Archaeal Pus10 proteins can produce both pseudouridine 54 and 55 in tRNA

Pus10, a recently identified pseudouridine (Ψ) synthase, does not belong to any of the five commonly identified families of Ψ synthases. Pyrococcus furiosus Pus10 has been shown to produce Ψ55 in tRNAs. However, in vitro studies have identified another mechanism for tRNA Ψ55 production in Archaea, which uses Cbf5 and other core proteins of the H/ACA ribonucleoprotein complex, in a guide RNA-independent manner. Pus10 homologs have been observed in nearly all sequenced archaeal genomes and in some higher eukaryotes, but not in yeast and bacteria. This coincides with the presence of Ψ54 in the tRNAs of Archaea and higher eukaryotes and its absence in yeast and bacteria. No tRNA Ψ54 synthase has been reported so far. Here, using recombinant Methanocaldococcus jannaschii and P. furiosus Pus10, we show that these proteins can function as synthase for both tRNA Ψ54 and Ψ55. The two modifications seem to occur independently. Salt concentration dependent variations in these activities of both proteins are observed. The Ψ54 synthase activity of M. jannaschii protein is robust, while the same activity of P. furiosus protein is weak. Probable reasons for these differences are discussed. Furthermore, unlike bacterial TruB and yeast Pus4, archaeal Pus10 does not require a U54•A58 reverse Hoogstein base pair and pyrimidine at position 56 to convert tRNA U55 to Ψ55. The homology of eukaryal Pus10 with archaeal Pus10 suggests that the former may also have a tRNA Ψ54 synthase activity.     

Pseudouridine formation in archaeal RNAs: The case of Haloferax volcanii

Pseudouridine (Ψ), the isomer of uridine, is commonly found at various positions of noncoding RNAs of all organisms. Ψ residues are formed by a number of single- or multisite specific Ψ synthases, which generally act as stand-alone proteins. In addition, in Eukarya and Archaea, specific ribonucleoprotein complexes, each containing a distinct box H/ACA guide RNA and four core proteins, can produce Ψ at many sites of different cellular RNAs. Cbf5 is the core Ψ synthase in these complexes. Using Haloferax volcanii as an archaeal model organism, we show that, contrary to eukaryotes, the Cbf5 homolog (HVO_2493) is not essential in this archaeon. The Cbf5-deleted strain of H. volcanii completely lacks Ψ at positions 1940, 1942, 2605, and 2591 (Escherichia coli positions 1915, 1917, 2572, and 2586) of its 23S rRNA, and contains reduced steady-state levels of some box H/ACA RNAs. Archaeal Cbf5 is known to have tRNA Ψ55 synthase activity in vitro but we could not confirm this activity in vivo in H. volcanii. Conversely, the Pus10 (previously PsuX) homolog (HVO_1979), which can produce tRNA Ψ55, as well as Ψ54 in vitro, is shown here to be essential in H. volcanii, whereas the corresponding tRNA Ψ55 synthases, Pus4 and TruB, are not essential in yeast and E. coli, respectively. Finally, we demonstrate that HVO_1852, the TruA/Pus3 homolog, is responsible for the pseudouridylation of position 39 in H. volcanii tRNAs and that the corresponding gene is not essential.     

An arginine-aspartate network in the active site of bacterial TruB is critical for catalyzing pseudouridine formation

Pseudouridine synthases introduce the most common RNA modification and likely use the same catalytic mechanism. Besides a catalytic aspartate residue, the contributions of other residues for catalysis of pseudouridine formation are poorly understood. Here, we have tested the role of a conserved basic residue in the active site for catalysis using the bacterial pseudouridine synthase TruB targeting U55 in tRNAs. Substitution of arginine 181 with lysine results in a 2500-fold reduction of TruB’s catalytic rate without affecting tRNA binding. Furthermore, we analyzed the function of a second-shell aspartate residue (D90) that is conserved in all TruB enzymes and interacts with C56 of tRNA. Site-directed mutagenesis, biochemical and kinetic studies reveal that this residue is not critical for substrate binding but influences catalysis significantly as replacement of D90 with glutamate or asparagine reduces the catalytic rate 30- and 50-fold, respectively. In agreement with molecular dynamics simulations of TruB wild type and TruB D90N, we propose an electrostatic network composed of the catalytic aspartate (D48), R181 and D90 that is important for catalysis by fine-tuning the D48-R181 interaction. Conserved, negatively charged residues similar to D90 are found in a number of pseudouridine synthases, suggesting that this might be a general mechanism.     

Structural and functional evidence of high specificity of Cbf5 for ACA trinucleotide

Cbf5 is the catalytic subunit of the H/ACA small nucleolar/Cajal body ribonucleoprotein particles (RNPs) responsible for site specific isomerization of uridine in ribosomal and small nuclear RNA. Recent evidence from studies on archaeal Cbf5 suggests its second functional role in modifying tRNA U55 independent of guide RNA. In order to act both as a stand-alone and a RNP pseudouridine synthase, Cbf5 must differentiate features in H/ACA RNA from those in tRNA or rRNA. Most H/ACA RNAs contain a hallmark ACA trinucleotide downstream of the H/ACA motif. Here we challenged an archaeal Cbf5 (in the form of a ternary complex with its accessory proteins Nop10 and Gar1) with T-stem-loop RNAs with or without ACA trinucleotide in the stem. Although these substrates were previously shown to be substrates for the bacterial stand-alone pseudouridine synthase TruB, the Cbf5-Nop10-Gar1 complex was only able to modify those without ACA trinucleotide. A crystal structure of Cbf5-Nop10-Gar1 trimer bound with an ACA-containing T-stem-loop revealed that the ACA trinucleotide detracted Cbf5 from the stand-alone binding mode, thereby suggesting that the H/ACA RNP-associated function of Cbf5 likely supersedes its stand-alone function.     

Differential roles of archaeal box H/ACA proteins in guide RNA-dependent and independent pseudouridine formation.

RNA-guided pseudouridine (Psi) synthesis in Archaea and Eukarya requires a four-protein one-RNA containing box H/ACA ribonucleoprotein (RNP) complex. The proteins in the archaeal RNP are aCbf5, aNop10, aGar1 and L7Ae. Pyrococcus aCbf5-aNop10 is suggested to be the minimal catalytic core in this synthesis and the activity is enhanced by L7Ae and aGar1. The protein aCbf5 is homologous to eukaryal Cbf5 (dyskerin, NAP57) as well as to bacterial TruB and eukaryal Pus4; the last two produce YPsi55 in tRNAs in a guide RNA-independent manner. Here, using recombinant Methanocaldococcus jannaschii proteins, we report that aCbf5 and aGar1 together can function as a tRNA Psi55 synthase in a guide RNA-independent manner. This activity is enhanced by aNop10, but not by L7Ae. The aCbf5 alone can also produce Psi55 in tRNAs that contain the canonical 3'-CCA sequence and this activity is stimulated by aGar1. These results suggest that the roles of accessory proteins are different in guide RNA-dependent and independent Psi synthesis by aCbf5. The presence of conserved C (or U) and A at tRNA positions 56 and 58, respectively, which are required for TruB/Pus4 activity, is not essential for aCbf5-mediated Psi55 formation. Conserved A58 in tRNA normally forms a tertiary reverse Hoogstein base pair with an equally conserved U54. This base pair is recognized by TruB. Apparently aCbf5 does not require this base pair to recognize U55 for conversion to Psi55.     

Formation of a stalled early intermediate of pseudouridine synthesis monitored by real-time FRET

Pseudouridine is the most abundant of more than 100 chemically distinct natural ribonucleotide modifications. Its synthesis consists of an isomerization reaction of a uridine residue in the RNA chain and is catalyzed by pseudouridine synthases. The unusual reaction mechanism has become the object of renewed research effort, frequently involving replacement of the substrate uridines with 5-fluorouracil (f⁵U). f⁵U is known to be a potent inhibitor of pseudouridine synthase activity, but the effect varies among the target pseudouridine synthases. Derivatives of f⁵U have previously been detected, which are thought to be either hydrolysis products of covalent enzyme-RNA adducts, or isomerization intermediates. Here we describe the interaction of pseudouridine synthase 1 (Pus1p) with f⁵U-containing tRNA. The interaction described is specific to Pus1p and position 27 in the tRNA anticodon stem, but the enzyme neither forms a covalent adduct nor stalls at a previously identified reaction intermediate of f⁵U. The f⁵U27 residue, as analyzed by a DNAzyme-based assay using TLC and mass spectrometry, displayed physicochemical properties unaltered by the reversible interaction with Pus1p. Thus, Pus1p binds an f⁵U-containing substrate, but, in contrast to other pseudouridine synthases, leaves the chemical structure of f⁵U unchanged. The specific, but nonproductive, interaction demonstrated here thus constitutes an intermediate of Pus turnover, stalled by the presence of f⁵U in an early state of catalysis. Observation of the interaction of Pus1p with fluorescence-labeled tRNA by a real-time readout of fluorescence anisotropy and FRET revealed significant structural distortion of f⁵U-tRNA structure in the stalled intermediate state of pseudouridine catalysis.     

Dye label interference with RNA modification reveals 5-fluorouridine as non-covalent inhibitor

The interest in RNA modification enzymes surges due to their involvement in epigenetic phenomena. Here we present a particularly informative approach to investigate the interaction of dye-labeled RNA with modification enzymes. We investigated pseudouridine (Ψ) synthase TruB interacting with an alleged suicide substrate RNA containing 5-fluorouridine (5FU). A longstanding dogma, stipulating formation of a stable covalent complex was challenged by discrepancies between the time scale of complex formation and enzymatic turnover. Instead of classic mutagenesis, we used differentially positioned fluorescent labels to modulate substrate properties in a range of enzymatic conversion between 6% and 99%. Despite this variegation, formation of SDS-stable complexes occurred instantaneously for all 5FU-substrates. Protein binding was investigated by advanced fluorescence spectroscopy allowing unprecedented simultaneous detection of change in fluorescence lifetime, anisotropy decay, as well as emission and excitation maxima. Determination of K_d values showed that introduction of 5FU into the RNA substrate increased protein affinity by 14× at most. Finally, competition experiments demonstrated reversibility of complex formation for 5FU-RNA. Our results lead us to conclude that the hitherto postulated long-term covalent interaction of TruB with 5FU tRNA is based on the interpretation of artifacts. This is likely true for the entire class of pseudouridine synthases.     

tRNA Binding,Positioning, and Modification by the Pseudouridine Synthase Pus10

Pus10 is the most recently identified pseudouridine synthase found in archaea and higher eukaryotes. It modifies uridine 55 in the TΨC arm of tRNAs. Here, we report the first quantitative biochemical analysis of tRNA binding and pseudouridine formation by Pyrococcus furiosus Pus10. The affinity of Pus10 for both substrate and product tRNA is high (K_d of 30 nM), and product formation occurs with a K_m of 400 nM and a k_cat of 0.9 s^− 1. Site-directed mutagenesis was used to demonstrate that the thumb loop in the catalytic domain is important for efficient catalysis; we propose that the thumb loop positions the tRNA within the active site. Furthermore, a new catalytic arginine residue was identified (arginine 208), which is likely responsible for triggering flipping of the target uridine into the active site of Pus10. Lastly, our data support the proposal that the THUMP-containing domain, found in the N-terminus of Pus10, contributes to binding of tRNA. Together, our findings are consistent with the hypothesis that tRNA binding by Pus10 occurs through an induced-fit mechanism, which is a prerequisite for efficient pseudouridine formation.     

Role of forefinger and thumb loops in production of Ψ54 and Ψ55 in tRNAs by archaeal Pus10

Pseudouridines (Ψ) are found in structurally and functionally important regions of RNAs. Six families of Ψ synthases, TruA, TruB, TruD, RsuA, RluA, and Pus10 have been identified. Pus10 is present in Archaea and Eukarya. While most archaeal Pus10 produce both tRNA Ψ54 and Ψ55, some produce only Ψ55. Interestingly, human PUS10 has been implicated in apoptosis and Crohn’s and Celiac diseases. Homology models of archaeal Pus10 proteins based on the crystal structure of human PUS10 reveal that there are subtle structural differences in all of these Pus10 proteins. These observations suggest that structural changes in homologous proteins may lead to loss, gain, or change of their functions, warranting the need to study the structure-function relationship of these proteins. Using comparison of structural models and a series of mutations, we identified forefinger loop (reminiscent of that of RluA) and an Arg and a Tyr residue of archaeal Pus10 as critical determinants for its Ψ54, but not for its Ψ55 activity. We also found that a Leu residue, in addition to the catalytic Asp, is essential for both activities. Since forefinger loop is needed for both rRNA and tRNA Ψ synthase activities of RluA, but only for tRNA Ψ54 activity of Pus10, archaeal Pus10 proteins must use a different mechanism of recognition for Ψ55 activity. We propose that archaeal Pus10 uses two distinct mechanisms for substrate uridine recognition and binding. However, since we did not observe any mutation that affected only Ψ55 activity, both mechanisms for archaeal Pus10 activities must share some common features.     

Pseudouridine-mediated translation control of mRNA by methionine aminoacyl tRNA synthetase

Modification of nucleotides within an mRNA emerges as a key path for gene expression regulation. Pseudouridine is one of the most common RNA modifications; however, only a few mRNA modifiers have been identified to date, and no one mRNA pseudouridine reader is known. Here, we applied a novel genome-wide approach to identify mRNA regions that are bound by yeast methionine aminoacyl tRNA^Met synthetase (MetRS). We found a clear enrichment to regions that were previously described to contain pseudouridine (Ψ). Follow-up in vitro and in vivo analyses on a prime target (position 1074 within YEF3 mRNA) demonstrated the importance of pseudouridine for MetRS binding. Furthermore, polysomal and protein analyses revealed that Ψ1074 mediates translation. Modification of this site occurs presumably by Pus6, a pseudouridine synthetase known to modify MetRS cognate tRNA. Consistently, the deletion of Pus6 leads to a decrease in MetRS association with both tRNA^Met and YEF3 mRNA. Furthermore, while global protein synthesis decreases in pus6Δ, translation of YEF3 increases. Together, our data imply that Pus6 ‘writes’ modifications on tRNA and mRNA, and both types of RNAs are ‘read’ by MetRS for translation regulation purposes. This represents a novel integrated path for writing and reading modifications on both tRNA and mRNA, which may lead to coordination between global and gene-specific translational responses.     

Pseudouridine mapping in the Saccharomyces cerevisiae spliceosomal U small nuclear RNAs (snRNAs) reveals that pseudouridine synthase pus1p exhibits a dual substrate specificity for U2 snRNA and tRNA

Pseudouridine (Psi) residues were localized in the Saccharomyces cerevisiae spliceosomal U small nuclear RNAs (UsnRNAs) by using the chemical mapping method. In contrast to vertebrate UsnRNAs, S. cerevisiae UsnRNAs contain only a few Psi residues, which are located in segments involved in intermolecular RNA-RNA or RNA-protein interactions. At these positions, UsnRNAs are universally modified. When yeast mutants disrupted for one of the several pseudouridine synthase genes (PUS1, PUS2, PUS3, and PUS4) or depleted in rRNA-pseudouridine synthase Cbf5p were tested for UsnRNA Psi content, only the loss of the Pus1p activity was found to affect Psi formation in spliceosomal UsnRNAs. Indeed, Psi44 formation in U2 snRNA was abolished. By using purified Pus1p enzyme and in vitro-produced U2 snRNA, Pus1p is shown here to catalyze Psi44 formation in the S. cerevisiae U2 snRNA. Thus, Pus1p is the first UsnRNA pseudouridine synthase characterized so far which exhibits a dual substrate specificity, acting on both tRNAs and U2 snRNA. As depletion of rRNA-pseudouridine synthase Cbf5p had no effect on UsnRNA Psi content, formation of Psi residues in S. cerevisiae UsnRNAs is not dependent on the Cbf5p-snoRNA guided mechanism.     

Domain organization and crystal structure of the catalytic domain of E.coli RluF, a pseudouridine synthase that acts on 23S rRNA

Pseudouridine synthases catalyze the isomerization of uridine to pseudouridine (Psi) in rRNA and tRNA. The pseudouridine synthase RluF from Escherichia coli (E.C. 4.2.1.70) modifies U2604 in 23S rRNA, and belongs to a large family of pseudouridine synthases present in all kingdoms of life. Here we report the domain architecture and crystal structure of the catalytic domain of E.coli RluF at 2.6A resolution. Limited proteolysis, mass spectrometry and N-terminal sequencing indicate that RluF has a distinct domain architecture, with the catalytic domain flanked at the N and C termini by additional domains connected to it by flexible linkers. The structure of the catalytic domain of RluF is similar to those of RsuA and TruB. RluF is a member of the RsuA sequence family of Psi-synthases, along with RluB and RluE. Structural comparison of RluF with its closest structural homologues, RsuA and TruB, suggests possible functional roles for the N-terminal and C-terminal domains of RluF.     

Pre-steady-state kinetic analysis of the three Escherichia coli pseudouridine synthases TruB, TruA, and RluA reveals uniformly slow catalysis

Pseudouridine synthases catalyze formation of the most abundant modification of functional RNAs by site-specifically isomerizing uridines to pseudouridines. While the structure and substrate specificity of these enzymes have been studied in detail, the kinetic and the catalytic mechanism of pseudouridine synthases remain unknown. Here, the first pre-steady-state kinetic analysis of three Escherichia coli pseudouridine synthases is presented. A novel stopped-flow absorbance assay revealed that substrate tRNA binding by TruB takes place in two steps with an overall rate of 6 sec(-1). In order to observe catalysis of pseudouridine formation directly, the traditional tritium release assay was adapted for the quench-flow technique, allowing, for the first time, observation of a single round of pseudouridine formation. Thereby, the single-round rate constant of pseudouridylation (k(Ψ)) by TruB was determined to be 0.5 sec(-1). This rate constant is similar to the k(cat) obtained under multiple-turnover conditions in steady-state experiments, indicating that catalysis is the rate-limiting step for TruB. In order to investigate if pseudouridine synthases are characterized by slow catalysis in general, the rapid kinetic quench-flow analysis was also performed with two other E. coli enzymes, RluA and TruA, which displayed rate constants of pseudouridine formation of 0.7 and 0.35 sec(-1), respectively. Hence, uniformly slow catalysis might be a general feature of pseudouridine synthases that share a conserved catalytic domain and supposedly use the same catalytic mechanism.     

Functional importance of Ψ38 and Ψ39 in distinct tRNAs,amplified for tRNAGln(UUG) by unexpected temperature sensitivity of the s2U modification in yeast

The numerous modifications of tRNA play central roles in controlling tRNA structure and translation. Modifications in and around the anticodon loop often have critical roles in decoding mRNA and in maintaining its reading frame. Residues U₃₈ and U₃₉ in the anticodon stem–loop are frequently modified to pseudouridine (Ψ) by members of the widely conserved TruA/Pus3 family of pseudouridylases. We investigate here the cause of the temperature sensitivity of pus3Δ mutants of the yeast Saccharomyces cerevisiae and find that, although Ψ₃₈ or Ψ₃₉ is found on at least 19 characterized cytoplasmic tRNA species, the temperature sensitivity is primarily due to poor function of tRNA^Gln(UUG), which normally has Ψ₃₈. Further investigation reveals that at elevated temperatures there are substantially reduced levels of the s²U moiety of mcm⁵s²U₃₄ of tRNA^Gln(UUG) and the other two cytoplasmic species with mcm⁵s²U₃₄, that the reduced s²U levels occur in the parent strain BY4741 and in the widely used strain W303, and that reduced levels of the s²U moiety are detectable in BY4741 at temperatures as low as 33°C. Additional examination of the role of Ψ_38,39 provides evidence that Ψ₃₈ is important for function of tRNA^Gln(UUG) at permissive temperature, and indicates that Ψ₃₉ is important for the function of tRNA^Trp(CCA) in trm10Δ pus3Δ mutants and of tRNA^Leu(CAA) as a UAG nonsense suppressor. These results provide evidence for important roles of both Ψ₃₈ and Ψ₃₉ in specific tRNAs, and establish that modification of the wobble position is subject to change under relatively mild growth conditions.     

Pseudouridine synthase 1: a site-specific synthase without strict sequence recognition requirements

Pseudouridine synthase 1 (Pus1p) is an unusual site-specific modification enzyme in that it can modify a number of positions in tRNAs and can recognize several other types of RNA. No consensus recognition sequence or structure has been identified for Pus1p. Human Pus1p was used to determine which structural or sequence elements of human tRNA(Ser) are necessary for pseudouridine (Ψ) formation at position 28 in the anticodon stem-loop (ASL). Some point mutations in the ASL stem of tRNA(Ser) had significant effects on the levels of modification and compensatory mutation, to reform the base pair, restored a wild-type level of Ψ formation. Deletion analysis showed that the tRNA(Ser) TΨC stem-loop was a determinant for modification in the ASL. A mini-substrate composed of the ASL and TΨC stem-loop exhibited significant Ψ formation at position 28 and a number of mutants were tested. Substantial base pairing in the ASL stem (3 out of 5 bp) is required, but the sequence of the TΨC loop is not required for modification. When all nucleotides in the ASL stem other than U28 were changed in a single mutant, but base pairing was retained, a near wild-type level of modification was observed.     

Partial activity is seen with many substitutions of highly conserved active site residues in human Pseudouridine synthase 1

Pseudouridine synthase 1 (Pus1p) is an enzyme that converts uridine to Pseudouridine (Ψ) in tRNA and other RNAs in eukaryotes. The active site of Pus1p is composed of stretches of amino acids that are highly conserved and it is hypothesized that mutation of select residues would impair the enzyme's ability to catalyze the formation of Ψ. However, most mutagenesis studies have been confined to substitution of the catalytic aspartate, which invariably results in an inactive enzyme in all Ψ synthases tested. To determine the requirements for particular amino acids at certain absolutely conserved positions in Pus1p, three residues (R116, Y173, R267) that correspond to amino acids known to compose the active site of TruA, a bacterial Ψ synthase that is homologous to Pus1p, were mutated in human Pus1p (hPus1p). The effects of those mutations were determined with three different in vitro assays of pseudouridylation and several tRNA substrates. Surprisingly, it was found that each of these components of the hPus1p active site could tolerate certain amino acid substitutions and in fact most mutants exhibited some activity. The most active mutants retained near wild-type activity at positions 27 or 28 in the substrate tRNA, but activity was greatly reduced or absent at other positions in tRNA readily modified by wild-type hPus1p.