Arginine methylation of a mitochondrial guide RNA binding protein from Trypanosoma brucei.

RBP16 is a mitochondrial Y-box protein from the parasitic protozoan Trypanosoma brucei that binds guide RNAs and ribosomal RNAs. It is comprised of an N-terminal cold-shock domain and a C-terminal domain rich in glycine and arginine residues, resembling the RGG RNA-binding motif. Arginine residues found within RGG domains are frequently asymmetrically dimethylated by a class of enzymes termed protein arginine methyltransferases (PRMTs). As Arg-93 of RBP16 exists in the context of a preferred sequence for asymmetric arginine dimethylation (G/FGGRGGG/F), we investigated whether modified arginines are present in native RBP16 by MALDI-TOF and post-source decay analyses. These analyses confirmed that Arg-93 is dimethylated. In addition, Arg-78 exists as an unmodified or as a monomethylated derivative, and Arg-85 is present in forms corresponding to the unmodified, di-, and tri-methylated state. While Arg-93 is apparently constitutively dimethylated, the methylation of Arg-78 and Arg-85 is mutually exclusive. Furthermore, whole cell extracts from procyclic form T. brucei are able to methylate bacterially expressed RBP16 (rRBP16), as well as endogenous proteins, in the presence of S-adenosyl-L-[methyl-3H]methionine. While assays of mitochondrial extracts suggest a small amount of PRMT may also be present in this subcellular compartment, the majority of trypanosome PRMT activity is extramitochondrial. We show that rRBP16 is methylated in trypanosome extracts through the action of a type I methyltransferase as well as serving as a substrate for heterologous mammalian type I PRMTs. In addition, we demonstrate the presence of type II PRMT activity in trypanosome cell extracts. These results suggest that protein arginine methylation is a common posttranslational modification in trypanosomes, and that it may regulate the function of RBP16.

The trypanosome homolog of human p32 interacts with RBP16 and stimulates its gRNA binding activity.

RBP16 is a guide RNA (gRNA)-binding protein that was shown through immunoprecipitation experiments to interact with approximately 30% of total gRNAs in Trypanosoma brucei mitochondria. To gain insight into the biochemical function of RBP16, we used affinity chromatography and immunoprecipitation to identify RBP16 protein binding partners. By these methods, RBP16 does not appear to stably interact with the core editing machinery. However, fractionation of mitochondrial extracts on MBP-RBP16 affinity columns consistently isolated proteins of 12, 16, 18 and 22 kDa that were absent from MBP control columns. We describe here our analysis of one RBP16-associated protein, p22. The predicted p22 protein has significant sequence similarity to a family of multimeric, acidic proteins that includes human p32 and Saccharomyces cerevisiae mam33p. Glutaraldehyde crosslinking of recombinant p22 identified homo-multimeric forms of the protein, further substantiating its homology to p32. We confirmed the p22-RBP16 interaction and demonstrated that the two proteins bind each other directly by ELISA utilizing recombinant p22 and RBP16. p32 family members have been reported to modulate viral and cellular pre-mRNA splicing, in some cases by perturbing interaction of their binding partners with RNA. To determine whether p22 similarly affects the gRNA binding properties of RBP16, we titrated recombinant p22 into UV crosslinking assays. These experiments revealed that p22 significantly stimulates the gRNA binding capacity of RBP16. Thus, p22 has the potential to be a regulatory factor in T.brucei mitochondrial gene expression by modulating the RNA binding properties of RBP16.

Transcriptional and post-transcriptional in organello labelling of Trypanosoma brucei mitochondrial RNA.

In organello labelling of Trypanosoma brucei mitochondrial (mt) RNA was characterised with respect to nucleotide requirements and drug sensitivity. Mitochondrial transcriptional activity is maximal in the presence of all ribonucleoside-triphosphate NTPs, and can be inhibited by UTP depletion. Mitochondrial transcription can also be partially inhibited by actinomycin D (actD) or ethidium bromide (EtBr). Post-transcriptional UTP incorporation is insensitive to actinomycin D or ethidium bromide. Proteins were identified that interact with transcriptional and post-transcriptionally labelled RNAs, and confirm the in vitro RNA-binding properties discovered for a number of T. brucei mt proteins. These experiments reveal new strategies for studying mt transcription and processing in T. brucei mitochondria.

UTP-dependent and -independent pathways of mRNA turnover in Trypanosoma brucei mitochondria.

Although primary transcripts are polycistronic in the mitochondria of Trypanosoma brucei, steady-state levels of mature, monocistronic RNAs change throughout the parasitic life cycle. This indicates that steady-state RNA abundance is controlled by posttranscriptional mechanisms involving differential RNA stability. In this study, in organello pulse-chase labeling experiments were used to analyze the stability of different T. brucei mitochondrial RNA populations. In this system, total RNA and rRNA are stable for many hours. In contrast, mRNAs can be degraded by two biochemically distinct turnover pathways. The first pathway results in the rapid degradation of mRNA (half-life [t(1/2)] of 11 to 18 min) and is dependent upon the presence of an mRNA poly(A) tail. Remarkably, this pathway also requires the addition of UTP and therefore is termed UTP dependent. The second pathway results in slow turnover of mitochondrial mRNA (t(1/2) of approximately 3 h) and is not dependent upon the presence of an mRNA poly(A) tail or the addition of exogenous UTP. In summary, these results demonstrate the presence of a novel, UTP-dependent degradation pathway for T. brucei mitochondrial mRNAs and reveal an unprecedented role for both UTP and mRNA polyadenylation in T. brucei mitochondrial gene expression.

RNA-binding properties of the mitochondrial Y-box protein RBP16.

We have previously identified a mitochondrial Y-box protein in Trypanosoma brucei that we designated RBP16. The predicted RBP16 amino acid sequence revealed the presence of a cold-shock domain at its N-terminus and a glycine- and arginine-rich C-terminus reminiscent of an RGG RNA-binding motif. Since RBP16 is capable of interacting with different guide RNAs (gRNAs) in vitro and in vivo primarily via the oligo(U) tail, as well as with ribosomal RNAs, possible functions of RBP16 may be in kinetoplastid RNA editing and/or translation. Herein, we report experiments that further define the RNA-binding properties of RBP16. RBP16 forms a single stable complex with the gRNA gA6[14] at low protein concentration, while at higher protein concentration two stable complexes that possibly represent two different conformations are observed. Both complexes are stable at relatively high salt and moderate heparin concentrations indicating that the binding of RBP16 to gA6[14] does not rely primarily on ionic interactions. Phenylglyoxal treatment of the protein indicates that arginine residues are important in RNA binding. The minimal length of RNA sequence necessary for the binding of RBP16 was assessed by gel retardation and UV cross-linking competition assays using oligo(U) ribonucleotides of varying lengths (4-40 nt). Although RBP16 can bind to oligonucleotides as small as U(4), its affinity increases with the length of the oligo(U) ribonucleotide, with a dramatic increase in binding efficiency observed when the length is increased to 10 nt. Gel retardation assays employing T.brucei mRNAs demonstrated that, although it acts as a major binding determinant, a 3' U tail is not an absolute requirement for efficient RBP16-RNA binding. Experiments with oligonucleotides containing U stretches embedded at different positions in oligo(dC) indicated that high-affinity binding requires both a uridine stretch, as well as 5' and 3' non-specific sequences. These results suggest a model for the molecular interactions involved in RBP16-RNA binding.

Cloning and characterisation of cDNA encoding the Trypanosoma brucei ribosomal protein L24.

A cDNA encoding ribosomal protein L24 was amplified by PCR from the protozoan parasite, Trypanosoma brucei. The 621 nucleotide cDNA had an open reading frame of 375 nucleotides, predicting a highly basic protein of 125 aa. Database searches revealed 33-40% identity between the T. brucei RPL24 protein and several eukaryotic RPL24 homologues. Southern blot analysis indicated that the gene was present as a single copy, and a transcript of approximately 620 nucleotides was detected in procyclic forms of the parasite. Interestingly, T. brucei PRL24 is the smallest eukaryotic RPL24 protein described to date. It is also the most divergent of the known kinetoplastid ribosomal proteins.

Trypanosoma brucei RBP16 is a mitochondrial Y-box family protein with guide RNA binding activity.

Trypanosoma brucei mitochondria possess a unique mechanism of mRNA maturation called RNA editing. In this process, uridylate residues are inserted and deleted posttranscriptionally into pre-mRNA to create translatable messages. The genetic information for RNA editing resides in small RNA molecules called guide RNAs (gRNAs). Thus, proteins in direct contact with gRNA are likely to catalyze or influence RNA editing. Herein we characterize an abundant gRNA-binding protein from T. brucei mitochondria. This protein, which we term RBP16 (for RNA-binding protein of 16 kDa), binds to different gRNA molecules. The major determinant of this interaction is the oligo(U) tail, present on the 3'-ends of gRNAs. RBP16 forms multiple, stable complexes with gRNA in vitro, and immunoprecipitation experiments provide evidence for an association between RBP16 and gRNA within T. brucei mitochondria. Mature RBP16 contains a cold shock domain at the N terminus and a C-terminal region rich in arginine and glycine. The presence of the cold shock domain places RBP16 as the first organellar member of the highly conserved Y-box protein family. The arginine and glycine rich C terminus in combination with the cold shock domain predicts that RBP16 will be involved in the regulation of gene expression at the posttranscriptional level.

Coordination of kRNA editing and polyadenylation in Trypanosoma brucei mitochondria: complete editing is not required for long poly(A) tract addition.

Mitochondrial RNAs in Trypanosoma brucei are post-transcriptionally modified by the addition and deletion of uridylate residues in a process called kRNA editing. Unedited, partially edited and fully edited RNAs exist in the steady-state RNA population. Previous experiments have demonstrated that T.brucei mitochondrial RNAs contain both short (approximately 20 nt) and long (120-200 nt) poly(A) tracts. However, it is unknown exactly what poly(A) tract lengths are present on unedited, partially edited and fully edited RNAs. To gain insight into the role of the poly(A) tract in T.brucei mitochondria, ribosomal protein S12 (RPS12) RNAs with short and long poly(A) tracts were purified by hybrid selection and analyzed by RT-PCR and DNA sequencing. Unedited RPS12 RNAs were found almost exclusively in populations with short poly(A) tracts. Both partially and fully edited RPS12 RNAs were found in populations with short and long poly(A) tracts. Therefore, there is a correlation between the presence of editing and the presence of the long poly(A) tract. Since a proportion of partially edited RPS12 RNAs contain long poly(A) tracts, it is unlikely that the long poly(A) tract is the sole signal for translation. Other implications for the role of polyadenylation in mitochondrial gene regulation are discussed.

Trypanosoma brucei poly(A) binding protein I cDNA cloning, expression, and binding to 5 untranslated region sequence elements.

Poly(A) binding protein I (PABPI) is a highly conserved eukaryotic protein that binds mRNA poly(A) tails and functions in the regulation of translational efficiency and mRNA stability. As a first step in our investigation of the role(s) of mRNA poly(A) tails in posttranscriptional gene regulation in Trypanosoma brucei, we have cloned the cDNA encoding PABPI from this organism. The cDNA predicts a protein homologous to PABPI from other organisms and displaying conserved features of these proteins, including four RNA binding domains that span the N-terminal two-thirds of the protein. Comparison of northern blot data with the cDNA sequence indicates an unusually long 3' untranslated region (UTR) of approximately three kilobases. The 5 UTR contains both A-rich and AU repeat regions, the former being a ubiquitous property of PABPI 5' UTRs. T. brucei PABPI, expressed as a glutathione-S-transferase fusion protein, bound to RNA comprised of its full length 5' UTR in UV cross-linking experiments. This suggests that PABPI may play an autoregulatory role in its own expression. Competition experiments indicate that the A-rich region, but not the AU repeats, are involved in this binding.

Experimental verification of the secondary structures of guide RNA-pre-mRNA chimaeric molecules in Trypanosoma brucei.

RNA editing in kinetoplastid organisms is an RNA-processing reaction that adds and deletes U nucleotides at specific sites in mitochondrial pre-mRNAs. The edited sequence is specified by guide RNAs and the processing presumably occurs within a high-molecular-mass ribonucleoprotein complex containing several enzymatic activities. Although the mechanism is not currently known, potential intermediates or by-products of the editing process are chimaeric RNAs where guide (g) RNAs are covalently attached, via their non-encoded U-tail, to their cognate pre-mRNAs. We determined the secondary structures of three different ATPase 6 chimaeras of Trypanosoma brucei using a set of structure-sensitive chemical and enzymatic probes. The experiments revealed a bipartite domain structure consisting of a gRNA/pre-mRNA interaction hairpin and an independently folding mRNA stem/loop in all three RNAs. The connecting U-tail was a determinant for the length of the interaction stems with the oligo(U) nucleotides base pairing to internal gRNA sequences. The probed structures have calculated delta G27o values of -92 kJ/ mol to -134 kJ/mol, somewhat less stable than the predicted minimal free energy structures and support previously proposed models for the interaction between gRNAs and pre-mRNAs. Optical melting studies indicated additional, higher order structural features for all three molecules with four defined melting transition between 10 degrees C and 90 degrees C. A comparison of CD spectra in the absence and presence of mitochondrial protein extracts demonstrated no gross structural changes of the RNA structures induced by the association with polypeptides.

Complexes from Trypanosoma brucei that exhibit deletion editing and other editing-associated properties.

Transcripts from many mitochondrial genes in kinetoplastids undergo RNA editing, a posttranscriptional process which inserts and deletes uridines. By assaying for deletion editing in vitro, we found that the editing activity from Trypanosoma brucei mitochondrial lysates (S.D. Seiwert and K.D. Stuart), Science 266:114-117,1994) sediments with a peak of approximately 20S. RNA helicase, terminal uridylyl transferase, RNA ligase, and adenylation activities, which may have a role in editing, cosediment in a broad distribution, with most of each activity at 35 to 40S. Most ATPase 6 (A6) guide RNA and unedited A6 mRNA sediments at 20 to 30S, with some sedimenting further into the gradient, while most edited A6 mRNA sediments at >35S. Several mitochondrial proteins which cross-link specifically with guide RNA upon UV treatment also sediment in glycerol gradients. Notably, a 65-kDa protein sediments primarily at approximately 20S, a 90-kDa protein sediments at 35 to 40S, and a 25-kDa protein is present at <10S. Most ribonucleoprotein complexes that form with gRNA in vitro sediment at 10 to 20S, except for one, which sediments at 30 to 45S. These results suggest that RNA editing takes place within a multicomponent complex. The potential functions of and relationships between the 20S and 35 to 40S complexes are discussed.

Developmental regulation of RNA editing and polyadenylation in four life cycle stages of Trypanosoma congolense.

The accumulation of many edited mRNAs is developmentally regulated in a transcript-specific fashion in Trypanosoma brucei. In addition, these transcripts are frequently present in two size classes which differ substantially in the lengths of their poly(A) tails, and poly(A) tail length is also developmentally regulated. Previously, these phenomena have only been studied in the mammalian bloodstream and insect procyclic forms (BF and PF, respectively) of T. brucei. In this paper, we examine developmental regulation of edited RNA abundance and poly(A) tail length of 3 mitochondrially encoded RNAs in mammalian BF and 3 insect stages (PF, epimastigotes, and metacyclics) of T. congolense. T. congolense BF and PF are similar, but not identical, to these stages of T. brucei with regard to edited RNA accumulation and poly(A) tail length. At the level of edited RNA, both epimastigotes and metacyclic stage parasites appear to be pre-adapted for the respiratory mechanisms of BF but not yet down-regulated from the cytochrome-based respiration of PF since edited RNAs encoding NADH dehydrogenase components are up-regulated and edited CYb RNA is abundant in these stages. Poly(A) tail lengths of mitochondrial mRNAs appear to be regulated independently of edited RNA abundance. These results indicate that multiple mechanisms for regulation of mitochondrial gene expression are active throughout the trypanosome life cycle.

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.

Editing of Trypanosoma brucei maxicircle CR5 mRNA generates variable carboxy terminal predicted protein sequences.

RNA editing post-transcriptionally modifies several mRNAs from the maxicircle of kinetoplastid parasites by addition and removal of uridine residues. We report here that maxicircle CR5 transcripts of Trypanosoma brucei are edited in two domains separated by an eight nucleotide sequence that remains unedited. The large 5' domain is edited to a consensus sequence while the smaller 3' domain is edited to multiple final sequences. In all, 205-217 Us are inserted and 13-16 encoded uridines are deleted from the CR5 mRNA, producing a mature transcript 75-80% larger than the unedited transcript. The edited RNAs predict small, highly hydrophobic proteins. The carboxy terminal 15-30% of these predicted proteins have multiple different amino acid sequences as a result of the variable edited 3' mRNA sequence, but these fall into two families of sequence. Limited amino acid sequence and hydrophobicity profile similarities suggest that the protein encoded by edited CR5 mRNA may be a subunit of NADH dehydrogenase.

Extensive editing of CR2 maxicircle transcripts of Trypanosoma brucei predicts a protein with homology to a subunit of NADH dehydrogenase.

Several genes of the Trypanosoma brucei mitochondrial genome (the maxicircle) encode mRNAs that are so extensively altered by RNA editing that the gene cannot be identified by analysis of the DNA sequence. The 322-nucleotide preedited RNA of one of these genes, CR2, is converted into a 647-nucleotide transcript by the addition of 345 uridines and the deletion of 20 genomically encoded uridines. The fully edited transcript has an open reading frame that predicts a 194-amino-acid protein. This protein, which we name ND9 (NADH dehydrogenase subunit 9), has homology to a subunit of NADH dehydrogenase (respiratory complex I). Seven guide RNAs that can specify edited CR2 sequence have been identified. Steady-state levels of unedited ND9 transcripts are greater in bloodstream than in procyclic forms, but edited ND9 mRNA is present in similar abundance in both life cycle stages.

Maxicircle DNA and edited mRNA sequences of closely related trypanosome species: implications of kRNA editing for evolution of maxicircle genomes.

kRNA editing produces functional mRNAs by uridine insertion and deletion. We analyzed portions of the apocytochrome b and NADH dehydrogenase subunits 7 and 8 (ND7 and 8) genes and their edited mRNAs in Trypanosoma congolense and compared these to the corresponding sequences in T.brucei. We find that these genes are highly diverged between the two species, especially in the positions of thymidines and in nucleotide transitions. Editing eliminates differences in encoded uridines producing edited mRNAs that are identical except for the nucleotide substitutions. The resulting predicted proteins are identical since all nucleotide substitutions are silent. A T.congolense minicircle-encoded gRNA which can specify editing of ND8 mRNA was identified. This gRNA can basepair with both T.congolense and T.brucei ND8 mRNA despite nucleotide transitions due to the flexibility of G:U base-pairing. These results illustrate how editing affects the characteristics of maxicircle sequence divergence and allows protein sequence conservation despite a level of DNA sequence divergence which would be predicted to be intolerable in the absence of editing.

Chimeric and truncated RNAs in Trypanosoma brucei suggest transesterifications at non-consecutive sites during RNA editing.

RNA editing adds and removes uridines at specific sites in several mitochondrial transcripts in kinetoplastid parasites probably as specified by guide RNAs (gRNAs) that are complementary to the final edited sequence. Editing has been postulated to involve transesterification which predicts (1) chimeric molecules with a gRNA covalently attached by its non-encoded oligo U tail to an internal editing site in the mRNA and (2) the corresponding truncated 5' portions of the mRNAs. We have characterized cDNAs representing a large number of both types of intermediates from Trypanosoma brucei. The lengths of both U tails and encoded gRNA sequences vary greatly in length. The majority of encoded gRNA sequences are shorter than predicted based on their minicircle coding sequences. Analysis of the predominant sites of gRNA attachment in chimeras suggests that the transesterifications that religate the truncated 5' mRNAs may proceed more rapidly at editing sites at the 5' end of an editing domain and at sites of U deletion. Partially edited sequences in the mRNA portion of chimeras and at the 3' ends of truncated 5' mRNAs also indicate a non-consecutive order of site selection during RNA editing.

Extensive editing of both processed and preprocessed maxicircle CR6 transcripts in Trypanosoma brucei.

Transcripts from several genes encoded in the Trypanosoma brucei maxicircle genome are altered by posttranscriptional uridine insertion and deletion through a process called RNA editing. We find that transcripts from the CR6 gene are extensively edited by addition of 132 uridines and deletion of 28 uridines to produce a fully edited mRNA 47% larger than unedited mRNA. Two open reading frames (ORFs) and their initiation and termination codons are created by editing of CR6 mRNA. Both ORFs specify small, hydrophobic proteins with no homology to proteins in three databases. Both unedited and edited CR6 transcripts are more abundant in bloodstream form than in procyclic form parasites. cDNA clones spanning both CR6 and the downstream NADH dehydrogenase subunit 5 (ND5) gene were isolated, indicating that mature CR6 and ND5 transcripts arise from a common precursor. Sequencing of these cDNAs revealed 37 nucleotides of overlap between the 3' end of CR6 and the 5' end of ND5. In addition, the CR6 portion of many of these molecules was extensively edited, indicating that RNA editing can precede precursor processing. These results provide the first clear demonstration of polycistronic transcription of maxicircle genes, and suggest new mechanisms by which both RNA editing and precursor processing may regulate maxicircle gene expression.