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Kinetoplastid RNA editing involves a 3' nucleotidyl phosphatase activity.

ABSTRACT: Mitochondrial pre-messenger RNAs (pre-mRNAs) in African trypanosomes require RNA editing in order to mature into functional transcripts. The process involves the addition and/or removal of U nucleotides and is mediated by a high-molecular-mass complex, the editosome. Editosomes catalyze the reaction through an enzyme-driven pathway that includes endo/exoribonuclease, terminal uridylate transferase and RNA ligase activities. Here we show that editing involves an additional reaction step, a 3' nucleotidyl phosphatase activity. The activity is associated with the editing complex and we demonstrate that the editosomal proteins TbMP99 and TbMP100 contribute to the activity. Both polypeptides contain endo-exonuclease-phosphatase domains and we show that gene ablation of either one of the two polypeptides is compensated by the other protein. However, simultaneous knockdown of both genes results in trypanosome cells with reduced 3' nucleotidyl phosphatase and reduced editing activity. The data provide a rationale for the exoUase activity of the editosomal protein TbMP42, which generates nonligatable 3' phosphate termini. Opposing phosphates at the two pre-mRNA cleavage fragments likely function as a roadblock to prevent premature ligation.

SUBMITTER: Niemann M

PROVIDER: S-EPMC2665232 | biostudies-literature | 2009 Apr

REPOSITORIES: biostudies-literature

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Kinetoplastid RNA editing involves a 3' nucleotidyl phosphatase activity.

Niemann Moritz M Kaibel Heike H Schlüter Elke E Weitzel Kerstin K Brecht Michael M Göringer H Ulrich HU

Nucleic acids research 20090203 6

Mitochondrial pre-messenger RNAs (pre-mRNAs) in African trypanosomes require RNA editing in order to mature into functional transcripts. The process involves the addition and/or removal of U nucleotides and is mediated by a high-molecular-mass complex, the editosome. Editosomes catalyze the reaction through an enzyme-driven pathway that includes endo/exoribonuclease, terminal uridylate transferase and RNA ligase activities. Here we show that editing involves an additional reaction step, a 3' nuc ...[more]

PMID: 19190092

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Project description:Adaptation and survival of Trypanosoma brucei requires editing of mitochondrial mRNA by uridylate (U) insertion and deletion. Hundreds of small guide RNAs (gRNAs) direct the mRNA editing at over 3,000 sites. RNA editing is controlled during the life cycle but the regulation of substrate and stage specificity remains unknown. Editing progresses in the 3' to 5' direction along the pre-mRNA in blocks, each targeted by a unique gRNA. A critical editing factor is the mitochondrial RNA binding complex 1 (MRB1) that binds gRNA and transiently interacts with the catalytic RNA editing core complex (RECC). MRB1 is a large and dynamic complex that appears to be comprised of distinct but related subcomplexes (termed here MRBs). MRBs seem to share a 'core' complex of proteins but differ in the composition of the 'variable' proteins. Since some proteins associate transiently the MRBs remain imprecisely defined. MRB1 controls editing by unknown mechanisms, and the functional relevance of the different MRBs is unclear. We previously identified two distinct MRBs, and showed that they carry mRNAs that undergo editing. We proposed that editing takes place in the MRBs because MRBs stably associate with mRNA and gRNA but only transiently interact with RECC, which is RNA free. Here, we identify the first specialized functions in MRBs: 1) 3010-MRB is a major scaffold for RNA editing, and 2) REH2-MRB contains a critical trans-acting RNA helicase (REH2) that affects multiple steps of editing function in 3010-MRB. These trans effects of the REH2 include loading of unedited mRNA and editing in the first block and in subsequent blocks as editing progresses. REH2 binds its own MRB via RNA, and conserved domains in REH2 were critical for REH2 to associate with the RNA and protein components of its MRB. Importantly, REH2 associates with a ~30 kDa RNA-binding protein in a novel ~15S subcomplex in RNA-depleted mitochondria. We use these new results to update our model of MRB function and organization.

Dataset Information

Kinetoplastid RNA editing involves a 3' nucleotidyl phosphatase activity.

Publications

Kinetoplastid RNA editing involves a 3' nucleotidyl phosphatase activity.

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