Roles for ligases in the RNA editing complex of Trypanosoma brucei: band IV is needed for U-deletion and RNA repair

Trypanosome RNA editing utilizes a seven polypeptide complex that includes two RNA ligases, band IV and band V. We now find that band IV protein contributes to the structural stability of the editing complex, so its lethal genetic knock-out could reflect structural or catalytic requirements. To assess the catalytic role in editing, we generated cell lines which inducibly replaced band IV protein with an enzymatically inactive but structurally conserved version. This induction halts cell growth, showing that catalytic activity is essential. These induced cells have impaired in vivo editing, specifically of RNAs requiring uridylate (U) deletion; unligated RNAs cleaved at U-deletion sites accumulated. Additionally, mitochondrial extracts of cells with reduced band IV activity were deficient in catalyzing U-deletion, specifically at its ligation step, but were not deficient in U-insertion. Thus band IV ligase is needed to seal RNAs in U-deletion. U-insertion does not appear to require band IV, so it might use the other ligase of the editing complex. Furthermore, band IV ligase was also found to serve an RNA repair function, both in vitro and in vivo.     

Distinct functions of two RNA ligases in active Trypanosoma brucei RNA editing complexes

Trypanosome RNA editing is a unique U insertion and U deletion process that involves cycles of pre-mRNA cleavage, terminal U addition or U removal, and religation. This editing can occur at massive levels and is directed by base pairing of trans-acting guide RNAs. Both U insertion and U deletion cycles are catalyzed by a single protein complex that contains only seven major proteins, band I through band VII. However, little is known about their catalytic functions, except that band IV and band V are RNA ligases and genetic analysis indicates that the former is important in U deletion. Here we establish biochemical approaches to distinguish the individual roles of these ligases, based on their distinctive ATP and pyrophosphate utilization. These in vitro analyses revealed that both ligases serve in RNA editing. Band V is the RNA editing ligase that functions very selectively to seal in U insertion (IREL), while band IV is the RNA editing ligase needed to seal in U deletion (DREL). In combination with our earlier findings about the cleavage and the U-addition/U-removal steps of U deletion and U insertion, these results show that all three steps of these editing pathways exhibit major differences and suggest that the editing complex could have physically separate regions for U deletion and U insertion.     

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.     

Purification of a functional enzymatic editing complex from Trypanosoma brucei mitochondria.

Kinetoplastid mitochondrial RNA editing, the insertion and deletion of U residues, is catalyzed by sequential cleavage, U addition or removal, and ligation reactions and is directed by complementary guide RNAs. We have purified a approximately 20S enzymatic complex from Trypanosoma brucei mitochondria that catalyzes a complete editing reaction in vitro. This complex possesses all four activities predicted to catalyze RNA editing: gRNA-directed endonuclease, terminal uridylyl transferase, 3' U-specific exonuclease, and RNA ligase. However, it does not contain other putative editing complex components: gRNA-independent endonuclease, RNA helicase, endogenous gRNAs or pre-mRNAs, or a 25 kDa gRNA-binding protein. The complex is composed of eight major polypeptides, three of which represent RNA ligase. These findings identify polypeptides representing catalytic editing factors, reveal the nature of this approximately 20S editing complex, and suggest a new model of editosome assembly.     

Association of two novel proteins, TbMP52 and TbMP48, with the Trypanosoma brucei RNA editing complex

RNA editing in kinetoplastid mitochondria inserts and deletes uridylates at multiple sites in pre-mRNAs as directed by guide RNAs. This occurs by a series of steps that are catalyzed by endoribonuclease, 3'-terminal uridylyl transferase, 3'-exouridylylase, and RNA ligase activities. A multiprotein complex that contains these activities and catalyzes deletion editing in vitro was enriched from Trypanosoma brucei mitochondria by sequential ion-exchange and gel filtration chromatography, followed by glycerol gradient sedimentation. The complex size is approximately 1,600 kDa, and the purified fraction contains 20 major polypeptides. A monoclonal antibody that was generated against the enriched complex reacts with an approximately 49-kDa protein and specifically immunoprecipitates in vitro deletion RNA editing activity. The protein recognized by the antibody was identified by mass spectrometry, and the corresponding gene, designated TbMP52, was cloned. Recombinant TbMP52 reacts with the monoclonal antibody. Another novel protein, TbMP48, which is similar to TbMP52, and its gene were also identified in the enriched complex. These results suggest that TbMP52 and TbMP48 are components of the RNA editing complex.     

The gene encoding the biotin-apoprotein ligase of Saccharomyces cerevisiae

Abstract We report the isolation, genomic mapping, and DNA sequence of the BPL1 gene encoding the biotin-apoprotein ligase of Saccharomyces cerevisiae . The gene was isolated by complementation of an Escherichia coli birA (biotin-apoprotein ligase) mutant indicating that the expressed yeast protein modified the essential biotinated protein of the bacterial host. The BPL1 gene encodes a protein of 690 residues ( M _r 76.4 kDa) with strong sequence similarites to the E. coli and human biotin-apoprotein ligases. BPL1 was mapped to chromosome IV, is allelic to the previously described ACC2 gene, and encodes the major (if not the only) biotin-apoprotein ligase activity of S. cerevisiae .     

Trypanosoma brucei mitochondrial guide RNA-mRNA chimera-forming activity cofractionates with an editing-domain-specific endonuclease and RNA ligase and is mimicked by heterologous nuclease and RNA ligase.

RNA editing in trypanosomes has been proposed to occur through transesterification or endonuclease cleavage and RNA ligation reactions. Both models involve a chimeric intermediate in which a guide RNA (gRNA) is joined through its 3' oligo(U) tail to an editing site of the corresponding mRNA. Velocity centrifugation of Trypanosoma brucei mitochondrial extracts had been reported to completely separate the gRNA-mRNA chimera-forming activity from endonuclease activity (V. W. Pollard, M. E. Harris, and S. L. Hajduk, EMBO J. 11:4429-4438, 1992), appearing to rule out the endonuclease-RNA ligase mechanism. However, we show that an editing-domain-specific endonuclease activity does cosediment with the chimera-forming activity, as does the RNA ligase activity, but detection of the specific endonuclease requires reducing assay conditions. This report further demonstrates that the T. brucei chimera-forming activity is mimicked by mung bean nuclease and T4 RNA ligase. Using cytochrome b (CYb) preedited mRNA and a model CYb gRNA, we found that these heterologous enzymes specifically generate CYb gRNA-mRNA chimeras analogous to those formed in the mitochondrial extract. These combined results provide support for the endonuclease-RNA ligase mechanism of chimera formation.     

The structure of the C-terminal domain of the largest editosome interaction protein and its role in promoting RNA binding by RNA-editing ligase L2

Trypanosomatids, such as the sleeping sickness parasite Trypanosoma brucei, contain a ～ 20S RNA-editing complex, also called the editosome, which is required for U-insertion/deletion editing of mitochondrial mRNAs. The editosome contains a core of 12 proteins including the large interaction protein A1, the small interaction protein A6, and the editing RNA ligase L2. Using biochemical and structural data, we identified distinct domains of T. brucei A1 which specifically recognize A6 and L2. We provide evidence that an N-terminal domain of A1 interacts with the C-terminal domain of L2. The C-terminal domain of A1 appears to be required for the interaction with A6 and also plays a key role in RNA binding by the RNA-editing ligase L2 in trans. Three crystal structures of the C-terminal domain of A1 have been elucidated, each in complex with a nanobody as a crystallization chaperone. These structures permitted the identification of putative dsRNA recognition sites. Mutational analysis of conserved residues of the C-terminal domain identified Arg703, Arg731 and Arg734 as key requirements for RNA binding. The data show that the editing RNA ligase activity is modulated by a novel mechanism, i.e. by the trans-acting RNA binding C-terminal domain of A1.     

TbMP81 is required for RNA editing in Trypanosoma brucei

Most mitochondrial mRNAs are edited in Trypano soma brucei by a series of steps that are catalyzed by a multienzyme complex that is in its initial stages of characterization. RNA interference (RNAi)-mediated repression of the expression of TbMP81, a zinc finger protein component of the complex, inhibited growth of bloodstream and insect forms, and blocked in vivo RNA editing. This repression preferentially inhibited insertion editing compared with deletion editing in vitro. It resulted in reduced specific endoribonucleolytic cleavage and a greater reduction of U addition and associated RNA ligation activities than U removal and associated RNA ligation activities. The repressed cells retained 20S editing complexes with several demonstrable proteins and adenylatable TbMP52 RNA ligase, but adenlyatable TbMP48 was not detected. Elimination of TbMP48 by RNAi repression did not inhibit cell growth or in vivo editing in either bloodstream or procyclic forms. These results indicate that TbMP81 is required for RNA editing and suggest that the editing complex is functionally partitioned.     

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.     

Structure and mechanism of RNA ligase

T4 RNA ligase 2 (Rnl2) exemplifies an RNA ligase family that includes the RNA editing ligases (RELs) of Trypanosoma and Leishmania. The Rnl2/REL enzymes are defined by essential signature residues and a unique C-terminal domain, which we show is essential for sealing of 3'-OH and 5'-PO4 RNA ends by Rnl2, but not for ligase adenylation or phosphodiester bond formation at a preadenylated AppRNA end. The N-terminal segment Rnl2(1-249) of the 334 aa Rnl2 protein comprises an autonomous adenylyltransferase/AppRNA ligase domain. We report the 1.9 A crystal structure of the ligase domain with AMP bound at the active site, which reveals a shared fold, catalytic mechanism, and evolutionary history for RNA ligases, DNA ligases, and mRNA capping enzymes.     

In Trypanosoma brucei RNA editing, TbMP18 (band VII) is critical for editosome integrity and for both insertional and deletional cleavages

In trypanosome RNA editing, uridylate (U) residues are inserted and deleted at numerous sites within mitochondrial pre-mRNAs by an approximately 20S protein complex that catalyzes cycles of cleavage, U addition/U removal, and ligation. We used RNA interference to deplete TbMP18 (band VII), the last unexamined major protein of our purified editing complex, showing it is essential. TbMP18 is critical for the U-deletional and U-insertional cleavages and for integrity of the approximately 20S editing complex, whose other major components, TbMP99, TbMP81, TbMP63, TbMP52, TbMP48, TbMP42 (bands I through VI), and TbMP57, instead sediment as approximately 10S associations. Additionally, TbMP18 augments editing substrate recognition by the TbMP57 terminal U transferase, possibly aiding the recognition component, TbMP81. The other editing activities and their coordination in precleaved editing remain active in the absence of TbMP18. These data are reminiscent of the data on editing subcomplexes reported by A. Schnaufer et al. (Mol. Cell 12:307-319, 2003) and suggest that these subcomplexes are held together in the approximately 20S complex by TbMP18, as was proposed previously. Our data additionally imply that the proteins are less long-lived in these subcomplexes than they are when held in the complete editing complex. The editing endonucleolytic cleavages being lost when the editing complex becomes fragmented, as upon TbMP18 depletion, should be advantageous to the trypanosome, minimizing broken mRNAs.     

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

Inhibition of RNA editing by down-regulation of expression of the mitochondrial RNA editing TUTase 1 by RNA interference had profound effects on kinetoplast biogenesis in Trypanosoma brucei procyclic cells. De novo synthesis of the apocytochrome b and cytochrome oxidase subunit I proteins was no longer detectable after 3 days of RNAi. The effect on protein synthesis correlated with a decline in the levels of the assembled mitochondrial respiratory complexes III and IV, and also cyanide-sensitive oxygen uptake. The steady-state levels of nuclear-encoded subunits of complexes III and IV were also significantly decreased. Because the levels of the corresponding mRNAs were not affected, the observed effect was likely due to an increased turnover of these imported mitochondrial proteins. This induced protein degradation was selective for components of complexes III and IV, because little effect was observed on components of the F(1).F(0)-ATPase complex and on several other mitochondrial proteins.