Summary In the mitochondria of trypanosomatids, the majority of mRNAs undergo massive U-insertion/deletion editing. Throughout the processes of pre-mRNA polyadenylation, guide RNA (gRNA) uridylylation and annealing to mRNA, and editing reactions, several multi-protein complexes must engage in transient interactions to produce a template for protein synthesis. Here we report the identification of a protein complex essential for gRNA stability. The gRNA binding complex (GRBC) interacts with gRNA processing, editing and polyadenylation machineries and with the mitochondrial edited mRNA stability (MERS1) factor. RNAi knockdown of the core subunits, GRBC1 and 2, led to the elimination of gRNAs thus inhibiting mRNA editing. Inhibition of MERS1 expression selectively abrogated edited mRNAs. Homologous proteins unique to the order of Kinetoplastida, GRBC1 and 2, form a stable 200 kDa particle which directly binds gRNAs. Systematic analysis of RNA-mediated and RNA-independent interactions involving GRBC and MERS1 suggests a unified model for RNA processing in the kinetoplast mitochondria.

SUMMARY The majority of trypanosomal mitochondrial pre-mRNAs undergo massive uridine insertion/deletion editing which creates open reading frames. Although the pre-editing addition of short 3′ A-tails is known to stabilize transcripts during and after the editing, the processing event committing the fully-edited mRNAs to translation remained unknown. Here we show that a heterodimer of pentatricopeptide repeat-containing (PPR) proteins, termed kinetoplast polyadenylation/ uridylation factors (KPAFs) 1 and 2, induces the post-editing addition of A/U-heteropolymers by KPAP1 poly(A) polymerase and RET1 terminal uridyltransferase. Edited transcripts bearing 200–300 nucleotide-long A/U-tails, but not short A-tails, were enriched in translating ribosomal complexes and affinity-purified ribosomal particles. KPAF1 repression led to a selective loss of A/U-tailed mRNAs and concomitant inhibition of protein synthesis. These results establish A/U extensions as the defining cis-elements of translation-competent mRNAs. Furthermore, we demonstrate that A/U-tailed mRNA preferentially interacts with the small ribosomal subunit, whereas edited substrates and complexes bind to the large subunit.

Enzymes embedded into the RNA editing core complex (RECC) catalyze the U-insertion/deletion editing cascade to generate open reading frames in trypanosomal mitochondrial mRNAs. The sequential reactions of mRNA cleavage, U-addition or removal, and ligation are directed by guide RNAs (gRNAs). We combined proteomic, genetic, and functional studies with sequencing of total and complex-bound RNAs to define a protein particle responsible for the recognition of gRNAs and pre-mRNA substrates, editing intermediates, and products. This approximately 23-polypeptide tripartite assembly, termed the RNA editing substrate binding complex (RESC), also functions as the interface between mRNA editing, polyadenylation, and translation. Furthermore, we found that gRNAs represent only a subset of small mitochondrial RNAs, and yet an inexplicably high fraction of them possess 3= U-tails, which correlates with gRNA's enrichment in the RESC. Although both gRNAs and mRNAs are associated with the RESC, their metabolic fates are distinct: gRNAs are degraded in an editing-dependent process, whereas edited mRNAs undergo 3= adenylation/uridylation prior to translation. Our results demonstrate that the well-characterized editing core complex (RECC) and the RNA binding particle defined in this study (RESC) typify enzymatic and substrate binding macromolecular constituents, respectively, of the ϳ40S RNA editing holoenzyme, the editosome.

Expression of the mitochondrial genome in protozoan parasite Trypanosoma brucei is controlled post‐transcriptionally and requires extensive U‐insertion/deletion mRNA editing. In mitochondrial extracts, 3′ adenylation reportedly influences degradation kinetics of synthetic edited and pre‐edited mRNAs. We have identified and characterized a mitochondrial poly(A) polymerase, termed KPAP1, and determined major polypeptides in the polyadenylation complex. Inhibition of KPAP1 expression abrogates short and long A‐tails typically found in mitochondrial mRNAs, and decreases the abundance of never‐edited and edited transcripts. Pre‐edited mRNAs are not destabilized by the lack of 3′ adenylation, whereas short A‐tails are required and sufficient to maintain the steady‐state levels of partially edited, fully edited, and never‐edited mRNAs. The editing directed by a single guide RNA is sufficient to impose a requirement for the short A‐tail in edited molecules. Upon completion of the editing process, the short A‐tails are extended as (A/U) heteropolymers into structures previously thought to be long poly(A) tails. These data provide the first direct evidence of functional interactions between 3′ processing and editing of mitochondrial mRNAs in trypanosomes.

A multiprotein, high molecular weight complex active in both U-insertion and U-deletion as judged by a pre-cleaved RNA editing assay was isolated from mitochondrial extracts of Leishmania tarentolae by the tandem af®nity puri®cation (TAP) procedure, using three different TAP-tagged proteins of the complex. This editing-or E-complex consists of at least three protein-containing components interacting via RNA: the RNA ligase-containing L-complex, a 3¢ TUTase (terminal uridylyltransferase) and two RNA-binding proteins, Ltp26 and Ltp28. Thirteen approximately stoichiometric components were identi®ed by mass spectrometric analysis of the core L-complex: two RNA ligases; homologs of the four Trypanosoma brucei editing proteins; and seven novel polypeptides, among which were two with RNase III, one with an AP endo/exonuclease and one with nucleotidyltransferase motifs. Three proteins have no similarities beyond kinetoplastids. Keywords: editosome/RNA editing/TAP/TUTase Introduction Uridine insertion/deletion RNA editing is a post-transcriptional RNA modi®cation phenomenon that occurs in the mitochondrion of kinetoplastid protists . The mechanism involves the initial hybridization to an mRNA of a complementary guide RNA (gRNA) which guides a speci®c endonuclease cleavage at the ®rst editing site . This is followed by either deletion of the unpaired uridines from the cleavage fragment or the 3¢ addition to the mRNA 5¢ cleavage fragment, hybridization of the added Us to the guiding nucleotides in the gRNA, and religation of the two mRNA cleavage fragments. Each gRNA speci®es the 3¢ to 5¢ editing of a small number of sites and, in the case of a multiple gRNA-mediated editing domain, creates the anchor sequence for hybridization of the adjacent upstream gRNA, thus producing an overall 3¢ to 5¢ progression of editing. A minimal non-progressive editing activity at one or two sites has been demonstrated in vitro using crude or partially puri®ed mitochondrial extract, and the reaction was shown to involve high molecular weight RNP complexes (Byrne et al., 1996;Cruz-Reyes and Sollner-Webb, 1996;Kable et al., 1996;Seiwert et al., 1996). The mechanism described above was proposed >12 years ago , and was veri®ed experimentally in 1996 for both Trypanosoma brucei and Leishmania tarentolae (Byrne et al., 1996;Cruz-Reyes and Sollner-Webb, 1996;Seiwert et al., 1996). However, progress in the identi®cation of speci®c proteins involved in editing has been hampered by their low abundance and by the low ef®ciency of the in vitro editing assays. A seven polypeptide complex from T.brucei mitochondria that supported in vitro insertion and deletion editing was isolated by two chromatographic steps and was proposed to represent a core editing complex (Rusche et al., 1997). An~20 polypeptide complex with similar activities was isolated in another laboratory by a similar fractionation (Panigrahi et al., 2001a,b).The genes for several of the major components of these complexes have been identi®ed, but only a few proteins so far have been ascribed ...

A 3' terminal RNA uridylyltransferase was purified from mitochondria of Leishmania tarentolae and the gene cloned and expressed from this species and from Trypanosoma brucei. The enzyme is specific for 3' U-addition in the presence of Mg(2+). TUTase is present in vivo in at least two stable configurations: one contains a approximately 500 kDa TUTase oligomer and the other a approximately 700 kDa TUTase complex. Anti-TUTase antiserum specifically coprecipitates a small portion of the p45 and p50 RNA ligases and approximately 40% of the guide RNAs. Inhibition of TUTase expression in procyclic T. brucei by RNAi downregulates RNA editing and appears to affect parasite viability.

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...

RNA uridylylation is critical for the expression of the mitochondrial genome in trypanosomes. Short U tails are added to guide RNAs and rRNAs, while long A/U heteropolymers mark 3 ends of most mRNAs. Three divergent mitochondrial terminal uridylyl transferases (TUTases) are known: RET1 catalyzes guide RNA (gRNA) uridylylation, RET2 executes U insertion mRNA editing, and MEAT1 associates with the editosomelike complex. However, the activities responsible for 3 uridylylation of rRNAs and mRNAs, and the roles of these modifications, are unclear. To dissect the functions of mitochondrial TUTases, we investigated the effects of their repression and overexpression on abundance, processing, 3-end status, and in vivo stability of major mitochondrially encoded RNA classes. We show that RET1 adds U tails to gRNAs, rRNAs, and select mRNAs and contributes U's into A/U heteropolymers. Furthermore, RET1's TUTase activity is required for the nucleolytic processing of gRNA, rRNA, and mRNA precursors. The U tail's presence does not affect the stability of gRNAs and rRNAs, while transcript-specific uridylylation triggers 3 to 5 mRNA decay. We propose that the minicircle-encoded antisense transcripts, which are stabilized by RET1-catalyzed uridylylation, may direct a nucleolytic cleavage of multicistronic precursors.