RNA editing provides a powerful means of altering gene expression in many systems, including humans, and results in RNA products which differ in highly specific ways from their original templates. Most RNAs present in the mitochondria of Physarum contain non-encoded residues, and insertion of each of the four nucleotides has been observed in this system. The long term goal of this project is to determine the mechanism by which this precise insertion of nucleotides is accomplished. Experiments with isolated mitochondria have demonstrated that editing in Physarum is closely associated with RNA synthesis. Transcription complexes isolated from Physarum mitochondria retain the ability to synthesize edited RNA, permitting the use of these complexes in mechanistic studies. The relationship between transcription and editing will be examined further by manipulating polymerase elongation rates and availability of nucleotide substrates, and through the use of chain-terminating nucleotide analogs. Both sets of experiments should yield information as to the point of nucleotide addition during synthesis of nascent RNAs. Isolated transcription complexes will also be subjected to strong washing conditions to determine whether there are dissociable trans-acting editing factors involved in the insertion of non-encoded nucleotides. Finally, further steps will be taken toward the establishment of a soluble in vitro transcription-editing system. Mitochondrial promoters will be identified and any necessary factor(s) required for their utilization will be isolated. The ultimate aim of these experiments is a molecular description of insertional editing in Physarum, including the identification of cis-acting sequences, trans-acting factors, and the chemical steps involved in this extraordinary process.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM054663-05
Application #
6525684
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Program Officer
Rhoades, Marcus M
Project Start
1998-08-01
Project End
2003-07-31
Budget Start
2002-08-01
Budget End
2003-07-31
Support Year
5
Fiscal Year
2002
Total Cost
$276,925
Indirect Cost
Name
Case Western Reserve University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
077758407
City
Cleveland
State
OH
Country
United States
Zip Code
44106
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Abad, Maria G; Long, Yicheng; Willcox, Allison et al. (2011) A role for tRNA(His) guanylyltransferase (Thg1)-like proteins from Dictyostelium discoideum in mitochondrial 5'-tRNA editing. RNA 17:613-23
Bundschuh, R; Altmuller, J; Becker, C et al. (2011) Complete characterization of the edited transcriptome of the mitochondrion of Physarum polycephalum using deep sequencing of RNA. Nucleic Acids Res 39:6044-55
Gott, Jonatha M; Somerlot, Benjamin H; Gray, Michael W (2010) Two forms of RNA editing are required for tRNA maturation in Physarum mitochondria. RNA 16:482-8
Bullerwell, Charles E; Burger, Gertraud; Gott, Jonatha M et al. (2010) Abundant 5S rRNA-like transcripts encoded by the mitochondrial genome in amoebozoa. Eukaryot Cell 9:762-73
Rhee, Amy C; Somerlot, Benjamin H; Parimi, Neeta et al. (2009) Distinct roles for sequences upstream of and downstream from Physarum editing sites. RNA 15:1753-65