The messenger RNA decapping and recapping pathway in Trypanosoma.
ABSTRACT: The 5' terminus of trypanosome mRNA is protected by a hypermethylated cap 4 derived from spliced leader (SL) RNA. Trypanosoma brucei nuclear capping enzyme with cap guanylyltransferase and methyltransferase activities (TbCgm1) modifies the 5'-diphosphate RNA (ppRNA) end to generate an m7G SL RNA cap. Here we show that T. brucei cytoplasmic capping enzyme (TbCe1) is a bifunctional 5'-RNA kinase and guanylyltransferase that transfers a ?-phosphate from ATP to pRNA to form ppRNA, which is then capped by transfer of GMP from GTP to the RNA ?-phosphate. A Walker A-box motif in the N-terminal domain is essential for the RNA kinase activity and is targeted preferentially to a SL RNA sequence with a 5'-terminal methylated nucleoside. Silencing of TbCe1 leads to accumulation of uncapped mRNAs, consistent with selective capping of mRNA that has undergone trans-splicing and decapping. We identify T. brucei mRNA decapping enzyme (TbDcp2) that cleaves m7GDP from capped RNA to generate pRNA, a substrate for TbCe1. TbDcp2 can also remove GDP from unmethylated capped RNA but is less active at a mature cap 4 end and thus may function in RNA cap quality surveillance. Our results establish the enzymology and relevant protein catalysts of a cytoplasmic recapping pathway that has broad implications for the functional reactivation of processed mRNA ends.
Project description:The ubiquitous m7G cap of eukaryotic mRNAs and of precursors to the spliceosomal small nuclear RNAs (snRNAs) is the result of an essential RNA modification acquired during transcript elongation. In trypanosomes, the m7G cap is restricted to the spliced leader (SL) RNA and the precursors of U2, U3, and U4 snRNAs. mRNA capping in these organisms occurs posttranscriptionally by trans splicing, which transfers the capped SL sequence to the 5' ends of all mRNAs. The SL cap is the most elaborate cap structure known in nature and has been shown to consist of an m7G residue followed by four methylated nucleotides. Using Crithidia fasciculata, we have characterized and purified the guanylyltransferase (capping enzyme), which transfers GMP from GTP to the diphosphate end of RNA. The corresponding gene codes for a protein of 697 amino acids, with the carboxy-terminal half of the C. fasciculata guanylyltransferase containing the six signature motifs previously identified in yeast capping enzymes. The amino-terminal half contains a domain that displays no resemblance to any other domain associated with capping enzymes. Intriguingly, this region harbors a consensus sequence for a phosphate-binding loop which is found in ATP- and GTP-binding proteins. This two-domain structure is also present in the Trypanosoma brucei capping enzyme, which shows 44% overall identity with the C. fasciculata capping enzyme. Thus, this structure appears to be common to all trypanosomatid protozoa and defines a novel class of capping enzymes.
Project description:The RNA-dependent RNA polymerase L protein of vesicular stomatitis virus (VSV), a prototypic nonsegmented negative strand (NNS) RNA virus classified into the Rhabdoviridae family, has been used to investigate the fundamental molecular mechanisms of NNS RNA viral mRNA synthesis and processing. In vitro studies on mRNA cap formation with the VSV L protein eventually led to the discovery of the unconventional mRNA capping pathway catalyzed by the guanosine 5'-triphosphatase and RNA:GDP polyribonucleotidyltransferase (PRNTase) activities. The PRNTase activity is a novel enzymatic activity, which transfers 5'-monophosphorylated (p-) RNA from 5'-triphosphorylated (ppp-) RNA to GDP to form 5'-capped RNA (GpppRNA) in a viral mRNA-start sequence-dependent manner. This unconventional capping (pRNA transfer) reaction with PRNTase can be experimentally distinguished from the conventional capping (GMP transfer) reaction with eukaryotic GTP:RNA guanylyltransferase (GTase) on the basis of the following differences in their substrate specificity for the cap formation: PRNTase uses GDP and pppRNA, but not ppRNA, whereas GTase employs GTP, but not GDP, and ppRNA. The pRNA transfer reaction with PRNTase proceeds through a covalent enzyme-pRNA intermediate with a phosphoamide bond. Hence, to prove the PRNTase activity, it is necessary to demonstrate the following consecutive steps separately: (1) the enzyme forms a covalent enzyme-pRNA intermediate, and (2) the intermediate transfers pRNA to GDP. This article describes the methods for in vitro transcription and capping with the recombinant VSV L protein, which permit detailed characterization of its enzymatic reactions and mapping of active sites of its enzymatic domains. It is expected that these systems are adaptable to rhabdoviruses and, by extension, other NNS RNA viruses belonging to different families.
Project description:The GDP polyribonucleotidyltransferase (PRNTase) domain of the multifunctional L protein of rhabdoviruses, such as vesicular stomatitis virus (VSV) and rabies virus, catalyzes the transfer of 5'-phospho-RNA (pRNA) from 5'-triphospho-RNA (pppRNA) to GDP via a covalent enzyme-pRNA intermediate to generate a 5'-cap structure (GpppA). Here, using an improved oligo-RNA capping assay with the VSV L protein, we showed that the Michaelis constants for GDP and pppAACAG (VSV mRNA-start sequence) are 0.03 and 0.4 μM, respectively. A competition assay between GDP and GDP analogues in the GpppA formation and pRNA transfer assay using GDP analogues as pRNA acceptors indicated that the PRNTase domain recognizes the C-2-amino group, but not the C-6-oxo group, N-1-hydrogen, or N-7-nitrogen, of GDP for the cap formation. 2,6-Diaminopurine-riboside (DAP), 7-deazaguanosine (7-deaza-G), and 7-methylguanosine (m7G) diphosphates efficiently accepted pRNA, resulting in the formation of DAPpppA, 7-deaza-GpppA, and m7GpppA (cap 0), respectively. Furthermore, either the 2'- or 3'-hydroxyl group of GDP was found to be required for efficient pRNA transfer. A 5'-diphosphate form of antiviral ribavirin weakly inhibited the GpppA formation but did not act as a pRNA acceptor. These results indicate that the PRNTase domain has a unique guanosine-binding mode different from that of eukaryotic mRNA capping enzyme, guanylyltransferase. IMPORTANCE mRNAs of nonsegmented negative-strand (NNS) RNA viruses, such as VSV, possess a fully methylated cap structure, which is required for mRNA stability, efficient translation, and evasion of antiviral innate immunity in host cells. GDP polyribonucleotidyltransferase (PRNTase) is an unconventional mRNA capping enzyme of NNS RNA viruses that is distinct from the eukaryotic mRNA capping enzyme, guanylyltransferase. In this study, we studied the pRNA acceptor specificity of VSV PRNTase using various GDP analogues and identified chemical groups of GDP as essential for the substrate activity. The findings presented here are useful not only for understanding the mechanism of the substrate recognition with PRNTase but also for designing antiviral agents targeting this enzyme.
Project description:The 5' cap structure (m(7)GpppX-) is an essential feature of eukaryotic mRNA required for mRNA stability and efficient translation. Influenza virus furnishes its mRNA with this structure by a cap-snatching mechanism, in which the viral polymerase cleaves host mRNA endonucleolytically 10-13 nucleotides from the 5' end and utilizes the capped fragment as a primer to synthesize viral transcripts. Here we report a unique cap-snatching mechanism by which the yeast double-stranded RNA totivirus L-A furnishes its transcript with a cap structure derived from mRNA. Unlike influenza virus, L-A transfers only m(7)Gp from the cap donor to the 5' end of the viral transcript, thus preserving the 5' ?- and ?-phosphates of the transcript in the triphosphate linkage of the final product. This in vitro capping reaction requires His154 of the coat protein Gag, a residue essential for decapping of host mRNA and known to form m(7)Gp-His adduct. Furthermore, the synthesis of capped viral transcripts in vivo and their expression were greatly compromised by the Arg154 mutation, indicating the involvement of Gag in the cap-snatching reaction. The overall reaction and the structure around the catalytic site in Gag resemble those of guanylyltransferase, a key enzyme of cellular mRNA capping, suggesting convergent evolution. Given that Pol of L-A is confined inside the virion and unable to access host mRNA in the cytoplasm, the structural protein Gag rather than Pol catalyzing this unique cap-snatching reaction exemplifies the versatility as well as the adaptability of eukaryotic RNA viruses.
Project description:The notion that decapping leads irreversibly to messenger RNA (mRNA) decay was contradicted by the identification of capped transcripts missing portions of their 5' ends and a cytoplasmic complex that can restore the cap on uncapped mRNAs. In this study, we used accumulation of uncapped transcripts in cells inhibited for cytoplasmic capping to identify the targets of this pathway. Inhibition of cytoplasmic capping results in the destabilization of some transcripts and the redistribution of others from polysomes to nontranslating messenger ribonucleoproteins, where they accumulate in an uncapped state. Only a portion of the mRNA transcriptome is affected by cytoplasmic capping, and its targets encode proteins involved in nucleotide binding, RNA and protein localization, and the mitotic cell cycle. The 3' untranslated regions of recapping targets are enriched for AU-rich elements and microRNA binding sites, both of which function in cap-dependent mRNA silencing. These findings identify a cyclical process of decapping and recapping that we term cap homeostasis.
Project description:The 5' RNA cap structure (m7GpppRNA) is a key feature of eukaryotic mRNAs with important roles in stability, splicing, polyadenylation, mRNA export, and translation. Higher eukaryotes can further modify this minimal cap structure with the addition of a methyl group on the ribose 2'-O position of the first transcribed nucleotide (m7GpppNmpRNA) and sometimes on the adjoining nucleotide (m7GpppNmpNmpRNA). In higher eukaryotes, the DXO protein was previously shown to be responsible for both decapping and degradation of RNA transcripts harboring aberrant 5' ends such as pRNA, pppRNA, GpppRNA, and surprisingly, m7GpppRNA. It was proposed that the interaction of the cap binding complex with the methylated cap would prevent degradation of m7GpppRNAs by DXO. However, the critical role of the 2'-O-methylation found in higher eukaryotic cap structures was not previously addressed. In the present study, we demonstrate that DXO possesses both decapping and exoribonuclease activities toward incompletely capped RNAs, only sparing RNAs with a 2'-O-methylated cap structure. Fluorescence spectroscopy assays also revealed that the presence of the 2'-O-methylation on the cap structure drastically reduces the affinity of DXO for RNA. Moreover, immunofluorescence and structure-function assays also revealed that a nuclear localisation signal is located in the amino-terminus region of DXO. Overall, these results are consistent with a quality control mechanism in which DXO degrades incompletely capped RNAs.
Project description:The spliced-leader (SL) RNA plays a key role in the biogenesis of mRNA in trypanosomes by providing the m(7)G-capped SL sequence to the 5' end of every mRNA. The cap structure of the SL RNA is unique in eukaryotes with 4 nucleotides after the cap carrying a total of seven methyl groups and by convention is referred to as "cap 4". Although the enzymatic machinery for cap addition has been characterized in several organisms, including Trypanosoma brucei, the identification of methyltransferases dedicated to the generation of higher order cap structures has lagged behind, except in viruses. Here we describe T. brucei MT57 (TbMT57), a primarily nuclear polypeptide with structural and functional similarities to vaccinia virus VP39, a bifunctional protein acting at the mRNA 5' end as a cap-specific 2'-O-methyltransferase. Down-regulation by RNAi or genetic ablation of TbMT57 resulted in the accumulation of SL RNA missing 2'-O-methyl groups at positions +3 and +4 and thus bearing a cap 2 rather than a cap 4. Furthermore, competitive binding studies indicated that modifications at the +3 and +4 positions are important for binding to the nuclear cap-binding complex. Genetic ablation of MT57 resulted in viable cells with no apparent defect in SL RNA trans-splicing, suggesting that MT57 is not essential or that trypanosomes have developed alternate mechanisms to counteract the absence of this protein. Interestingly, MT57 homologs are only found in trypanosomatid protozoa that have a cap 4 structure and in poxviruses, of which vaccinia virus is a prototype.
Project description:Flaviviruses are small, capped positive sense RNA viruses that replicate in the cytoplasm of infected cells. Dengue virus and other related flaviviruses have evolved RNA capping enzymes to form the viral RNA cap structure that protects the viral genome and directs efficient viral polyprotein translation. The N-terminal domain of NS5 possesses the methyltransferase and guanylyltransferase activities necessary for forming mature RNA cap structures. The mechanism for flavivirus guanylyltransferase activity is currently unknown, and how the capping enzyme binds its diphosphorylated RNA substrate is important for deciphering how the flavivirus guanylyltransferase functions. In this report we examine how flavivirus NS5 N-terminal capping enzymes bind to the 5' end of the viral RNA using a fluorescence polarization-based RNA binding assay. We observed that the K(D) for RNA binding is approximately 200 nM Dengue, Yellow Fever, and West Nile virus capping enzymes. Removal of one or both of the 5' phosphates reduces binding affinity, indicating that the terminal phosphates contribute significantly to binding. RNA binding affinity is negatively affected by the presence of GTP or ATP and positively affected by S-adensyl methoninine (SAM). Structural superpositioning of the dengue virus capping enzyme with the Vaccinia virus VP39 protein bound to RNA suggests how the flavivirus capping enzyme may bind RNA, and mutagenesis analysis of residues in the putative RNA binding site demonstrate that several basic residues are critical for RNA binding. Several mutants show differential binding to 5' di-, mono-, and un-phosphorylated RNAs. The mode of RNA binding appears similar to that found with other methyltransferase enzymes, and a discussion of diphosphorylated RNA binding is presented.
Project description:The multifunctional RNA-dependent RNA polymerase L protein of vesicular stomatitis virus catalyzes unconventional pre-mRNA capping via the covalent enzyme-pRNA intermediate formation, which requires the histidine-arginine (HR) motif in the polyribonucleotidyltransferase domain. Here, the effects of cap-defective mutations in the HR motif on transcription were analyzed using an in vitro reconstituted transcription system. The wild-type L protein synthesized the leader RNA from the 3'-end of the genome followed by 5'-capped and 3'-polyadenylated mRNAs from internal genes by a stop-start transcription mechanism. Cap-defective mutants efficiently produced the leader RNA, but displayed aberrant stop-start transcription using cryptic termination and initiation signals within the first gene, resulting in sequential generation of ?40-nucleotide transcripts with 5'-ATP from a correct mRNA-start site followed by a 28-nucleotide transcript and long 3'-polyadenylated transcript initiated with non-canonical GTP from atypical start sites. Frequent transcription termination and re-initiation within the first gene significantly attenuated the production of downstream mRNAs. Consistent with the inability of these mutants in in vitro mRNA synthesis and capping, these mutations were lethal to virus replication in cultured cells. These findings indicate that viral mRNA capping is required for accurate stop-start transcription as well as mRNA stability and translation and, therefore, for virus replication in host cells.
Project description:mRNA cap 1 2'-O-ribose methylation is a widespread modification that is implicated in processing, trafficking, and translational control in eukaryotic systems. The eukaryotic enzyme has yet to be identified. In kinetoplastid flagellates trans-splicing of spliced leader (SL) to polycistronic precursors conveys a hypermethylated cap 4, including a cap 0 m7G and seven additional methylations on the first 4 nucleotides, to all nuclear mRNAs. We report the first eukaryotic cap 1 2'-O-ribose methyltransferase, TbMTr1, a member of a conserved family of viral and eukaryotic enzymes. Recombinant TbMTr1 methylates the ribose of the first nucleotide of an m7G-capped substrate. Knockdowns and null mutants of TbMTr1 in Trypanosoma brucei grow normally, with loss of 2'-O-ribose methylation at cap 1 on substrate SL RNA and U1 small nuclear RNA. TbMTr1-null cells have an accumulation of cap 0 substrate without further methylation, while spliced mRNA is modified efficiently at position 4 in the absence of 2'-O-ribose methylation at position 1; downstream cap 4 methylations are independent of cap 1. Based on TbMTr1-green fluorescent protein localization, 2'-O-ribose methylation at position 1 occurs in the nucleus. Accumulation of 3'-extended SL RNA substrate indicates a delay in processing and suggests a synergistic role for cap 1 in maturation.