Native mRNA editing complexes from Trypanosoma brucei mitochondria.

The aim of this study was to identify multicomponent complexes involved in kinetoplastid mitochondrial mRNA editing. Mitochondrial extracts from Trypanosoma brucei were fractionated on 10-30% glycerol gradients and assayed for RNAs and activities potentially involved in editing, including pre-edited mRNA, guide RNA (gRNA), endonuclease, terminal uridylyltransferase (TUTase), RNA ligase and gRNA-mRNA chimera-forming activities. These experiments suggest that two distinct editing complexes exist. Complex I (19S) consists of gRNA, TUTase, RNA ligase and chimera-forming activity. Complex II (35-40S) is composed of gRNA, preedited mRNA, RNA ligase and chimera-forming activity. These studies provide the first evidence that editing occurs in a multicomponent complex. The possible roles of complex I, complex II and RNA ligase in editing are discussed.     

A 100-kD complex of two RNA-binding proteins from mitochondria of Leishmania tarentolae catalyzes RNA annealing and interacts with several RNA editing components

A stable 100-kD complex from mitochondria of Leishmania tarentolae containing two RNA-binding proteins, Ltp26 and Ltp28, was identified by cross-linking to unpaired 4-thiouridine nucleotides in a partially duplex RNA substrate. The genes were cloned and expressed and the complex was reconstituted from recombinant proteins in the absence of RNA or additional factors. The Ltp26 and Ltp28 proteins are homologs of gBP27 and gBP29 from Crithidia fasciculata and gBP25 and gBP21 from Trypanosoma brucei, respectively. The purified Ltp26/Ltp28 complex, the individual recombinant proteins, and the reconstituted complex are each capable of catalyzing the annealing of complementary RNAs, as was previously shown for gBP21 from T. brucei. A high-molecular-weight RNP complex consisting of the Ltp26/Ltp28 complex and several 55-60-kD proteins together with guide RNA could be purified from mitochondrial extract of L. tarentolae transfected with Ltp28-TAP. This complex also interacted in a less stable manner with the RNA ligase-containing L-complex and with the 3' TUTase. The Ltp26/Ltp28 RNP complex is a candidate for catalyzing the annealing of guide RNA and pre-edited mRNA in the initial step of RNA editing.     

Trypanosome mitochondrial 3' terminal uridylyl transferase (TUTase): the key enzyme in U-insertion/deletion RNA editing

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.     

Trypanosoma brucei guide RNA poly(U) tail formation is stabilized by cognate mRNA

Guide RNAs (gRNAs) are small RNAs that provide specificity for uridine addition and deletion during mRNA editing in trypanosomes. Terminal uridylyl transferase (TUTase) adds uridines to pre-mRNAs during RNA editing and adds a poly(U) tail to the 3' end of gRNAs. The poly(U) tail may stabilize the association of gRNAs with cognate mRNA during editing. Both TUTase and gRNAs associate with two ribonucleoprotein complexes, I (19S) and II (35S to 40S). Complex II is believed to be the fully assembled active editing complex, since it contains pre-edited mRNA and enzymes thought necessary for editing. Purification of TUTase from mitochondrial extracts resulted in the identification of two chromatographically distinct TUTase activities. Stable single-uridine addition to different substrate RNAs is performed by the 19S complex, despite the presence of a uridine-specific 3' exonuclease within this complex. Multiple uridines are added to substrate RNAs by a 10S particle that may be an unstable subunit of complex I lacking the uridine-specific 3' exonuclease. Multiple uridines could be stably added onto gRNAs by complex I when the cognate mRNA is present. We propose a model in which the purine-rich region of the cognate mRNA protects the uridine tail from a uridine exonuclease activity that is present within the complex. To test this model, we have mutated the purine-rich region of the pre-mRNA to abolish base-pairing interaction with the poly(U) tail of the gRNA. This RNA fails to protect the uridine tail of the gRNA from exoribonucleolytic trimming and is consistent with a role for the purine-rich region of the mRNA in gRNA maturation.     

Mechanism of U-Insertion RNA Editing in Trypanosome Mitochondria: Characterization of RET2 Functional Domains by Mutational Analysis

3′-Terminal uridylyl transferases (TUTases) selectively bind uridine 5′-triphosphate (UTP) and catalyze the addition of uridine 5′-monophosphate to the 3′-hydroxyl of RNA substrates in a template-independent manner. RNA editing TUTase 1 and RNA editing TUTase 2 (RET2) play central roles in uridine insertion/deletion RNA editing, which is an essential part of mitochondrial RNA processing in trypanosomes. Although the conserved N-terminal (catalytic) domain and C-terminal (nucleotide base recognition) domain are readily distinguished in all known TUTases, nucleotide specificity, RNA substrate preference, processivity, quaternary structures, and auxiliary domains vary significantly among enzymes of divergent biological functions. RET2 acts as a subunit of the RNA editing core complex to carry out guide-RNA-dependent U-insertion into mitochondrial mRNA. By correlating mutational effects on RET2 activity as recombinant protein and as RNA editing core complex subunit with RNAi-based knock-in phenotypes, we have assessed the UTP and RNA binding sites in RET2. Here we demonstrate functional conservation of key UTP-binding and metal-ion-coordinating residues and identify amino acids involved in RNA substrate recognition. Invariant arginine residues 144 and 435 positioned in the vicinity of the UTP binding site are critical for RET2 activity on single-stranded and double-stranded RNAs, as well as function in vivo. Recognition of a double-stranded RNA, which resembles a guide RNA/mRNA duplex, is further facilitated by multipoint contacts across the RET2-specific middle domain.     

Assembly of mitochondrial ribonucleoprotein complexes involves specific guide RNA (gRNA)-binding proteins and gRNA domains but does not require preedited mRNA.

RNA editing in kinetoplastids probably employs a macromolecular complex, the editosome, that is likely to include the guide RNAs (gRNAs) which specify the edited sequence. Specific ribonucleoprotein (RNP) complexes which form in vitro with gRNAs (H. U. Göringer, D. J. Koslowsky, T. H. Morales, and K. D. Stuart, Proc. Natl. Acad. Sci. USA, in press) are potential editosomes or their precursors. We find that several factors are important for in vitro formation of these RNP complexes and identify specific gRNA-binding proteins present in the complexes. Preedited mRNA promotes the in vitro formation of the four major gRNA-containing RNP complexes under some conditions but is required for the formation of only a subcomponent of one complex. The 5' gRNA sequence encompassing the RYAYA and anchor regions and the 3' gRNA oligo(U) tail are both important in complex formation, since their deletion results in a dramatic decrease of some complexes and the absence of others. UV cross-linking experiments identify several proteins which are in contact with gRNA and preedited mRNA in mitochondrial extracts. Proteins of 25 and 90 kDa are highly specific for gRNAs, and the 90-kDa protein binds specifically to gRNA oligo(U) tails. The gRNA-binding proteins exhibit a differential distribution between the four in vitro-formed complexes. These experiments reveal several proteins potentially involved in RNA editing and indicate that multiple recognition elements in gRNAs are used for complex formation.     

H/ACA guide RNAs, proteins and complexes

H/ACA guide RNAs direct site-specific pseudouridylation of substrate RNAs by forming ribonucleoprotein (RNP) complexes with pseudouridine synthase Cbf5 and three accessory proteins. Recently determined crystal structures of H/ACA protein complexes and a fully assembled H/ACA RNP complex have provided significant insights into the architecture, assembly and mechanism of action of RNA-guided pseudouridine synthase. The binding of guide RNA is directed by its conserved secondary structure and sequence motifs, which enables guide RNA with different sequences to be incorporated into the same protein complex. Accessory proteins and peripheral domains crucially coordinate the position of guide RNA, and possibly regulate the reaction process.     

TbMP57 is a 3' terminal uridylyl transferase (TUTase) of the Trypanosoma brucei editosome

RNA editing produces mature trypanosome mitochondrial mRNAs by uridylate (U) insertion and deletion. In insertion editing, Us are added to the pre-mRNA by a 3' terminal uridylyl transferase (TUTase) activity. We report the identification of a TUTase activity that copurifies with in vitro editing and is catalyzed by the integral editosome protein TbMP57. TbMP57 catalyzes the addition of primarily a single U to single-stranded (ss) RNA and adds the number of Us specified by a guide RNA to insertion editing-like substrates. TbMP57 is distinct from a previously identified TUTase that adds many Us to ssRNA and which we find is neither a stable editosome component nor does it add Us to editing-like substrates. Recombinant TbMP57 specifically interacts with the editosome protein TbMP81, and this interaction enhances the TUTase activity. These results suggest that TbMP57 catalyzes U addition to pre-mRNA during editing.     

Kinetoplastid RNA-editing-associated protein 1 (REAP-1): a novel editing complex protein with repetitive domains.

Kinetoplastid RNA editing consists of the addition or deletion of uridines at specific sites within mitochondrial mRNAs. This unusual RNA processing event is catalyzed by a ribonucleoprotein (RNP) complex that includes editing site-specific endoribonuclease, RNA ligase and terminal uridylnucleotidyl transferase (Tutase) among its essential enzymatic activities. To identify the components of this RNP, monoclonal antibodies were raised against partially purified editing complexes. One antibody reacts with a mitochondrially located 45 kDa polypeptide (p45) which contains a conserved repetitive amino acid domain. p45 co-purifies with RNA ligase and Tutase in a large ( approximately 700 kDa) RNP, and anti-p45 antibody inhibits in vitro RNA editing. Thus, p45 is the first kinetoplastid RNA-editing-associated protein (REAP-1) that has been cloned and identified as a protein component of a functional editing complex.     

The spatial-functional coupling of box C/D and C'/D' RNPs is an evolutionarily conserved feature of the eukaryotic box C/D snoRNP nucleotide modification complex

Box C/D ribonucleoprotein particles guide the 2'-O-ribose methylation of target nucleotides in both archaeal and eukaryotic RNAs. These complexes contain two functional centers, assembled around the C/D and C'/D' motifs in the box C/D RNA. The C/D and C'/D' RNPs of the archaeal snoRNA-like RNP (sRNP) are spatially and functionally coupled. Here, we show that similar coupling also occurs in eukaryotic box C/D snoRNPs. The C/D RNP guided 2'-O-methylation when the C'/D' motif was either mutated or ablated. In contrast, the C'/D' RNP was inactive as an independent complex. Additional experiments demonstrated that the internal C'/D' RNP is spatially coupled to the terminal box C/D complex. Pulldown experiments also indicated that all four core proteins are independently recruited to the box C/D and C'/D' motifs. Therefore, the spatial-functional coupling of box C/D and C'/D' RNPs is an evolutionarily conserved feature of both archaeal and eukaryotic box C/D RNP complexes.     

PPR polyadenylation factor defines mitochondrial mRNA identity and stability in trypanosomes

In Trypanosoma brucei, most mitochondrial mRNAs undergo internal changes by RNA editing and 3′ end modifications. The temporally separated and functionally distinct modifications are manifested by adenylation prior to editing, and by post‐editing extension of a short A‐tail into a long A/U‐heteropolymer. The A‐tail stabilizes partially and fully edited mRNAs, while the A/U‐tail enables mRNA binding to the ribosome. Here, we identify an essential pentatricopeptide repeat‐containing RNA binding protein, kinetoplast polyadenylation factor 3 (KPAF3), and demonstrate its role in protecting pre‐mRNA against degradation by the processome. We show that KPAF3 recruits KPAP1 poly(A) polymerase to the 3′ terminus, thus leading to pre‐mRNA stabilization, or decay depending on the occurrence and extent of editing. In vitro, KPAF3 stimulates KPAP1 activity and inhibits mRNA uridylation by RET1 TUTase. Our findings indicate that KPAF3 selectively directs pre‐mRNA toward adenylation rather than uridylation, which is a default post‐trimming modification characteristic of ribosomal and guide RNAs. As a quality control mechanism, KPAF3 binding ensures that mRNAs entering the editing pathway are adenylated and, therefore, competent for post‐editing A/U‐tailing and translational activation.