Transcripts from many mitochondrial genes in kinetoplastids undergo RNA editing, a posttranscriptional process which inserts and deletes uridines. By assaying for deletion editing in vitro, we found that the editing activity from Trypanosoma brucei mitochondrial lysates (S.D. Seiwert and K.D. Stuart), Science 266:114-117,1994) sediments with a peak of approximately 20S. RNA helicase, terminal uridylyl transferase, RNA ligase, and adenylation activities, which may have a role in editing, cosediment in a broad distribution, with most of each activity at 35 to 40S. Most ATPase 6 (A6) guide RNA and unedited A6 mRNA sediments at 20 to 30S, with some sedimenting further into the gradient, while most edited A6 mRNA sediments at >35S. Several mitochondrial proteins which cross-link specifically with guide RNA upon UV treatment also sediment in glycerol gradients. Notably, a 65-kDa protein sediments primarily at approximately 20S, a 90-kDa protein sediments at 35 to 40S, and a 25-kDa protein is present at <10S. Most ribonucleoprotein complexes that form with gRNA in vitro sediment at 10 to 20S, except for one, which sediments at 30 to 45S. These results suggest that RNA editing takes place within a multicomponent complex. The potential functions of and relationships between the 20S and 35 to 40S complexes are discussed.

Analysis of the JSRV env gene revealed a conserved tyrosine (597) and methionine (600) residue in the cytoplasmic tail within the transmembrane domain of the envelope, which creates a known binding site of SH2 domains in the p85 subunit of phosphatidylinositol 3-kinase. However, when this tyrosine residue was mutated to serine or alanine, transformation was not affected. Furthermore, mutation of the methionine residue to valine or leucine also failed to eliminate JSRV env-mediated transformation. These results are in contrast to mutational analysis performed in JSRV env-transformed murine NIH-3T3 cells in which both the tyrosine and methionine residues are necessary for transformation. These findings suggest that more than one mechanism may be involved in JSRV env-mediated transformation.

RNA editing in Trypanosoma brucei mitochondria produces mature mRNAs by a series of enzyme-catalyzed reactions that specifically insert or delete uridylates in association with a macromolecular complex. Using a mitochondrial fraction enriched for in vitro RNA editing activity, we produced several monoclonal antibodies that are specific for a 21-kDa guide RNA (gRNA) binding protein initially identified by UV cross-linking. Immunofluorescence studies localize the protein to the mitochondrion, with a preference for the kinetoplast. The antibodies cause a supershift of previously identified gRNA-specific ribonucleoprotein complexes and immunoprecipitate in vitro RNA editing activities that insert and delete uridylates. The immunoprecipitated material also contains gRNA-specific endoribonuclease, terminal uridylyltransferase, and RNA ligase activities as well as gRNA and both edited and unedited mRNA. The immunoprecipitate contains numerous proteins, of which the 21-kDa protein, a 90-kDa protein, and novel 55-and 16-kDa proteins can be UV cross-linked to gRNA. These studies indicate that the 21-kDa protein associates with the ribonucleoprotein complex (or complexes) that catalyze RNA editing.RNA editing produces mature mRNAs in the mitochondria of trypanosomatids by guide RNA (gRNA)-directed posttranscriptional insertion and deletion of uridylates (U's) (2). This process can be so extensive that most of the coding sequence, as well as the initiation and termination codons, results from RNA editing (1,11,27,28,30). Stage-specific RNA editing appears to regulate mitochondrial respiration in the different life stages of African trypanosomes (9, 29). The mRNAs for components of respiratory complex I are preferentially edited in the mammalian stage of the life cycle, where the trypanosomes lack cytochromes, rely on glycolysis for energy production, and utilize complex I and alternate oxidase for terminal respiration. In contrast, the invertebrate stage predominantly utilizes cytochrome-mediated oxidative phosphorylation for energy generation while editing cytochrome mRNAs only in this stage.The edited mRNA sequence is specified by trans-acting small RNA molecules called gRNAs, which are complementary to their edited cognate mRNAs (4). The gRNAs have three distinct regions. A 5-to 15-nucleotide (nt) region at the 5Ј end of gRNAs is complementary to the sequence of its cognate preedited mRNA that is immediately 3Ј to the region that will be edited. Formation of a duplex between these regions, called the anchor duplex, is an essential prelude to editing. A 55-to 70-nt guiding region, immediately 3Ј to the anchor region, contains the sequence information that can specify the insertion or deletion of U's at 1 to 20 internucleotide sites in the pre-mRNA. The third region contains the 3Ј end of the gRNA, which has a 5-to 24-nt oligo(U) tail that is added posttranscriptionally, presumably by a terminal uridylyltransferase (TUTase) activity. The function of the oligo(U) tail is unknown, but it may enhance the interaction between t...

Ovine pulmonary carcinoma (OPC) is a contagious neoplasm of alveolar epithelial type II (ATII) or