A tale of two TUTases

Abstract

The insertion and deletion of U residues at specific sites in mRNAs in trypanosome mitochondria is thought to involve 3 terminal uridylyl transferase (TUTase) activity. TUTase activity is also required to create the nonencoded 3 oligo[U] tails of the transacting guide RNAs (gRNAs). We have described two TUTases, RET1 (RNA editing TUTase 1) and RET2 (RNA editing TUTase 2) as components of different editing complexes. Tandem affinity purificationtagged Trypanosoma brucei RET2 (TbRET2) was expressed and localized to the cytosol in Leishmania tarentolae cells by removing the mitochondrial signal sequence. Double-affinity isolation yielded tagged TbRET2, together with a few additional proteins. This material exhibits a U-specific transferase activity in which a single U is added to the 3 end of a single-stranded RNA, thereby confirming that RET2 is a 3 TUTase. We also found that RNA interference of RET2 expression in T. brucei inhibits in vitro Uinsertion editing and has no effect on the length of the 3 oligo[U] tails of the gRNAs, whereas down-regulation of RET1 has a minor effect on in vitro U-insertion editing, but produces a decrease in the average length of the oligo[U] tails. This finding suggests that RET2 is responsible for U-insertions at editing sites and RET1 is involved in gRNA 3 end maturation, which is essential for creating functional gRNAs. From these results we have functionally relabeled the previously described TUT-II complex containing RET1 as the guide RNA processing complex.U -insertion͞deletion RNA editing in trypanosome mitochondria involves the annealing of a guide RNA (gRNA) to an mRNA, endonuclease cleavage at a non-base-paired editing site adjacent to the RNA duplex, addition or deletion of Us to or from the 3Ј end of the 5Ј cleavage fragment, and religation of the two mRNA fragments (1-4). Each gRNA mediates the editing of 1-5 sites in a 3Ј to 5Ј polarity, and multiple overlapping gRNAs mediate the editing of an entire domain, also in a 3Ј to 5Ј polarity (5).We previously isolated the RNA editing terminal uridylyl transferase (TUTase) 1 (RET1) and showed it to be present both as a free tetramer and as a component of a high molecular weight complex (TUT II) (6). This complex migrates on a native gel at Ϸ700 kDa and interacts in an RNase-sensitive manner with the larger ligase-containing complex (L-complex) (7). Downregulation of RET1 expression in Trypanosoma brucei by conditional RNA interference (RNAi) caused a decrease in the steady-state abundance of edited mRNAs without any effect on transcription (6). The core L-complex and associated proteins, capable of both U-insertion and U-deletion editing activity in vitro, was isolated from Leishmania tarentolae mitochondrial extract by tandem affinity purification (TAP) (8), followed by glycerol gradient sedimentation (7). The L-complex contains two adenylatable RNA ligases, REL1 and REL2, three related zinc finger-containing proteins, two proteins with RNase III motifs, two proteins with an AP endonuclease exonuclease phosphatase motif, a se...

The Effect of RNA Interference Down-regulation of RNA Editing 3′-Terminal Uridylyl Transferase (TUTase) 1 on Mitochondrial de Novo Protein Synthesis and Stability of Respiratory Complexes in Trypanosoma brucei

“…Conditional down-regulation of RET1 expression in procyclic T. brucei by RNAi was shown previously to be lethal (16). The role of RET1 was shown to involve the addition of untemplated U's to the 3Ј ends of guide RNAs, thereby creating a 3Ј oligo-U tail, which is apparently required for editing (37). In this paper, we have presented evidence that the effect on viability is due, at least in part, to an effect on the assembly or stability of the cytochrome bc 1 complex (respiratory complex III) and the cytochrome c oxidase complex (respiratory complex IV), both of which contain some subunits translated from edited mRNAs.…”

“…Down-regulation of expression of RET1 by conditional RNAi affected editing of maxicircle-derived mRNAs (16) by affecting the length of the guide RNA 3Ј oligo-U tails (37). It follows that RET1 RNAi should also affect synthesis of proteins such as Cyb, where the mRNAs require editing to be translatable.…”

“…These RNA molecules have 3Ј oligo-U tails (15), which are added posttranscriptionally by the RET1 TUTase (16,17). Down-regulation of RET1 expression by RNA interference (RNAi) results in a decrease in the steady-state abundance of edited transcripts (16) because of an effect on the 3Ј oligo-U tail of the guide RNAs (37).…”

Functional complementation of Trypanosoma brucei RNA in vitro editing with recombinant RNA ligase

“…The RNAediting terminal uridylyl transferase 1 (TUTase 1) (RET1) complex contains the RET1 tetramer and one or more unidentified components (10). RET1 has been shown to be involved in the addition of nonencoded Us to the 3Ј ends of guide RNAs (11). The RNA ligase-containing L-complex was initially detected as an ␣-32 P-ATP-labeled particle sedimenting Ϸ20S in glycerol gradients and migrating as a single band with an approximate size of 1,200-2,000 kDa in native gels (12,13).…”

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

“…L-complex proteins that have been expressed in active form and characterized include the RNA editing ligases 1 and 2 (REL1 and REL2) (4)(5)(6)(7), the RNA editing 3Ј-5Ј U-specific exonucleases (REX1 and REX2) (8)(9)(10), and the RET2Ј3Ј TUTase (11)(12)(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).…”