Editing Complexes Research Articles

Post-transfer editing mechanism of a D-aminoacyl-tRNA deacylase-like domain in threonyl-tRNA synthetase from archaea

To ensure a high fidelity during translation, threonyl-tRNA synthetases (ThrRSs) harbor an editing domain that removes noncognate L-serine attached to tRNAThr. Most archaeal ThrRSs possess a unique editing domain structurally similar to D-aminoacyl-tRNA deacylases (DTDs) found in eubacteria and eukaryotes that specifically removes D-amino acids attached to tRNA. Here, we provide mechanistic insights into the removal of noncognate L-serine from tRNAThr by a DTD-like editing module from Pyrococcus abyssi ThrRS (Pab-NTD). High-resolution crystal structures of Pab-NTD with pre- and post-transfer substrate analogs and with L-serine show mutually nonoverlapping binding sites for the seryl moiety. Although the pre-transfer editing is excluded, the analysis reveals the importance of main chain atoms in proper positioning of the post-transfer substrate for its hydrolysis. A single residue has been shown to play a pivotal role in the inversion of enantioselectivity both in Pab-NTD and DTD. The study identifies an enantioselectivity checkpoint that filters opposite chiral molecules and thus provides a fascinating example of how nature has subtly engineered this domain for the selection of chiral molecules during translation.

Identification, cloning, and functional analysis of the human U6 snRNA-specific terminal uridylyl transferase

Mammalian cells contain a highly specific terminal uridylyl transferase (TUTase) that exclusively accepts U6 snRNA as substrate. This enzyme, termed U6-TUTase, was purified from HeLa cell extracts and analyzed by microsequencing. All sequenced peptides matched a unique human cDNA coding for a previously unknown protein. Domain structure analysis revealed that the U6-TUTase also belongs to the well-characterized poly(A) polymerase protein superfamily. However, by amino acid sequence as well as RNA-binding motifs, human U6-TUTase is highly divergent from both the poly(A) polymerases and from the TUTases identified within the editing complexes of trypanosomes. After cloning, the recombinant U6-TUTase was expressed in HeLa cells. Analysis of its catalytical activity confirmed the identity of the cloned protein as U6-TUTase, exhibiting the same exclusive substrate specificity for U6 snRNA as the endogenous enzyme. That unique selectivity even excluded as substrate U6atac RNA, the functional homolog of the minor spliceosome. Finally, RNAi knockdown experiments revealed that U6-TUTase is essential for cell proliferation. Surprisingly, large amounts of the recombinant enzyme were found to accumulate within nucleoli.

Preferential interaction of a 25 kDa protein with an A6 pre-mRNA substrate for RNA editing in Trypanosoma brucei

Mitochondrial gene expression in kinetoplastids is controlled after transcription, potentially at the levels of RNA maturation, stability and translation. Among these processes, RNA editing by U-insertion/deletion catalysed by multi-subunit editing complexes is best characterised at the molecular level. Nevertheless, mitochondrial RNA metabolism overall remains poorly understood, including the potential regulatory factors that may interact with the relevant catalytic molecular machines and/or RNA substrates. Here we report on a ∼25 kDa polypeptide in mitochondrial extracts that exhibits a preferential “zero-distance” photo-crosslinking interaction with an A6 pre-mRNA model substrate for RNA editing containing a single [ 32P] at the first editing site. The ∼25 kDa polypeptide purified away from editosomes upon ion-exchange chromatography and glycerol gradient sedimentation. Competition assays with homologous and heterologous transcripts suggest that the preferential recognition of the A6 substrate is based on relatively low-specificity RNA-protein contacts. Our mapping and substrate truncation analyses suggest that the crosslinking activity primarily targeted a predicted stem-loop region containing the first editing sites. Consistent with the notion that pre-mRNA folding may be required, pre-annealing with guide RNA abolished crosslinking. Interestingly, this preferential protein interaction with the A6 substrate seemed to require adenosine 5′-triphosphate but not hydrolysis. As in other biological systems, fine regulation in vivo may be brought about by transient networks of relatively low-specificity interactions in which multiple auxiliary factors bind to mRNAs and/or editing complexes in unique higher-order assemblies.

RNA editing complex interactions with a site for full-round U deletion in Trypanosoma brucei

Trypanosome U insertion and U deletion RNA editing of mitochondrial pre-mRNAs is catalyzed by multisubunit editing complexes as directed by partially complementary guide RNAs. The basic enzymatic activities and protein composition of these high-molecular mass complexes have been under intense study, but their specific protein interactions with functional pre-mRNA/gRNA substrates remains unknown. We show that editing complexes purified through extensive ion-exchange chromatography and immunoprecipitation make specific cross-linking interactions with A6 pre-mRNA containing a single 32P and photoreactive 4-thioU at the scissile bond of a functional site for full-round U deletion. At least four direct protein-RNA contacts are detected at this site by cross-linking. All four interactions are stimulated by unpaired residues just 5' of the pre-mRNA/gRNA anchor duplex, but strongly inhibited by pairing of the editing site region. Furthermore, competition analysis with homologous and heterologous transcripts suggests preferential contacts of the editing complex with the mRNA/gRNA duplex substrate. This apparent structural selectivity suggests that the RNA-protein interactions we observe may be involved in recognition of editing sites and/or catalysis in assembled complexes.

Minimal pre-mRNA substrates with natural and converted sites for full-round U insertion and U deletion RNA editing in trypanosomes

Trypanosome RNA editing by uridylate insertion or deletion cycles is a mitochondrial mRNA maturation process catalyzed by multisubunit complexes. A full-round of editing entails three consecutive steps directed by partially complementary guide RNAs: pre-mRNA cleavage, U addition or removal, and ligation. The structural and functional composition of editing complexes is intensively studied, but their molecular interactions in and around editing sites are not completely understood. In this study, we performed a systematic analysis of distal RNA requirements for full-round insertion and deletion by purified editosomes. We define minimal substrates for efficient editing of A6 and CYb model transcripts, and established a new substrate, RPS12. Important differences were observed in the composition of substrates for insertion and deletion. Furthermore, we also showed for the first time that natural sites can be artificially converted in both directions: from deletion to insertion or from insertion to deletion. Our site conversions enabled a direct comparison of the two editing kinds at common sites during substrate minimization and demonstrate that all basic determinants directing the editosome to carry out full-round insertion or deletion reside within each editing site. Surprisingly, we were able to engineer a deletion site into CYb, which exclusively undergoes insertion in nature.

Thermodynamic Dissection of the Polymerizing and Editing Modes of a DNA Polymerase

DNA polymerases with intrinsic proofreading activity interact with DNA primer/templates in two distinct modes, corresponding to the complexes formed during the 5′–3′ polymerization or 3′–5′ editing of a nascent DNA chain. Thermodynamic measurements designed to quantify the energetic contributions of individual DNA–protein contacts in either the polymerizing or editing complexes are complicated by the fact that both species exist in solution and are not resolved in conventional DNA–protein binding assays. To overcome this problem, we have developed a new binding analysis that combines information from steady-state and time-resolved fluorescence experiments and uses the Klenow fragment of Escherichia coli DNA polymerase I (KF) and fluorescently labeled primer/template oligonucleotides as a model polymerase–DNA system. Steady-state fluorescence titrations are used to evaluate the overall affinity of KF for the primer/template, while time-resolved fluorescence anisotropy is used to quantify the equilibrium fractions of the primer/template bound in the polymerizing and editing modes. From a combined analysis of both data, the equilibrium constant and hence standard free energy change associated with each binding mode can be obtained unequivocally. This method is initially used to determine the equilibrium constants describing binding of a correctly base-paired primer/template to the 5′–3′ polymerase and 3′–5′ exonuclease sites of KF. It is then extended to quantify the extent to which these parameters are affected by the introduction of mismatches into the primer/template, and by rearrangement of specific side-chains in the exonuclease domain of the protein. While these perturbants were originally designed to demonstrate the utility of our new approach, they are also relevant in their own right since they have helped identify some hitherto unknown determinants of polymerase fidelity.

A tale of two TUTases.

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 purification-tagged 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 U-insertion 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.

Dyskinetoplastic Trypanosoma brucei contains functional editing complexes.

Mitochondrial pre-mRNAs undergo posttranscriptional RNA editing as directed by small guide RNAs (gRNAs) to produce functional mRNAs in kinetoplastid protozoa. The pre-mRNAs and gRNAs are encoded in the maxicircle and minicircle components, respectively, of the kinetoplastid mitochondrial DNA (kDNA), and editing is catalyzed by a multienzyme protein complex. Trypanosoma evansi AnTat3/3, which lacks maxicircles but retains a single class of minicircles, and a dyskinetoplastic mutant of Trypanosoma brucei EATRO164, which is devoid of kDNA, were both shown to retain genes and proteins for the editing complex. The proteins are present in complexes that immunoprecipitate and sediment indistinguishably from wild-type complexes. The complexes catalyze precleaved insertion and deletion editing as well as full-round deletion editing in vitro. Thus, mutants which lack the natural substrates for RNA editing and all or most gRNAs retain editing complexes that contain the four primary catalytic activities of editing and function in editing, at least in vitro. Therefore neither pre-mRNA nor gRNA is required to form functional RNA-editing complexes.

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.

Role of uridylate-specific exoribonuclease activity in Trypanosoma brucei RNA editing.

Editing of mitochondrial mRNAs in kinetoplastid protozoa occurs by a series of enzymatic steps that insert and delete uridylates (U's) as specified by guide RNAs (gRNAs). The characteristics of the 3' exonuclease activity that removes the U's following cleavage during deletion editing were determined by using an in vitro precleaved deletion assay that is based on ATPase subunit 6 pre-mRNA and gA6[14] gRNA. The exonuclease in partially purified editing complexes is specific for U's. The specificity occurs in the absence of gRNA, but its activity is enhanced by the presence of gRNA. The 3' pre-mRNA fragment enhances the specificity, but not the efficiency, of U removal. The activity is sensitive to the 5' phosphate of the 3' fragment, which is not required for U removal. The ability of the 3' U's to base pair with purines in the gRNA protects them from removal, suggesting that the U-specific 3' exonuclease (exoUase) is specific for U's which are not base paired. ExoUase is stereospecific and cannot remove (Rp)alpha-thio-U. The specificity of the exoUase activity thus contributes to the precision of RNA editing.

Composition of the editing complex of Trypanosoma brucei.

The RNA editing that produces most functional mRNAs in trypanosomes is catalysed by a multiprotein complex. This complex catalyses the endoribonucleolytic cleavage, uridylate addition and removal, and RNA ligation steps of the editing process. Enzymatic and in vitro editing analyses reveal that each catalytic step contributes to the specificity of the editing and, together with the interaction between gRNA and the mRNA, results in precisely edited mRNAs. Tandem mass spectrometric analysis was used to identify the genes for several components of biochemically purified editing complexes. Their identity and presence in the editing complex were confirmed using immunochemical analyses utilizing mAbs specific to the editing complex components. The genes for two RNA ligases were identified. Genetic studies show that some, but not all, of the components of the complex are essential for editing. The TbMP52 RNA ligase is essential for editing while the TbMP48 RNA ligase is not. Editing was found to be essential in bloodstream form trypanosomes. This is surprising because mutants devoid of genes encoding RNAs that become edited survive as bloodstream forms but encouraging since editing complex components may be targets for chemotherapy.

Four related proteins of the Trypanosoma brucei RNA editing complex.

RNA editing in kinetoplastid mitochondria occurs by a series of enzymatic steps that is catalyzed by a macromolecular complex. Four novel proteins and their corresponding genes were identified by mass spectrometric analysis of purified editing complexes from Trypanosoma brucei. These four proteins, TbMP81, TbMP63, TbMP42, and TbMP18, contain conserved sequences to various degrees. All four proteins have sequence similarity in the C terminus; TbMP18 has considerable sequence similarity to the C-terminal region of TbMP42, and TbMP81, TbMP63, and TbMP42 contain zinc finger motif(s). Monoclonal antibodies that are specific for TbMP63 and TbMP42 immunoprecipitate in vitro RNA editing activities. The proteins are present in the immunoprecipitates and sediment at 20S along with the in vitro editing, and RNA editing ligases TbMP52 and TbMP48. Recombinant TbMP63 and TbMP52 coimmunoprecipitate. These results indicate that these four proteins are components of the RNA editing complex and that TbMP63 and TbMP52 can interact.

Cloning and characterization of two guide RNA-binding proteins from mitochondria of Crithidia fasciculata: gBP27, a novel protein, and gBP29, the orthologue of Trypanosoma brucei gBP21.

In kinetoplastid protozoa, mitochondrial (mt) mRNAs are post-transcriptionally edited by insertion and deletion of uridylate residues, the information being provided by guide (g)RNAs. Currently popular mechanisms for the editing process envisage a series of consecutive 'cut-and-paste' reactions, carried out by a complex RNP machinery. Here we report on the purification, cloning and functional analysis of two gRNA-binding proteins of 28.8 (gBP29) and 26.8 kDa (gBP27) from mitochondria of the insect trypanosome Crithidia fasciculata. gBP29 and gBP27 proved to be similar, Arg + Ala-rich proteins, with pI values of approximately 10.0. gBP27 has no homology to known proteins, but gBP29 is the C.fasciculata orthologue of gBP21 from Trypanosoma brucei, a gRNA-binding protein that associates with active RNA editing complexes. As measured in UV cross-linking assays, His-tagged recombinant gBP29 and gBP27 bind to radiolabelled poly(U) and synthetic gRNAs, while competition experiments suggest a role for the gRNA 3'-(U)-tail in binding to these proteins. Immunoprecipitates of mt extracts generated with antibodies against gBP29 also contained gBP27 and vice versa. The immunoprecipitates further harbored a large proportion of the cellular content of four different gRNAs and of edited and pre-edited NADH dehydrogenase subunit 7 mRNAs, but only small amounts of mt rRNAs. In addition, the bulk of gBP29 and gBP27 co-eluted with gRNAs from gel filtration columns in the high molecular weight range. Together, these results suggest that the proteins are part of a large macromolecular complex(es). We infer that gBP29 and gBP27 are components of the C.fasciculata editing machinery that may interact with gRNAs.

RNA Editing–Associated Protein 1 Is an RNA Binding Protein with Specificity for Preedited mRNA

RNA editing in the mitochondria of kinetoplastids involves the addition and deletion of uridines at specific sites as directed by guide RNAs (gRNAs). Ample evidence shows that ribonucleoprotein (RNP) complexes carry out this posttranscriptional processing. One component of RNA editing complexes is REAP-1, a protein of previously unknown function found primarily in mRNA containing editing complexes. We now show that REAP-1 is an RNA binding protein and map the binding activity to the amino-terminal third of the protein. REAP-1 binds to poly(G) and single-stranded guanosine rich RNAs. Data presented here demonstrates that preedited RNAs are the preferred substrate for REAP-1. The results suggest a model in which the role of REAP-1 is to bring preedited mRNAs into the editing complex.

Identification of candidate mitochondrial RNA editing ligases from Trypanosoma brucei.

Most mitochondrial genes of Trypanosoma brucei do not contain the necessary information to make translatable mRNAs. These transcripts must undergo RNA editing, a posttranscriptional process by which uridine residues are added and deleted from mitochondrial mRNAs. RNA editing is believed to be catalyzed by a ribonucleoprotein complex containing endonucleolytic, terminal uridylyl transferase (TUTase), 3' uridine-specific exonucleolytic (U-exo), and ligase activities. None of the catalytic enzymes for RNA editing have been identified. Here we describe the identification of two candidate RNA ligases (48 and 52 kDa) that are core catalytic components of the T. brucei ribonucleoprotein editing complex. Both enzymes share homology to the covalent nucleotidyl transferase superfamily and contain five key signature motifs, including the active site KXXG. In this report, we present data on the proposed 48 kDa RNA editing ligase. We have prepared polyclonal antibodies against recombinant 48 kDa ligase that specifically recognize the trypanosome enzyme. When expressed in trypanosomes as an epitope-tagged fusion protein, the recombinant ligase localizes to the mitochondrion, associates with RNA editing complexes, and adenylates with ATP. These findings provide strong support for the enzymatic cascade model for kinetoplastid RNA editing.

Processing of polycistronic guide RNAs is associated with RNA editing complexes in Trypanosoma brucei.

In kinetoplastid mitochondrial mRNA editing, post-transcriptional insertion or deletion of uridines is templated by guide RNAs (gRNAs). Pre-mRNAs are encoded by maxicircles, while gRNAs are encoded by both maxicircles and minicircles. We have investigated minicircle transcription and the processing of gRNAs in Trypanosoma brucei. We find that minicircles are transcribed polycistronically and that transcripts are accurately processed by an approximately 19S complex. This gRNA processing activity co-purifies with RNA editing complexes, and both remain associated in 19S complexes. Furthermore, we show that RNA editing complexes associate preferentially with a polycistronic gRNA over non-processed RNAs. We propose that the approximately 19S complexes initially described as RNA editing complex I are gRNA processing complexes that cleave polycistronic gRNA transcripts into monocistrons.

APOBEC-1 dependent cytidine to uridine editing of apolipoprotein B RNA in yeast.

Cytidine to uridine editing of apolipoprotein B (apoB) mRNA requires the cytidine deaminase APOBEC-1 as well as a tripartite sequence motif flanking a target cytidine in apoB mRNA and an undefined number of auxiliary proteins that mediate RNA recognition and determine site-specific editing. Yeast engineered to express APOBEC-1 and apoB mRNA supported editing under conditions of late log phase growth and stationary phase. The cis -acting sequence requirements and the intracellular distribution of APOBEC-1 in yeast were similar to those described in mammalian cells. These findings suggest that auxiliary protein functions necessary for the assembly of editing complexes, or 'editosomes', are expressed in yeast and that the distribution of editing activity is to the cell nucleus.

The involvement of gRNA-binding protein gBP21 in RNA editing-an in vitro and in vivo analysis.

RNA editing in the parasitic organism Trypanosoma brucei is characterised by the insertion and deletion of uridylate residues into otherwise incomplete primary transcripts. The processing reaction is a required pathway for the expression of most mitochondrial genes and proceeds by a cascade of enzyme-catalysed steps. RNA editing involves one or more macromolecular ribonucleoprotein complexes which are likely to interact with additional components as the reaction proceeds. Here we examined the involvement of the gRNA-binding polypeptide gBP21, a protein which has been demonstrated to be associated with active RNA editing complexes. We show that in vitro RNA editing can be suppressed by the addition of a gBP21-specific antibody or by immunodepletion of the protein. By creating a gBP21 knockout mutant we analysed the requirement for the protein in vivo. gBP21(-) trypanosomes are viable as bloodstream stage cells and contain edited mRNAs. However, the knockout mutant is not capable of differentiating from the bloodstream to the insect life cycle stage in vitro. Moreover, mutant cells are characterised by a low mitochondrial transcript abundance. Together, these data establish that gBP21 contributes a non-essential function to the RNA editing reaction and further suggest that the protein is involved in additional mitochondrial processes which impact a larger pool of mitochondrial transcripts.

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.

Purification of Native and Recombinant Double-Stranded RNA-Specific Adenosine Deaminases

ADAR1 and ADAR2 are members of a family of enzymes that catalyze the conversion of adenosine to inosine in double-stranded RNA. Unlike the other types of RNA editing that involve multiprotein editing complexes, the site-specific deamination of an adenosine to inosine is catalyzed by single enzymes. ADAR1 and ADAR2 have been purified and the genes cloned from various sources. Each gene encodes multiple splice variants. As it is crucial to have an adequate supply of pure protein to investigate this type of RNA editing, we describe in this article methods for both the purification and the overexpression of either full-length or partial ADAR1 and ADAR2 isoforms.