Modifications in the T arm of tRNA globally determine tRNA maturation, function, and cellular fitness.

Almost all elongator tRNAs (Transfer RNAs) harbor 5-methyluridine 54 and pseudouridine 55 in the T arm, generated by the enzymes TrmA and TruB, respectively, in Escherichia coli. TrmA and TruB both act as tRNA chaperones, and strains lacking trmA or truB are outcompeted by wild type. Here, we investigate how TrmA and TruB contribute to cellular fitness. Deletion of trmA and truB in E. coli causes a global decrease in aminoacylation and alters other tRNA modifications such as acp3U47. While overall protein synthesis is not affected in ΔtrmA and ΔtruB strains, the translation of a subset of codons is significantly impaired. As a consequence, we observe translationally reduced expression of many specific proteins, that are either encoded with a high frequency of these codons or that are large proteins. The resulting proteome changes are not related to a specific growth phenotype, but overall cellular fitness is impaired upon deleting trmA and truB in accordance with a general protein synthesis impact. In conclusion, we demonstrate that universal modifications of the tRNA T arm are critical for global tRNA function by enhancing tRNA maturation, tRNA aminoacylation, and translation, thereby improving cellular fitness irrespective of the growth conditions which explains the conservation of trmA and truB.

RNA modifying enzymes shape tRNA biogenesis and function.

Transfer RNAs (tRNAs) are the most highly modified cellular RNAs, both with respect to the proportion of nucleotides that are modified within the tRNA sequence and with respect to the extraordinary diversity in tRNA modification chemistry. However, the functions of many different tRNA modifications are only beginning to emerge. tRNAs have two general clusters of modifications. The first cluster is within the anticodon stem-loop including several modifications essential for protein translation. The second cluster of modifications is within the tRNA elbow, and roles for these modifications are less clear. In general, tRNA elbow modifications are typically not essential for cell growth, but nonetheless several tRNA elbow modifications have been highly conserved throughout all domains of life. In addition to forming modifications, many tRNA modifying enzymes have been demonstrated or hypothesized to additionally play an important role in folding tRNA acting as tRNA chaperones. In this review, we summarize the known functions of tRNA modifying enzymes throughout the lifecycle of a tRNA molecule, from transcription to degradation. Thereby, we describe how tRNA modification and folding by tRNA modifying enzymes enhance tRNA maturation, tRNA aminoacylation, and tRNA function during protein synthesis, ultimately impacting cellular phenotypes and disease.

Fluorescent labeling of tRNA for rapid kinetic interaction studies with tRNA-binding proteins.

Transfer RNA (tRNA) plays a critical role during translation and interacts with numerous proteins during its biogenesis, functional cycle and degradation. In particular, tRNA is extensively post-transcriptionally modified by various tRNA modifying enzymes which each target a specific nucleotide at different positions within tRNAs to introduce different chemical modifications. Fluorescent assays can be used to study the interaction between a protein and tRNA. Moreover, rapid mixing fluorescence stopped-flow assays provide insights into the kinetics of the tRNA-protein interaction in order to elucidate the tRNA binding mechanism for the given protein. A prerequisite for these studies is a fluorescently labeled molecule, such as fluorescent tRNA, wherein a change in fluorescence occurs upon protein binding. In this chapter, we discuss the utilization of tRNA modifications in order to introduce fluorophores at particular positions within tRNAs. Particularly, we focus on in vitro thiolation of a uridine at position 8 within tRNAs using the tRNA modification enzyme ThiI, followed by labeling of the thiol group with fluorescein. As such, this fluorescently labeled tRNA is primarily unmodified, with the exception of the thiolation modification to which the fluorophore is attached, and can be used as a substrate to study the binding of different tRNA-interacting factors. Herein, we discuss the example of studying the tRNA binding mechanism of the tRNA modifying enzymes TrmB and DusA using internally fluorescein-labeled tRNA.

Molecular mechanism of tRNA binding by the Escherichia coli N7 guanosine methyltransferase TrmB.

Among the large and diverse collection of tRNA modifications, 7-methylguanosine (m7G) is frequently found in the tRNA variable loop at position 46. This modification is introduced by the TrmB enzyme, which is conserved in bacteria and eukaryotes. However, the molecular determinants and the mechanism for tRNA recognition by TrmB are not well understood. Complementing the report of various phenotypes for different organisms lacking TrmB homologs, we report here hydrogen peroxide sensitivity for the Escherichia coli ΔtrmB knockout strain. To gain insight into the molecular mechanism of tRNA binding by E. coli TrmB in real time, we developed a new assay based on introducing a 4-thiouridine modification at position 8 of in vitro transcribed tRNAPhe enabling us to fluorescently label this unmodified tRNA. Using rapid kinetic stopped-flow measurements with this fluorescent tRNA, we examined the interaction of WT and single substitution variants of TrmB with tRNA. Our results reveal the role of S-adenosylmethionine for rapid and stable tRNA binding, the rate-limiting nature of m7G46 catalysis for tRNA release, and the importance of residues R26, T127, and R155 across the entire surface of TrmB for tRNA binding.

Synergistic interaction network between the snR30 RNP, Utp23, and ribosomal RNA during ribosome synthesis.

snR30/U17 is a highly conserved H/ACA RNA that is required for maturation of the small ribosomal subunit in eukaryotes. By base-pairing to the expansion segment 6 (ES6) of 18S ribosomal RNA (rRNA), the snR30 H/ACA Ribonucleoprotein (RNP) indirectly facilitates processing of the precursor rRNA (pre-rRNA) together with other proteins such as Utp23 and other RNAs acting as ribosome assembly factors. However, the details of the molecular interaction network of snR30 and its binding partners and how these interactions contribute to pre-rRNA processing remains unknown. Here, we report the in vitro reconstitution of a Saccharomyces cerevisiae snR30 RNP and quantitative characterization of the interactions of snR30, H/ACA proteins, the Utp23 protein and ES6 of the 18S rRNA. The snR30 RNA is bound tightly by both H/ACA proteins and Utp23. We dissected the importance of different 18S rRNA regions for snR30 RNP binding and demonstrated that the snR30 complex is tightly anchored on the pre-rRNA through base-pairing to ES6 whereas other reported rRNA binding sites do not contribute to the affinity of the snR30 RNP. On its own, the ribosome assembly factor Utp23 binds in a tight, but unspecific manner to RNA. However, in complex with the snR30 RNP, Utp23 increases the affinity of the RNP for rRNA revealing synergies between snR30 RNP and Utp23 which are enhancing specificity and affinity for rRNA, respectively. Together, these findings provide mechanistic insights how the snR30 RNP and Utp23 cooperate to interact tightly and specifically with rRNA during the early stages of ribosome biogenesis.

Multi-wavelength analytical ultracentrifugation of biopolymer mixtures and interactions.

Multi-wavelength analytical ultracentrifugation (MW-AUC) is a recent development made possible by new analytical ultracentrifuge optical systems. MW-AUC extends the basic hydrodynamic information content of AUC and provides access to a wide range of new applications for biopolymer characterization, and is poised to become an essential analytical tool to study macromolecular interactions. It adds an orthogonal spectral dimension to the traditional hydrodynamic characterization by exploiting unique chromophores in analyte mixtures that may or may not interact. Here we illustrate the utility of MW-AUC for experimental investigations where the benefit of the added spectral dimension provides critical information that is not accessible, and impossible to resolve with traditional AUC methods. We demonstrate the improvements in resolution and information content obtained by this technique compared to traditional single- or dual-wavelength approaches, and discuss experimental design considerations and limitations of the method. We further address the advantages and disadvantages of the two MW optical systems available today, and the differences in data analysis strategies between the two systems.

The human pseudouridine synthase PUS7 recognizes RNA with an extended multi-domain binding surface.

The human pseudouridine synthase PUS7 is a versatile RNA modification enzyme targeting many RNAs thereby playing a critical role in development and brain function. Whereas all target RNAs of PUS7 share a consensus sequence, additional recognition elements are likely required, and the structural basis for RNA binding by PUS7 is unknown. Here, we characterize the structure-function relationship of human PUS7 reporting its X-ray crystal structure at 2.26 Å resolution. Compared to its bacterial homolog, human PUS7 possesses two additional subdomains, and structural modeling studies suggest that these subdomains contribute to tRNA recognition through increased interactions along the tRNA substrate. Consistent with our modeling, we find that all structural elements of tRNA are required for productive interaction with PUS7 as the consensus sequence of target RNA alone is not sufficient for pseudouridylation by human PUS7. Moreover, PUS7 binds several, non-modifiable RNAs with medium affinity which likely enables PUS7 to screen for productive RNA substrates. Following tRNA modification, the product tRNA has a significantly lower affinity for PUS7 facilitating its dissociation. Taken together our studies suggest a combination of structure-specific and sequence-specific RNA recognition by PUS7 and provide mechanistic insight into its function.

Partially modified tRNAs for the study of tRNA maturation and function.

Transfer RNA (tRNA) is the most highly and diversely modified class of RNA in all domains of life. However, we still have only a limited understanding of the concerted action of the many enzymes that modify tRNA during tRNA maturation and the synergistic functions of tRNA modifications for protein synthesis. Here, we describe the preparation of in vitro transcribed tRNAs with a partial set of defined modifications and the use of partially modified tRNAs in biochemical assays. By comparing the affinity and activity of tRNA modification enzymes for partially modified and unmodified tRNAs, we gain insight into the preferred pathways of tRNA maturation. Additionally, partially modified tRNAs will be highly useful to investigate the importance of tRNA modifications for tRNA function during translation including the interaction with aminoacyl-tRNA synthases, translation factors and the ribosome. Thereby, the methods described here lay the foundation for understanding the mechanistic function of tRNA modifications.

Assaying the Molecular Determinants and Kinetics of RNA Pseudouridylation by H/ACA snoRNPs and Stand-Alone Pseudouridine Synthases.

Posttranscriptional modifications of RNA play an important role in promoting the maturation and functional diversity of many RNA species. Accordingly, understanding the enzymes and mechanisms that underlie RNA modifications is an important aspect in advancing our knowledge of the continually expanding RNA modification field. However, of the more than 160 currently identified RNA modifications, a large portion remains without quantitative detection assays for their biochemical characterization. Here, we describe the tritium release assay as a convenient tool allowing for the quantitative assessment of in vitro RNA pseudouridylation by stand-alone or box H/ACA RNA-guided pseudouridine synthases. This assay enables quantification of RNA pseudouridylation over a time course to effectively compare pseudouridylation activity between different substrates and/or different recombinant enzymes as well as to determine kinetic parameters. With the help of a quench-flow apparatus, the tritium release assay can be adapted for rapid kinetic measurements under single-turnover conditions to dissect reaction mechanisms. As examples, we show the formation of pseudouridines by a reconstituted Saccharomyces cerevisiae H/ACA small ribonucleoprotein (snoRNP) and an Escherichia coli stand-alone pseudouridine synthase.

H/ACA Small Ribonucleoproteins: Structural and Functional Comparison Between Archaea and Eukaryotes.

During ribosome synthesis, ribosomal RNA is modified through the formation of many pseudouridines and methylations which contribute to ribosome function across all domains of life. In archaea and eukaryotes, pseudouridylation of rRNA is catalyzed by H/ACA small ribonucleoproteins (sRNPs) utilizing different H/ACA guide RNAs to identify target uridines for modification. H/ACA sRNPs are conserved in archaea and eukaryotes, as they share a common general architecture and function, but there are also several notable differences between archaeal and eukaryotic H/ACA sRNPs. Due to the higher protein stability in archaea, we have more information on the structure of archaeal H/ACA sRNPs compared to eukaryotic counterparts. However, based on the long history of yeast genetic and other cellular studies, the biological role of H/ACA sRNPs during ribosome biogenesis is better understood in eukaryotes than archaea. Therefore, this review provides an overview of the current knowledge on H/ACA sRNPs from archaea, in particular their structure and function, and relates it to our understanding of the roles of eukaryotic H/ACA sRNP during eukaryotic ribosome synthesis and beyond. Based on this comparison of our current insights into archaeal and eukaryotic H/ACA sRNPs, we discuss what role archaeal H/ACA sRNPs may play in the formation of ribosomes.

Revisiting tRNA chaperones: New players in an ancient game.

tRNAs undergo an extensive maturation process including post-transcriptional modifications that influence secondary and tertiary interactions. Precursor and mature tRNAs lacking key modifications are often recognized as aberrant and subsequently targeted for decay, illustrating the importance of modifications in promoting structural integrity. tRNAs also rely on tRNA chaperones to promote the folding of misfolded substrates into functional conformations. The best characterized tRNA chaperone is the La protein, which interacts with nascent RNA polymerase III transcripts to promote folding and offers protection from exonucleases. More recently, certain tRNA modification enzymes have also been demonstrated to possess tRNA folding activity distinct from their catalytic activity, suggesting that they may act as tRNA chaperones. In this review, we will discuss pioneering studies relating post-transcriptional modification to tRNA stability and decay pathways, present recent advances into the mechanism by which the RNA chaperone La assists pre-tRNA maturation, and summarize emerging research directions aimed at characterizing modification enzymes as tRNA chaperones. Together, these findings shed light on the importance of tRNA folding and how tRNA chaperones, in particular, increase the fraction of nascent pre-tRNAs that adopt a folded, functional conformation.

snR30/U17 Small Nucleolar Ribonucleoprotein: A Critical Player during Ribosome Biogenesis.

The small nucleolar RNA snR30 (U17 in humans) plays a unique role during ribosome synthesis. Unlike most members of the H/ACA class of guide RNAs, the small nucleolar ribonucleoprotein (snoRNP) complex assembled on snR30 does not direct pseudouridylation of ribosomal RNA (rRNA), but instead snR30 is critical for 18S rRNA processing during formation of the small subunit (SSU) of the ribosome. Specifically, snR30 is essential for three pre-rRNA cleavages at the A0/01, A1/1, and A2/2a sites in yeast and humans, respectively. Accordingly, snR30 is the only essential H/ACA guide RNA in yeast. Here, we summarize our current knowledge about the interactions and functions of snR30, discuss what remains to be elucidated, and present two non-exclusive hypotheses on the possible molecular function of snR30 during ribosome biogenesis. First, snR30 might be responsible for recruiting other proteins including endonucleases to the SSU processome. Second, snR30 may contribute to the refolding of pre-rRNA into a required conformation that serves as a checkpoint during ribosome biogenesis facilitating pre-rRNA cleavage. In both scenarios, the snR30 snoRNP may have scaffolding and RNA chaperoning activity. In conclusion, the snR30 snoRNP is a crucial player with an unknown molecular mechanism during ribosome synthesis, posing many interesting future research questions.

The methyltransferase TrmA facilitates tRNA folding through interaction with its RNA-binding domain.

tRNAs are the most highly modified RNAs in all cells, and formation of 5-methyluridine (m5U) at position 54 in the T arm is a common RNA modification found in all tRNAs. The m5U modification is generated by the methyltransferase TrmA. Here, we test and prove the hypothesis that Escherichia coli TrmA has dual functions, acting both as a methyltransferase and as a tRNA chaperone. We identify two conserved residues, F106 and H125, in the RNA-binding domain of TrmA, which interact with the tRNA elbow and are critical for tRNA binding. Co-culture competition assays reveal that the catalytic activity of TrmA is important for cellular fitness, and that substitutions of F106 or H125 impair cellular fitness. We directly show that TrmA enhances tRNA folding in vitro independent of its catalytic activity. In conclusion, our study suggests that F106 and H125 in the RNA-binding domain of TrmA act as a wedge disrupting tertiary interactions between tRNA's D arm and T arm; this tRNA unfolding is the mechanistic basis for TrmA's tRNA chaperone activity. TrmA is the second tRNA modifying enzyme next to the pseudouridine synthase TruB shown to act as a tRNA chaperone supporting a functional link between RNA modification and folding.

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tRNA elbow modifications affect the tRNA pseudouridine synthase TruB and the methyltransferase TrmA.

tRNAs constitute the most highly modified class of RNA. Every tRNA contains a unique set of modifications, and Ψ55, m5U54, and m7G46 are frequently found within the elbow of the tRNA structure. Despite the abundance of tRNA modifications, we are only beginning to understand the orchestration of modification enzymes during tRNA maturation. Here, we investigated whether pre-existing modifications impact the binding affinity or catalysis by tRNA elbow modification enzymes. Specifically, we focused on the Escherichia coli enzymes TruB, TrmA, and TrmB which generate Ψ55, m5U54, and m7G46, respectively. tRNAs containing a single modification were prepared, and the binding and activity preferences of purified E. coli TrmA, TruB, and TrmB were examined in vitro. TruB preferentially binds and modifies unmodified tRNA. TrmA prefers to modify unmodified tRNA, but binds most tightly to tRNA that already contains Ψ55. In contrast, binding and modification by TrmB is insensitive to the tRNA modification status. Our results suggest that TrmA and TruB are likely to act on mostly unmodified tRNA precursors during the early stages of tRNA maturation whereas TrmB presumably acts on later tRNA intermediates that are already partially modified. In conclusion, we uncover the mechanistic basis for the preferred modification order in the E. coli tRNA elbow region.

Proceedings of the 14th annual RiboWest conference: perspectives and outcome.

The RiboWest Conference brings together RNA researchers in Canada with the 2-fold goals of fostering internationally competitive RNA research and of training the next generation of scientists. The 14th Annual RiboWest conference (RiboWest 2018) was held at the University of Lethbridge (Lethbridge, Alberta) from June 10th to 13th, 2018. This meeting was focused on all major aspects of RNA research, ranging from understanding the cellular role of RNA, studying RNA interactions and structures, and employing them as a therapeutic tool. The invited keynote speakers (5) provided insights into the wide-range of RNA-based research. One of the unique features of this conference was that the majority of the oral presentations were given by the trainees (undergraduate/graduate students and postdoctoral researchers). Hosted by the Alberta RNA Research and Training Institute (ARRTI) at the University of Lethbridge as the leading center of RNA research in Western Canada, the RiboWest 2018 was well attended by researchers from across the country (>110 attendees in total). This conference proceedings editorial presents the overview of the conference, and briefly introduces articles published in this special issue of Biochemistry and Cell Biology.

Base-pairing interactions between substrate RNA and H/ACA guide RNA modulate the kinetics of pseudouridylation, but not the affinity of substrate binding by H/ACA small nucleolar ribonucleoproteins.

H/ACA small nucleolar ribonucleoproteins (snoRNPs) pseudouridylate RNA in eukaryotes and archaea. They target many RNAs site-specifically through base-pairing interactions between H/ACA guide and substrate RNA. Besides ribosomal RNA (rRNA) and small nuclear RNA (snRNA), H/ACA snoRNPs are thought to also modify messenger RNA (mRNA) with potential impacts on gene expression. However, the base pairing between known target RNAs and H/ACA guide RNAs varies widely in nature, and therefore the rules governing substrate RNA selection are still not fully understood. To provide quantitative insight into substrate RNA recognition, we systematically altered the sequence of a substrate RNA target by the Saccharomyces cerevisiae H/ACA guide RNA snR34. Time courses measuring pseudouridine formation revealed a gradual decrease in the initial velocity of pseudouridylation upon reducing the number of base pairs between substrate and guide RNA. Changing or inserting nucleotides close to the target uridine severely impairs pseudouridine formation. Interestingly, filter binding experiments show that all substrate RNA variants bind to H/ACA snoRNPs with nanomolar affinity. Next, we showed that binding of inactive, near-cognate RNAs to H/ACA snoRNPs does not inhibit their activity for cognate RNAs, presumably because near-cognate RNAs dissociate rapidly. We discuss that the modulation of initial velocities by the base-pairing strength might affect the order and efficiency of pseudouridylation in rRNA during ribosome biogenesis. Moreover, the binding of H/ACA snoRNPs to near-cognate RNAs may be a mechanism to search for cognate target sites. Together, our data provide critical information to aid in the prediction of productive H/ACA guide-substrate RNA pairs.

Molecular Determinants for 23S rRNA Recognition and Modification by the E. coli Pseudouridine Synthase RluE.

The isomerization of uridine to pseudouridine is the most common type of RNA modification found in RNAs across all domains of life and is performed by RNA-dependent and RNA-independent enzymes. The Escherichia coli pseudouridine synthase RluE acts as a stand-alone, highly specific enzyme forming the universally conserved pseudouridine at position 2457, located in helix 89 (H89) of the 23S rRNA in the peptidyltransferase center. Here, we conduct a detailed structure-function analysis to determine the structural elements both in RluE and in 23S rRNA required for RNA-protein interaction and pseudouridine formation. We determined that RluE recognizes a large part of 23S rRNA comprising both H89 and the single-stranded flanking regions which explains the high substrate specificity of RluE. Within RluE, the target RNA is recognized through sequence-specific contacts with loop L7-8 as well as interactions with loop L1-2 and the flexible N-terminal region. We demonstrate that RluE is a faster pseudouridine synthase than other enzymes which likely enables it to act in the early stages of ribosome formation. In summary, our biochemical characterization of RluE provides detailed insight into the molecular mechanism of RluE forming a highly conserved pseudouridine during ribosome biogenesis.

Efficient RNA pseudouridylation by eukaryotic H/ACA ribonucleoproteins requires high affinity binding and correct positioning of guide RNA.

H/ACA ribonucleoproteins (H/ACA RNPs) are responsible for introducing many pseudouridines into RNAs, but are also involved in other cellular functions. Utilizing a purified and reconstituted yeast H/ACA RNP system that is active in pseudouridine formation under physiological conditions, we describe here the quantitative characterization of H/ACA RNP formation and function. This analysis reveals a surprisingly tight interaction of H/ACA guide RNA with the Cbf5p-Nop10p-Gar1p trimeric protein complex whereas Nhp2p binds comparably weakly to H/ACA guide RNA. Substrate RNA is bound to H/ACA RNPs with nanomolar affinity which correlates with the GC content in the guide-substrate RNA base pairing. Both Nhp2p and the conserved Box ACA element in guide RNA are required for efficient pseudouridine formation, but not for guide RNA or substrate RNA binding. These results suggest that Nhp2p and the Box ACA motif indirectly facilitate loading of the substrate RNA in the catalytic site of Cbf5p by correctly positioning the upper and lower parts of the H/ACA guide RNA on the H/ACA proteins. In summary, this study provides detailed insight into the molecular mechanism of H/ACA RNPs.

Eukaryotic stand-alone pseudouridine synthases - RNA modifying enzymes and emerging regulators of gene expression?

For a long time, eukaryotic stand-alone pseudouridine synthases (Pus enzymes) were neglected as non-essential enzymes adding seemingly simple modifications to tRNAs and small nuclear RNAs. Most studies were limited to the identification and initial characterization of the yeast Pus enzymes. However, recent transcriptome-wide mapping of pseudouridines in yeast and humans revealed pervasive modification of mRNAs and other non-coding RNAs by Pus enzymes which is dynamically regulated in response to cellular stress. Moreover, mutations in at least 2 genes encoding human Pus enzymes cause inherited diseases affecting muscle and brain function. Together, the recent findings suggest a broader-than-anticipated role of the Pus enzymes which are emerging as potential regulators of gene expression. In this review, we summarize the current knowledge on Pus enzymes, generate hypotheses regarding their cellular function and outline future areas of research of pseudouridine synthases.