Uridylate-specific 3′–5′-Exoribonucleases Involved in Uridylate-deletion RNA Editing in Trypanosomatid Mitochondria

Abstract

In kinetoplastid protists, maturation of mitochondrial premRNAs involves the insertion and deletion of uridylates (Us) within coding regions, as specified by mitochondrial DNA-encoded guide RNAs. U-deletion editing involves endonucleolytic cleavage of the pre-mRNA at the editing site followed by U-specific 3-5-exonucleolytic removal of nonbase-paired Us prior to ligation of the two mRNA cleavage fragments. We showed previously that an exonuclease/endonuclease/phosphatase (EEP) motif protein from Leishmania major, designated RNA editing exonuclease 1 (REX1) (Kang, X., Rogers, K., Gao, G., Falick, A. M., Zhou, S.-L., and Simpson, L. (2005) Proc. Natl. Acad. Sci. U. S. A. 102, 1017-1022), exhibits 3-5-exonuclease activity. Two EEP motif proteins have also been identified in the Trypanosoma brucei editing complex. TbREX1 is a homologue of LmREX1, and TbREX2 shows homology to another editing protein in L. major, which lacks the EEP motif (LmREX2*). Here we have expressed the T. brucei EEP motif proteins in insect cells and purified them to homogeneity. We showed that these are U-specific 3-5-exonucleases that are inhibited by base pairing of 3 Us. The recombinant EEP motif alone also showed 3-5 U-specific exonuclease activity, and mutations of the REX EEP motifs greatly reduced exonuclease activity. The absence of enzymatic activity in LmREX2* was confirmed with a purified recombinant protein. We showed that pre-cleaved U-deletion editing could be reconstituted with either TbREX1 or TbREX2 in combination with either RNA ligase, LmREL1, or LmREL2. Down-regulation of TbREX2 expression by conditional RNA interference had little effect on parasite viability or sedimentation of the L-complex, suggesting either that TbREX2 is inactive in vivo or that TbREX1 can compensate for the loss of TbREX2 function in down-regulated cells.The majority of mitochondrial pre-mRNAs in kinetoplastid protists are edited through the insertion and deletion of uridylate residues (Us).3 The editing process is directed by guide RNAs (gRNAs) that initially anneal to the pre-edited mRNA at the first downstream editing site via a region of complementarity termed the "anchor sequence." An editing cycle involves an enzyme cascade (2) initiated by specific RNA editing endonucleases (REN), one of which (REN1) cleaves the mRNA immediately 5Ј to the mRNA/gRNA duplex at U-deletion sites and the other (REN2) cleaves at U-insertion sites (3-5). The upstream gRNA sequence directs the modification of the 3Ј end of the 5Ј mRNA fragment by either the insertion of complementary Us by an RNA editing terminal uridylyltransferase (RET2) or by the removal of nonbase-paired Us by an RNA editing exonuclease (REX). Ligation of the mRNA fragments by an RNA editing ligase (REL) terminates the single site editing cycle. Editing then proceeds to the adjacent upstream site until all sites mediated by the single gRNA are edited resulting in an edited mRNA/gRNA duplex. Multiple overlapping gRNAs are required for extensive editing (pan-editing). Editing is catalyzed by a ribon...

Novel TUTase associates with an editosome-like complex in mitochondria of Trypanosoma brucei

“…degrades synthetic ssRNA ending with Us (Aphasizhev and Simpson 2001). In agreement with the report that both REX1 and REX2 are active in vitro as U-specific exonucleases (Rogers et al 2007), this activity was indeed readily detectable in the MEAT1 complex (Fig. 6C).…”

“…Although the resultant phenotypes and effects on editing in vivo varied considerably, to our knowledge only REL2 (Gao and Simpson 2003;O'Hearn et al 2003) and REX2 (Rogers et al 2007) knockdowns had no effect on T. brucei viability. Puzzled by the apparently intact editing process but inhibited cell growth in MEAT1-depleted cells, we developed a functional RNAi knock-in system to address the questions of possible RNAi off-targeting and whether MEAT1's enzymatic activity is responsible for the observed phenotype.…”

“…Two U-specific exonucleases have been identified in the 20S editosome from T. brucei : REX1, which is essential for parasite viability (Kang et al 2005) and has an ortholog in Leishmania (Aphasizhev et al 2003a), and apparently nonessential REX2 (Rogers et al 2007). Because only the latter was detected in the MEAT1 complex (Table 2), we next tested the TAP-purified fraction for a 39-59 exonuclease activity that selectively FIGURE 6.…”

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

“…7), and Schnaufer et al (2003) observed active precleaved U-deletion from protein associations that contain minimal TbMP42. Furthermore, TbMP99 Worthey et al 2003) and/or TbMP100 Rogers et al 2007) have been reported to be enzymes catalyzing the 39-U-exo activity of U-deletion. Nonetheless, editing could utilize another 39-U-exo activity.…”

“…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). Finally, the ligation steps are catalyzed by the TbMP52/KREL1/band IV and TbMP48/KREL2/band V RNA ligases (Rusche et al 1997(Rusche et al , 2001Schnaufer et al 2001; see also Sabatini and Hajduk 1995;McManus et al 2001), which preferentially seal in U-deletion and Uinsertion, respectively Cruz-Reyes et al 2002;Schnaufer et al 2003; see also Gao and Simpson 2003).…”