ARD-1 cDNA from human cells encodes a site-specific single-strand endoribonuclease that functionally resembles Escherichia coli RNase E. J Biol Chem 272:13823-8
McDowall, K J; Cohen, S N (1996) The N-terminal domain of the rne gene product has RNase E activity and is non-overlapping with the arginine-rich RNA-binding site. J Mol Biol 255:349-55
Wang, M; Cohen, S N (1994) ard-1: a human gene that reverses the effects of temperature-sensitive and deletion mutations in the Escherichia coli rne gene and encodes an activity producing RNase E-like cleavages. Proc Natl Acad Sci U S A 91:10591-5
McDowall, K J; Lin-Chao, S; Cohen, S N (1994) A+U content rather than a particular nucleotide order determines the specificity of RNase E cleavage. J Biol Chem 269:10790-6
Lin-Chao, S; Wong, T T; McDowall, K J et al. (1994) Effects of nucleotide sequence on the specificity of rne-dependent and RNase E-mediated cleavages of RNA I encoded by the pBR322 plasmid. J Biol Chem 269:10797-803
McDowall, K J; Hernandez, R G; Lin-Chao, S et al. (1993) The ams-1 and rne-3071 temperature-sensitive mutations in the ams gene are in close proximity to each other and cause substitutions within a domain that resembles a product of the Escherichia coli mre locus. J Bacteriol 175:4245-9
Xu, F; Lin-Chao, S; Cohen, S N (1993) The Escherichia coli pcnB gene promotes adenylylation of antisense RNAI of ColE1-type plasmids in vivo and degradation of RNAI decay intermediates. Proc Natl Acad Sci U S A 90:6756-60
Klug, G; Cohen, S N (1991) Effects of translation on degradation of mRNA segments transcribed from the polycistronic puf operon of Rhodobacter capsulatus. J Bacteriol 173:1478-84
Lin-Chao, S; Cohen, S N (1991) The rate of processing and degradation of antisense RNAI regulates the replication of ColE1-type plasmids in vivo. Cell 65:1233-42
Klug, G; Cohen, S N (1990) Combined actions of multiple hairpin loop structures and sites of rate-limiting endonucleolytic cleavage determine differential degradation rates of individual segments within polycistronic puf operon mRNA. J Bacteriol 172:5140-6

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