Trypanosome mRNA recapping is triggered by hypermethylation originating from cap 4.

RNA methylation adjacent to the 5' cap plays a critical role in controlling mRNA stability and protein synthesis. In trypanosomes the 5'-terminus of mRNA is protected by hypermethylated cap 4. Trypanosomes encode a cytoplasmic recapping enzyme TbCe1 which possesses an RNA kinase and guanylyltransferase activities that can convert decapped 5'-monophosphate-terminated pRNA into GpppRNA. Here, we demonstrated that the RNA kinase activity is stimulated by two orders of magnitude on a hypermethylated pRNA derived from cap 4. The N6, N6-2'-O trimethyladenosine modification on the first nucleotide was primarily accountable for enhancing both the RNA kinase and the guanylyltransferase activity of TbCe1. In contrast, N6 methyladenosine severely inhibits the guanylyltransferase activity of the mammalian capping enzyme. Furthermore, we showed that TbCmt1 cap (guanine N7) methyltransferase was localized in the cytoplasm, and its activity was also stimulated by hypermethylation at 2'-O ribose, suggesting that TbCe1 and TbCmt1 act together as a recapping enzyme to regenerate translatable mRNA from decapped mRNA. Our result establishes the functional role of cap 4 hypermethylation in recruitment and activation of mRNA recapping pathway. Methylation status at the 5'-end of transcripts could serve as a chemical landmark to selectively regulate the level of functional mRNA by recapping enzymes.

Crystal structures of the RNA triphosphatase from Trypanosoma cruzi provide insights into how it recognizes the 5'-end of the RNA substrate.

RNA triphosphatase catalyzes the first step in mRNA cap formation, hydrolysis of the terminal phosphate from the nascent mRNA transcript. The RNA triphosphatase from the protozoan parasite Trypanosoma cruzi, TcCet1, belongs to the family of triphosphate tunnel metalloenzymes (TTMs). TcCet1 is a promising antiprotozoal drug target because the mechanism and structure of the protozoan RNA triphosphatases are completely different from those of the RNA triphosphatases found in mammalian and arthropod hosts. Here, we report several crystal structures of the catalytically active form of TcCet1 complexed with a divalent cation and an inorganic tripolyphosphate in the active-site tunnel at 2.20-2.51 Å resolutions. The structures revealed that the overall structure, the architecture of the tunnel, and the arrangement of the metal-binding site in TcCet1 are similar to those in other TTM proteins. On the basis of the position of three sulfate ions that cocrystallized on the positively charged surface of the protein and results obtained from mutational analysis, we identified an RNA-binding site in TcCet1. We conclude that the 5'-end of the triphosphate RNA substrate enters the active-site tunnel directionally. The structural information reported here provides valuable insight into designing inhibitors that could specifically block the entry of the triphosphate RNA substrate into the TTM-type RNA triphosphatases of T. cruzi and related pathogens.

Structural and mutational analysis of archaeal ATP-dependent RNA ligase identifies amino acids required for RNA binding and catalysis.

An ATP-dependent RNA ligase from Methanobacterium thermoautotrophicum (MthRnl) catalyzes intramolecular ligation of single-stranded RNA to form a closed circular RNA via covalent ligase-AMP and RNA-adenylylate intermediate. Here, we report the X-ray crystal structures of an MthRnl•ATP complex as well as the covalent MthRnl-AMP intermediate. We also performed structure-guided mutational analysis to survey the functions of 36 residues in three component steps of the ligation pathway including ligase-adenylylation (step 1), RNA adenylylation (step 2) and phosphodiester bond synthesis (step 3). Kinetic analysis underscored the importance of motif 1a loop structure in promoting phosphodiester bond synthesis. Alanine substitutions of Thr117 or Arg118 favor the reverse step 2 reaction to deadenylate the 5'-AMP from the RNA-adenylate, thereby inhibiting step 3 reaction. Tyr159, Phe281 and Glu285, which are conserved among archaeal ATP-dependent RNA ligases and are situated on the surface of the enzyme, are required for RNA binding. We propose an RNA binding interface of the MthRnl based on the mutational studies and two sulfate ions that co-crystallized at the active site cleft in the MthRnl-AMP complex.

The messenger RNA decapping and recapping pathway in Trypanosoma.

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 γ-phosphate from ATP to pRNA to form ppRNA, which is then capped by transfer of GMP from GTP to the RNA ß-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.

Genetic and Functional Analyses of Archaeal ATP-Dependent RNA Ligase in C/D Box sRNA Circularization and Ribosomal RNA Processing.

RNA ligases play important roles in repairing and circularizing RNAs post-transcriptionally. In this study, we generated an allelic knockout of ATP-dependent RNA ligase (Rnl) in the hyperthermophilic archaeon Thermococcus kodakarensis to identify its biological targets. A comparative analysis of circular RNA reveals that the Rnl-knockout strain represses circularization of C/D box sRNAs without affecting the circularization of tRNA and rRNA processing intermediates. Recombinant archaeal Rnl could circularize C/D box sRNAs with a mutation in the conserved C/D box sequence element but not when the terminal stem structures were disrupted, suggesting that proximity of the two ends could be critical for intramolecular ligation. Furthermore, T. kodakarensis accumulates aberrant RNA fragments derived from ribosomal RNA in the absence of Rnl. These results suggest that Rnl is responsible for C/D box sRNA circularization and may also play a role in ribosomal RNA processing.

Archaeal RNA ligase is a homodimeric protein that catalyzes intramolecular ligation of single-stranded RNA and DNA.

RNA ligases participate in repair, splicing and editing pathways that either reseal broken RNAs or alter their primary structure. Here, we report the characterization of an RNA ligase from the thermophilic archaeon, Methanobacterium thermoautotrophicum. The 381-amino acid Methanobacterium RNA ligase (MthRnl) catalyzes intramolecular ligation of 5'-PO(4) single-strand RNA to form a covalently closed circular RNA molecule through ligase-adenylylate and RNA-adenylylate (AppRNA) intermediates. At the optimal temperature of 65 degrees C, AppRNA was predominantly ligated to a circular product. In contrast, at 35 degrees C, phosphodiester bond formation was suppressed and the majority of the AppRNA was deadenylylated. Sedimentation analysis indicates that MthRnl is a homodimer in solution. The C-terminal 127-amino acid segment is required for dimerization, is itself capable of oligomeization and acts in trans to inhibit the ligation activity of native MthRnl. MthRnl can also join single-stranded DNA to form a circular molecule. The lack of specificity for RNA and DNA by MthRnl may exemplify an undifferentiated ancestral stage in the evolution of ATP-dependent ligases.

Functional characterization of a 48 kDa Trypanosoma brucei cap 2 RNA methyltransferase.

Kinetoplastid mRNAs possess a unique hypermethylated cap 4 structure derived from the standard m7GpppN cap structure, with 2'-O methylations on the first four ribose sugars and additional base methylations on the first adenine and the fourth uracil. While the enzymes responsible for m7GpppN cap 0 formations has been characterized in Trypanosoma brucei, the mechanism of cap 4 methylation and the role of the hypermethylated structure remain unclear. Here, we describe the characterization of a 48 kDa T.brucei 2'-O nucleoside methyltransferase (TbCom1). Recombinant TbCom1 transfers the methyl group from S-adenosylmethionine (AdoMet) to the 2'-OH of the second nucleoside of m7GpppNpNp-RNA to form m7GpppNpNmp-RNA. TbCom1 is also capable of converting cap 1 RNA to cap 2 RNA. The methyl transfer reaction is dependent on the m7GpppN cap, as the enzyme does not form a stable interaction with GpppN-terminated RNA. Mutational analysis establishes that the TbCom1 and vaccinia virus VP39 methyltransferases share mechanistic similarities in AdoMet- and cap-recognition. Two aromatic residues, Tyr18 and Tyr187, may participate in base-stacking interactions with the guanine ring of the cap, as the removal of each of these aromatic side-chains abolishes cap-specific RNA-binding.

Detection and Identification of Uncapped RNA by Ligation-Mediated Reverse Transcription Polymerase Chain Reaction.

The 5'-cap structure is an essential feature in eukaryotic mRNA required for mRNA stability and enhancement of translation. Ceratin transcripts are selectively silenced by decapping in the cytoplasm and later become translationally active again by acquiring the cap structure to regenerate translatable mRNAs. Identification of uncapped mRNA transcripts will reveal how gene expression is regulated by the mRNA recapping pathway. What follows is a sensitive method to detect and identify the uncapped mRNA from the cells. The technique consists of three parts: selective ligation of anchor RNA to the 5'-end of monophosphate RNA by double-strand RNA ligase, conversion of ligated RNA product into cDNA by reverse transcription, and amplification of a specific cDNA by polymerase chain reaction.

Cleavage of 3'-terminal adenosine by archaeal ATP-dependent RNA ligase.

Methanothermobacter thermoautotrophicus RNA ligase (MthRnl) catalyzes formation of phosphodiester bonds between the 5'-phosphate and 3'-hydroxyl termini of single-stranded RNAs. It can also react with RNA with a 3'-phosphate end to generate a 2',3'-cyclic phosphate. Here, we show that MthRnl can additionally remove adenosine from the 3'-terminus of the RNA to produce 3'-deadenylated RNA, RNA(3'-rA). This 3'-deadenylation activity is metal-dependent and requires a 2'-hydroxyl at both the terminal adenosine and the penultimate nucleoside. Residues that contact the ATP/AMP in the MthRnl crystal structures are essential for the 3'-deadenylation activity, suggesting that 3'-adenosine may occupy the ATP-binding pocket. The 3'-end of cleaved RNA(3'-rA) consists of 2',3'-cyclic phosphate which protects RNA(3'-rA) from ligation and further deadenylation. These findings suggest that ATP-dependent RNA ligase may act on a specific set of 3'-adenylated RNAs to regulate their processing and downstream biological events.

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.

Nanomolar Inhibitors of Trypanosoma brucei RNA Triphosphatase.

UNLABELLED: Eukaryal taxa differ with respect to the structure and mechanism of the RNA triphosphatase (RTPase) component of the mRNA capping apparatus. Protozoa, fungi, and certain DNA viruses have a metal-dependent RTPase that belongs to the triphosphate tunnel metalloenzyme (TTM) superfamily. Because the structures, active sites, and chemical mechanisms of the TTM-type RTPases differ from those of mammalian RTPases, the TTM RTPases are potential targets for antiprotozoal, antifungal, and antiviral drug discovery. Here, we employed RNA interference (RNAi) knockdown methods to show that Trypanosoma brucei RTPase Cet1 (TbCet1) is necessary for proliferation of procyclic cells in culture. We then conducted a high-throughput biochemical screen for small-molecule inhibitors of the phosphohydrolase activity of TbCet1. We identified several classes of chemicals-including chlorogenic acids, phenolic glycopyranosides, flavonoids, and other phenolics-that inhibit TbCet1 with nanomolar to low-micromolar 50% inhibitory concentrations (IC50s). We confirmed the activity of these compounds, and tested various analogs thereof, by direct manual assays of TbCet1 phosphohydrolase activity. The most potent nanomolar inhibitors included tetracaffeoylquinic acid, 5-galloylgalloylquinic acid, pentagalloylglucose, rosmarinic acid, and miquelianin. TbCet1 inhibitors were less active (or inactive) against the orthologous TTM-type RTPases of mimivirus, baculovirus, and budding yeast (Saccharomyces cerevisiae). Our results affirm that a TTM RTPase is subject to potent inhibition by small molecules, with the caveat that parallel screens against TTM RTPases from multiple different pathogens may be required to fully probe the chemical space of TTM inhibition. IMPORTANCE: The stark differences between the structure and mechanism of the RNA triphosphatase (RTPase) component of the mRNA capping apparatus in pathogenic protozoa, fungi, and viruses and those of their metazoan hosts highlight RTPase as a target for anti-infective drug discovery. Protozoan, fungal, and DNA virus RTPases belong to the triphosphate tunnel metalloenzyme family. This study shows that a protozoan RTPase, TbCet1 from Trypanosoma brucei, is essential for growth of the parasite in culture and identifies, via in vitro screening of chemical libraries, several classes of potent small-molecule inhibitors of TbCet1 phosphohydrolase activity.

Trypanosoma brucei encodes a bifunctional capping enzyme essential for cap 4 formation on the spliced leader RNA.

The 5' end of kinetoplastid mRNA possesses a hypermethylated cap 4 structure, which is derived from standard m7GpppN (cap 0) with additional methylations at seven sites within the first four nucleosides on the spliced leader RNA. In addition to TbCe1 guanylyltransferase and TbCmt1 (guanine N-7) methyltransferase, Trypanosoma brucei encodes a second cap 0 forming enzyme. TbCgm1 (T. brucei cap guanylyltransferase-methyltransferase) is a novel bifunctional capping enzyme consisting of an amino-terminal guanylyltransferase domain and a carboxyl-terminal methyltransferase domain. Recombinant TbCgm1 transfers the GMP to spliced leader RNA (SL RNA) via a covalent enzyme-GMP intermediate, and methylates the guanine N-7 position of the GpppN-terminated RNA to form cap 0 structure. The two domains can function autonomously in vitro. TbCGM1 is essential for parasite growth. Silencing of TbCGM1 by RNA interference increased the abundance of uncapped SL RNA and lead to accumulation of hypomethylated SL RNA. In contrast, silencing of TbCE1 and TbCMT1 did not affect parasite growth or SL RNA capping. We conclude that TbCgm1 specifically cap SL RNA, and cap 0 is a prerequisite for subsequent methylation events leading to the formation of mature SL RNA.

Distinct RNA elements confer specificity to flavivirus RNA cap methylation events.

The 5' end of the flavivirus plus-sense RNA genome contains a type 1 cap (m(7)GpppAmG), followed by a conserved stem-loop structure. We report that nonstructural protein 5 (NS5) from four serocomplexes of flaviviruses specifically methylates the cap through recognition of the 5' terminus of viral RNA. Distinct RNA elements are required for the methylations at guanine N-7 on the cap and ribose 2'-OH on the first transcribed nucleotide. In a West Nile virus (WNV) model, N-7 cap methylation requires specific nucleotides at the second and third positions and a 5' stem-loop structure; in contrast, 2'-OH ribose methylation requires specific nucleotides at the first and second positions, with a minimum 5' viral RNA of 20 nucleotides. The cap analogues GpppA and m(7)GpppA are not active substrates for WNV methytransferase. Footprinting experiments using Gppp- and m(7)Gppp-terminated RNAs suggest that the 5' termini of RNA substrates interact with NS5 during the sequential methylation reactions. Cap methylations could be inhibited by an antisense oligomer targeting the first 20 nucleotides of WNV genome. The viral RNA-specific cap methylation suggests methyltransferase as a novel target for flavivirus drug discovery.

Characterization of a Trypanosoma brucei RNA cap (guanine N-7) methyltransferase.

The m7GpppN cap structure of eukaryotic mRNA is formed by the sequential action of RNA triphosphatase, guanylyltransferase, and (guanine N-7) methyltransferase. In trypanosomatid protozoa, the m7GpppN is further modified by seven methylation steps within the first four transcribed nucleosides to form the cap 4 structure. The RNA triphosphatase and guanylyltransferase components have been characterized in Trypanosoma brucei. Here we describe the identification and characterization of a T. brucei (guanine N-7) methyltransferase (TbCmt1). Sequence alignment of the 324-amino acid TbCmt1 with the corresponding enzymes from human (Hcm1), fungal (Abd1), and microsporidian (Ecm1) revealed the presence of conserved residues known to be essential for methyltransferase activity. Purified recombinant TbCmt1 catalyzes the transfer of a methyl group from S-adenosylmethionine to the N-7 position of the cap guanine in GpppN-terminated RNA to form the m7GpppN cap. TbCmt1 also methylates GpppG and GpppA but not GTP or dGTP. Mutational analysis of individual residues of TbCmt1 that were predicted-on the basis of the crystal structure of Ecm1--to be located at or near the active site identified six conserved residues in the putative AdoMet- or cap-binding pocket that caused significant reductions in TbCmt1 methyltransferase activity. We also report the identification of a second T. brucei RNA (guanine N-7) cap methyltransferase (named TbCgm1). The 1050-amino acid TbCgm1 consists of a C-terminal (guanine N-7) methyltransferase domain, which is homologous with TbCmt1, and an N-terminal guanylyltransferase domain, which contains signature motifs found in the nucleotidyl transferase superfamily.

Identification of microRNAs of the herpesvirus family.

Epstein-Barr virus (EBV or HHV4), a member of the human herpesvirus (HHV) family, has recently been shown to encode microRNAs (miRNAs). In contrast to most eukaryotic miRNAs, these viral miRNAs do not have close homologs in other viral genomes or in the genome of the human host. To identify other miRNA genes in pathogenic viruses, we combined a new miRNA gene prediction method with small-RNA cloning from several virus-infected cell types. We cloned ten miRNAs in the Kaposi sarcoma-associated virus (KSHV or HHV8), nine miRNAs in the mouse gammaherpesvirus 68 (MHV68) and nine miRNAs in the human cytomegalovirus (HCMV or HHV5). These miRNA genes are expressed individually or in clusters from either polymerase (pol) II or pol III promoters, and share no substantial sequence homology with one another or with the known human miRNAs. Generally, we predicted miRNAs in several large DNA viruses, and we could neither predict nor experimentally identify miRNAs in the genomes of small RNA viruses or retroviruses.

Bacteriophage T4 RNA ligase 2 (gp24.1) exemplifies a family of RNA ligases found in all phylogenetic domains.

RNA ligases participate in repair, splicing, and editing pathways that either reseal broken RNAs or alter their primary structure. Bacteriophage T4 RNA ligase (gp63) is the best-studied member of this class of enzymes, which includes yeast tRNA ligase and trypanosome RNA-editing ligases. Here, we identified another RNA ligase from the bacterial domain--a second RNA ligase (Rnl2) encoded by phage T4. Purified Rnl2 (gp24.1) catalyzes intramolecular and intermolecular RNA strand joining through ligase-adenylate and RNA-adenylate intermediates. Mutational analysis identifies amino acids required for the ligase-adenylation or phosphodiester synthesis steps of the ligation reaction. The catalytic residues of Rnl2 are located within nucleotidyl transferase motifs I, IV, and V that are conserved in DNA ligases and RNA capping enzymes. Rnl2 has scant amino acid similarity to T4 gp63. Rather, Rnl2 exemplifies a distinct ligase family, defined by variant motifs, that includes the trypanosome-editing ligases and a group of putative RNA ligases encoded by eukaryotic viruses (baculoviruses and an entomopoxvirus) and many species of archaea. These findings have implications for the evolution of covalent nucleotidyl transferases and virus-host dynamics based on RNA restriction and repair.

Portability and fidelity of RNA-repair systems.

Yeast tRNA ligase (Trl1) is an essential enzyme that converts cleaved tRNA half-molecules into spliced tRNAs containing a 2'-PO(4), 3'-5' phosphodiester at the splice junction. Trl1 also catalyzes splicing of HAC1 mRNA during the unfolded protein response. Trl1 performs three reactions: the 2',3'-cyclic phosphate of the proximal RNA fragment is hydrolyzed to a 3'-OH, 2'-PO(4) by a cyclic phosphodiesterase; the 5'-OH of the distal RNA fragment is phosphorylated by a GTP-dependent polynucleotide kinase; and the 3'-OH, 2'-PO(4), and 5'-PO(4) ends are then sealed by an ATP-dependent RNA ligase. The removal of the 2'-PO(4) at the splice junction is catalyzed by the essential enzyme Tpt1, which transfers the RNA 2'-PO(4) to NAD(+) to form ADP-ribose 1"-2"-cyclic phosphate. Here, we show that the bacteriophage T4 enzymes RNA ligase 1 and polynucleotide kinase/phosphatase can fulfill the tRNA and HAC1 mRNA splicing functions of yeast Trl1 in vivo and bypass the requirement for Tpt1. These results attest to the portability of RNA-repair systems, notwithstanding the significant differences in the specificities, mechanisms, and reaction intermediates of the individual yeast and T4 enzymes responsible for the RNA healing and sealing steps. We surmise that Tpt1 and its unique metabolite ADP-ribose 1"-2"-cyclic phosphate do not play essential roles in yeast independent of the tRNA-splicing reaction. Our finding that one-sixth of spliced HAC1 mRNAs in yeast cells containing the T4 RNA-repair system suffered deletion of a single nucleotide at the 3' end of the splice-donor site suggests a model whereby the yeast RNA-repair system evolved a requirement for the 2'-PO(4) for RNA ligation to suppress inappropriate RNA recombination.

Structure-function analysis of T4 RNA ligase 2.

Bacteriophage T4 RNA ligase 2 (Rnl2) exemplifies a polynucleotide ligase family that includes the trypanosome RNA-editing ligases and putative RNA ligases encoded by eukaryotic viruses and archaea. Here we analyzed 12 individual amino acids of Rnl2 that were identified by alanine scanning as essential for strand joining. We determined structure-activity relationships via conservative substitutions and examined mutational effects on the isolated steps of ligase adenylylation and phosphodiester bond formation. The essential residues of Rnl2 are located within conserved motifs that define a superfamily of nucleotidyl transferases that act via enzyme-(lysyl-N)-NMP intermediates. Our mutagenesis results underscore a shared active site architecture in Rnl2-like ligases, DNA ligases, and mRNA capping enzymes. They also highlight two essential signature residues, Glu(34) and Asn(40), that flank the active site lysine nucleophile (Lys(35)) and are unique to the Rnl2-like ligase family.

RNA substrate specificity and structure-guided mutational analysis of bacteriophage T4 RNA ligase 2.

Here we report that bacteriophage T4 RNA ligase 2 (Rnl2) is an efficient catalyst of RNA ligation at a 3'-OH/5'-PO(4) nick in a double-stranded RNA or an RNA.DNA hybrid. The critical role of the template strand in approximating the reactive 3'-OH and 5'-PO(4) termini is underscored by the drastic reductions in the RNA-sealing activity of Rnl2 when the duplex substrates contain gaps or flaps instead of nicks. RNA nick joining requires ATP and a divalent cation cofactor (either Mg or Mn). Neither dATP, GTP, CTP, nor UTP can substitute for ATP. We identify by alanine scanning seven functionally important amino acids (Tyr-5, Arg-33, Lys-54, Gln-106, Asp-135, Arg-155, and Ser-170) within the N-terminal nucleotidyl-transferase domain of Rnl2 and impute specific roles for these residues based on the crystal structure of the AMP-bound enzyme. Mutational analysis of 14 conserved residues in the C-terminal domain of Rnl2 identifies 3 amino acids (Arg-266, Asp-292, and Glu-296) as essential for ligase activity. Our findings consolidate the evolutionary connections between bacteriophage Rnl2 and the RNA-editing ligases of kinetoplastid protozoa.