Association of Guide RNA Binding Protein gBP21 with Active RNA Editing Complexes in Trypanosoma brucei

Abstract

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...

A 100-kD complex of two RNA-binding proteins from mitochondria of Leishmania tarentolae catalyzes RNA annealing and interacts with several RNA editing components

“…We have confirmed and extended previous results from T. brucei (Koller et al 1997;Allen et al 1998;Lambert et al 1999;Blom et al 2001;Muller et al 2001) and C. fasciculata (Blom et al 2001) for the presence of two small mitochon- drial RNA-binding proteins, with L. tarentolae. Several features of the L. tarentolae proteins are similar to those reported previously, but there are several novel findings.…”

“…However, as mentioned above, previous evidence for involvement of the T. brucei gBP21 protein in RNA editing is somewhat equivocal. gBP21 was reported to coprecipitate with a 20S in vitro editing activity (Allen et al 1998), whereas gBP21 null mutants of bloodstream T. brucei showed an overall decrease in the abundance of mitochondrial transcripts but no effect on editing in vivo (Lambert et al 1999). One explanation of these earlier results could be the observed redundancy of the two proteins of the Ltp26/Ltp28 complex.…”

“…brucei gPB21 has been shown to coimmunoprecipitate with in vitro RNA editing activity, ATP-labeled RNA ligase proteins, and TUTase activity, and the interaction was sensitive to micrococcal nuclease treatment, suggesting involvement of an RNA component (Allen et al 1998). The C. fasciculata gPB27 and gPB29 proteins, however, were reported not to interact with any identified editing components or RNA (Blom et al 2001).…”

“…The RNA-binding mitochondrial protein gBP21, from T. brucei, has been extensively studied (Köller et al 1994;Allen et al 1998;Lambert et al 1999;Muller et al 2001) and has several properties that make it a very likely candidate for indirect involvement in editing. These involve high-affinity binding to gRNA and coimmunoprecipitation with in vitro editing activity.…”

Disruption of the Zinc Finger Motifs in the Leishmania tarentolae LC-4 (=TbMP63) L-complex Editing Protein Affects the Stability of the L-complex

“…At least 16 polypeptides are present in this complex, the stability of which is determined entirely by protein-protein interactions (2,7,8). Two additional complexes were shown to interact with the L-complex via RNA linkers (2,9,10). Three L-complex proteins, LC-1 (MP81), LC-4 (MP63, Band III), and LC-7b (MP42) contain zinc finger motifs that could be involved in protein-protein interactions or protein-RNA/DNA interactions (2,5).…”