The overall goal of this project is to understand the process of mitochondrial biogenesis in the parasite kinetoplastids and the role of the kinetoplast DNA in this process. Specific emphasis is on the unusual RNA processing phenomenon termed RNA editing which occurs in the mitochondrion of these cells.
The specific aims are as follows: #1. In vivo and in vitro pulse-chase kinetics of transcription and editing. Unedited, edited and partially edited oligomers will be used in Sl and RNase A protection experiments to determine the relative rates of RNA transcription and RNA editing, and to determine if the partially edited RNAs are true intermediates. #2. Test of several models involving putative editing templates. #3. Analysis of the in vitro editing-like system: (1) Optimization of the in vitro RNA editing-like system using preedited cytochrome b RNA. Various parameters will be tested in an attempt to increase the extent of internal U addition versus 3' end U addition. (2) Deletion analysis of substrate CYb RNA in order to ascertain the putative signal for specific cleavage and possibly for U addition. (3) Testing additional pre-edited and non-edited RNAs in the TL reaction for internal U. The COIII, MURF2 and MURF3 5' ends will be used as pre-edited RNAs, and the ND1, COI, ND4, ND5 and a bacterial plasmid sequence will be used as non-edited controls. GC-rich intergenic region RNA will also be tested. (4) Localization of internal U's in the in vitro edited RNA. Fingerprints of specific RNase digests will be used, as well as biotin-avidin selection of labeled fragments and direct cloning and sequencing. (5) Can the internal U addition activity be reconstituted by addition of mitochondrial membrane fractions to the clarified Triton extract? If so, this fraction will be fractionated and the components necessary for reconstitution determined. (6) Determination of sites of cleavage of heparin-treated CYb RNA in the in vitro system. Is cleavage due to an endonuclease or to self-cleavage? #4. Search for an exonuclease that could act as a """"""""trimming"""""""" enzyme to remove extra U's at sites of editing. #5. Purification of the mitochondrial TUTase and RNA ligase enzymes. Production of specific antisera. #6. Isolation of mitochondrial proteins produced from edited mRNAs. Amino acid sequences corresponding to edited regions will be obtained. #7. Evolution of RNA editing in the kinetoplastids. Detection of pan-editing in kinetoplastids from other genera. #8. Computer analysis of pre-edited sequences. Search for additional edited genes in GC-rich intergenic regions and for signals or secondary structures that could be involved in editing.
