An essential RNase III insertion editing endonuclease in Trypanosoma brucei

Abstract

RNA editing adds and deletes uridine nucleotides in many preedited mRNAs to create translatable mRNAs in the mitochondria of the parasite Trypanosoma brucei. Kinetoplastid RNA editing protein B3 (KREPB3, formerly TbMP61) is part of the multiprotein complex that catalyzes editing in T. brucei and contains an RNase III motif that suggests nuclease function. Repression of KREPB3 expression, either by RNA interference in procyclic forms (PFs) or by conditional inactivation of an ectopic KREPB3 allele in bloodstream forms (BFs) that lack both endogenous alleles, strongly inhibited growth and in vivo editing in PFs and completely blocked them in BFs. KREPB3 repression inhibited cleavage of insertion editing substrates but not deletion editing substrates in vitro, whereas the terminal uridylyl transferase, U-specific exoribonuclease, and ligase activities of editing were unaffected, and Ϸ20S editosomes were retained. Expression of KREPB3 alleles with single amino acid mutations in the RNase III motif had similar consequences. These data indicate that KREPB3 is an RNA editing endonuclease that is specific for insertion sites and is accordingly renamed KREN2 (kinetoplastid RNA editing endonuclease 2).any mitochondrial mRNAs in trypanosomatids are edited by the insertion and deletion of uridine nucleotides (Us) as directed by guide RNAs (gRNAs) (1). The key steps of RNA editing are the coordinated endonucleolytic cleavage of preedited mRNA (pre-mRNA); terminal uridylyl transferase or U-specific exoribonuclease-mediated insertion or deletion of Us, respectively; and ligation. These enzymatic activities are in an Ϸ20S multiprotein complex, the editosome, which contains at least 20 proteins (2). The proteins responsible for U insertion (KRET2), deletion (KREX1), and ligation (KREL1 and KREL2) have been identified (3-7), but the protein (or proteins) responsible for endonucleolytic cleavage remain unidentified.The RNA editing endonuclease activity by editosomecontaining extracts cleaves synthetic ATPase subunit 6 (A6) or cytochrome b (CYb) pre-mRNAs in vitro in a gRNA-directed manner (8-10). The cleavage occurs 5Ј to the pre-mRNA:gRNA anchor duplex, leaving a 3Ј hydroxyl and 5Ј phosphate at the cleavage site. Cleavage of insertion sites is inhibited by adenosine nucleotides, whereas cleavage of deletion sites is stimulated by these nucleotides (11), suggesting that there may be distinct endonucleases. Several editosome proteins identified by mass spectrometry have motifs suggestive of nuclease function and may be RNA editing endonucleases (2).Kinetoplastid RNA editing protein (KREP) B1, KREPB2, and KREPB3 have RNase III, U1-like Zn 2ϩ finger, and dsRNAbinding motifs indicative of endonucleases. RNase III and dsRNA-binding motifs are typical in bacterial and eukaryotic endonucleases, and U1-like Zn 2ϩ finger motifs imply RNA and protein interactions in a complex (12). The RNase III motifs of KREPB1, KREPB2, and KREPB3 all conserve the amino acids that are critical to endonuclease function (13). A report indicating that KREPA3 ...

The Architecture of Trypanosoma brucei editosomes

“…S1 and Table S1). However, they are compositionally and functionally distinct in that each contains a different single RNase III endonuclease with a uniquely associated specific partner protein, and a different editing site cleavage specificity (22)(23)(24)(25)(26)29) (SI Appendix, Fig. S1 and Table S1).…”

“…Editing occurs by rounds of coordinated catalytic steps that require a number of different enzymes: cleavage of the mRNA substrate by endonucleases, addition of Us by 3′ terminal uridylytransferase (TUTase) or removal of Us by U-specific 3′ exonuclease (exoUase) at insertion or deletion editing sites, respectively, and rejoining of mRNA fragments by RNA ligases. The enzymes that catalyze RNA editing in T. brucei are in ∼20S editosome complexes that also contain proteins with no known catalytic functions (16,(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28).…”

Reconstitution of full-round uridine-deletion RNA editing with three recombinant proteins

“…It was suggested that MP90 is a U-deletion site-specific endonuclease (which was labeled KREN1 for kinetoplast RNA editing nuclease), and MP61 is a U-insertion site endonuclease (which was labeled KREN2). However, recombinant proteins were enzymatically inactive (18,19).In this study, we provide both indirect and direct evidence for a role of the MP90 L-complex protein in the initial cleavage at preedited mRNA U-deletion editing sites, and we show that a full-round cycle of in vitro U-deletion editing can be reconstituted with just three recombinant proteins: REX1, REL1, and MP90. MP90 has been functionally renamed as RNA editing nuclease 1 (REN1).…”

“…Carnes et al (19) provided indirect evidence for a role of the MP61 or TbREN2 L-complex protein in cleavage of preedited mRNA at U-insertion sites, but the roles of the MP67 and LC8 (MP44) RNase III L-complex proteins are still uncertain, especially in view of the lack of any phenotype of MP67-downregulated cells. And, of course, the precise topology and stoichiometry of the various L-complex proteins, including the REL1 and REL2 subcomplexes and the internal core L-complex proteins and their interaction with mRNAs and gRNAs (29), remain open questions that are critical for understanding this type of RNA editing in detail (30).…”

“…It was suggested that MP90 is a U-deletion site-specific endonuclease (which was labeled KREN1 for kinetoplast RNA editing nuclease), and MP61 is a U-insertion site endonuclease (which was labeled KREN2). However, recombinant proteins were enzymatically inactive (18,19).…”