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

Abstract

Uridine (U)-insertion͞deletion RNA editing in trypanosome mitochondria involves an initial cleavage of the preedited mRNA at specific sites determined by the annealing of partially complementary guide RNAs. An involvement of two RNase III-containing core editing complex (L-complex) proteins, MP90 (KREPB1) and MP61 (KREPB3) in, respectively, U-deletion and U-insertion editing, has been suggested, but these putative enzymes have not been characterized or expressed in active form. Recombinant MP90 proteins from Trypanosoma brucei and Leishmania major were expressed in insect cells and cytosol of Leishmania tarentolae, respectively. These proteins were active in specifically cleaving a model Udeletion site and not a U-insertion site. Deletion or mutation of the RNase III motif abolished this activity. Full-round guide RNA (gRNA)-mediated in vitro U-deletion editing was reconstituted by a mixture of recombinant MP90 and recombinant RNA editing exonuclease I from L. major, and recombinant RNA editing RNA ligase 1 from L. tarentolae. MP90 is designated REN1, for RNAediting nuclease 1.mitochondria involves the participation of at least three RNAlinked multiprotein complexes, the Ϸ19-polypeptide ligasecontaining core editing complex (L-complex), the mitochondrial RNA-binding protein (MRP) complex, and the RNA editing 3Ј terminal uridylyl transferase (TUTase) (RET)1 complex(es) (1-3). L-complex proteins that have been expressed in active form and characterized include the RNA editing ligases 1 and 2 (REL1 and REL2) (4-7), the RNA editing 3Ј-5Ј U-specific exonucleases (REX1 and REX2) (8-10), and the RET2Ј3Ј TUTase (11-13). Characterization of the protein components of the L-complex led to several nuclease candidates: LC6A, or MP61, MP90, and MP67 contain RNase III motifs, and LC8, or MP44, has a highly diverged RNase III motif (11,(14)(15)(16). Although TbMP90 and TbMP61 were stated in recent reviews (1, 16) to also contain double-strand RNA-binding motifs, such motifs are not readily identifiable (L.S., unpublished data). In addition, the MP42 L-complex protein, which has only zinc finger (ZnF C2H2) and single-strand RNA-binding (SSB) motifs, nevertheless exhibited both exonuclease and endonuclease activities, but the activities identified did not show the required specificity, and the role of this protein remains an open question (17). Trotter et al. (18) and Carnes et al. (19) recently provided in vivo evidence for a specific role in cleavage at mRNA U-deletion and U-insertion editing sites for, respectively, the essential TbMP90 and TbMP61 RNase III motif-containing 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 sh...

Trypanosoma brucei RNA editing protein TbMP42 (band VI) is crucial for the endonucleolytic cleavages but not the subsequent steps of U-deletion and U-insertion

“…4A), indicating that TbMP42 is needed for any cleavage by the TbMP90 endonuclease in trypanosome extracts. (However, studies using recombinant proteins have shown that rTbMP90 cleaves an editing site in the presence of rTbMP63, without rTbMP42 [Kang et al 2006], suggesting that in vivo TbMP42 likely serves to retain the presence of the active TbMP90.) In contrast to its effects on the U-deletional endonuclease, depletion of TbMP42 (at 3 d of RNAi induction) does not affect the basic cleavage activity of the U-insertional endonuclease (Fig.…”

“…In some references, the initial ''K'' is omitted, and, additionally, TbMP52 has also been referred to as DREL (Cruz-Reyes et al 2002) and TbMP48 as IREL (Cruz-Reyes et al 2002). Purification of editing complexes from L. tarentolae yields many homologous proteins, initially named LmLC# ) then shortened to LC# (Kang et al 2006). For uniformity with the bulk of the editing literature, after their initial mention, we use the TbMP# designation.…”

“…We also provide potential explanations for the previous, differing conclusions. Rusche et al (1997) (see Law et al 2007 for additional verification of the reproducibility of this purification), the ''TbMP'' designation (representing T. brucei mitochondrial protein followed by the molecular weight of its cytoplasmic precursor) from Panigrahi et al (2001a), and a more recent kinetoplastid RNA editing protein nomenclature (''KREPX#,'' representing kinetoplastid RNA editing protein followed by a letter for the class of protein and a number indicating approximate ascending size within the group), that is being replaced with a KREØ# functional nomenclature (KRE as above, followed by a letter for the biological role of protein and a number indicating approximate ascending size within the group) from the Stuart laboratory (see references in the text; for reviews, see Worthey et al 2003;Simpson et al 2004;Stuart et al 2005;Kang et al 2006;Panigrahi et al 2006). The proteins shown in brackets appear quite unabundant and/or are only obtained from some purification procedures (e.g., Panigrahi et al 2006).…”

“…1B). The U-deletional and U-insertional cleavage steps are catalyzed by TbMP90/KREN1 and TbMP61/ KREN2, respectively Trotter et al 2005;Kang et al 2006) (or by TbMP67/KREPB2/KREN3 for the U-insertion that uses a cis-located gRNA; Carnes et al 2008), although it has additionally been inferred that TbMP42/KREPA3/band VI may catalyze editing endonucleolytic cleavage (Brecht et al 2005). The U-removal step is catalyzed by TbMP100/KREX1 (Worthey et al 2003;Kang et al 2005) and TbMP99/KREPC2/KREX2/band I Worthey et al 2003;Rogers et al 2007) and/or possibly TbMP42/KREPA3/band VI (Brecht et al 2005); the U-addition step is catalyzed by the TbMP57/KRET2 TUTase (Aphasizhev et al 2003c;Ernst et al 2003).…”

Guide RNA biogenesis involves a novel RNase III family endoribonuclease inTrypanosoma brucei

“…Several mRNAs are edited at over a hundred sites, and at each site, editing starts by a nuclease cleavage directed by a complementary gRNA (Rusche et al 1997;Stuart et al 2005;Li et al 2009;Simpson et al 2010). There are three accepted specialized editing endonucleases, REN1, REN2, and REN3 Trotter et al 2005;Kang et al 2006;Simpson et al 2010), although only REN1 has been produced in an active recombinant form (Kang et al 2006). RENs have a conserved RNase III domain and two domains of unknown function (Worthey et al 2003).…”