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 also 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.

Bacterial RNA-free RNase P: Structural and functional characterization of multiple oligomeric forms of a minimal protein-only ribonuclease P.

tRNAs are typically transcribed with extended 5' and 3' ends that must be removed before they attain their active form. One of the first steps of tRNA processing in nearly every organism is the removal of the 5' leader sequence by ribonuclease P (RNase P). Here, we investigate a recently discovered class of RNase P enzymes, Homologs of Aquifex RNase P (HARPs). In contrast to other RNase Ps, HARPs consist only of a metallonuclease domain and lack the canonical substrate recognition domain essential in other classes of proteinaceous RNase P. We determined the cryo-EM structure of Aquifex aeolicus HARP (Aq880) and two crystal structures of Hydrogenobacter thermophilus HARP (Hth1307) to reveal that both enzymes form large ring-like assemblies: a dodecamer in Aq880 and a tetradecamer in Hth1307. In both oligomers, the enzyme active site is 42 Å away from a positively charged helical region, as seen in other protein-only RNase P enzymes, which likely serves to recognize and bind the elbow region of the pre-tRNA substrate. In addition, we use native mass spectrometry to confirm and characterize the previously unreported tetradecamer state. Notably, we find that multiple oligomeric states of Hth1307 are able to cleave pre-tRNAs. Furthermore, our single-turnover kinetic studies indicate that Hth1307 cleaves pre-tRNAs from multiple species with a preference for native substrates. These data provide a closer look at the nuanced similarities and differences in tRNA processing across disparate classes of RNase P.

Minimal protein-only RNase P structure reveals insights into tRNA precursor recognition and catalysis.

Ribonuclease P (RNase P) is an endoribonuclease that catalyzes the processing of the 5' leader sequence of precursor tRNA (pre-tRNA). Ribonucleoprotein RNase P and protein-only RNase P (PRORP) in eukaryotes have been extensively studied, but the mechanism by which a prokaryotic nuclease recognizes and cleaves pre-tRNA is unclear. To gain insights into this mechanism, we studied homologs of Aquifex RNase P (HARPs), thought to be enzymes of approximately 23 kDa comprising only this nuclease domain. We determined the cryo-EM structure of Aq880, the first identified HARP enzyme. The structure unexpectedly revealed that Aq880 consists of both the nuclease and protruding helical (PrH) domains. Aq880 monomers assemble into a dimer via the PrH domain. Six dimers form a dodecamer with a left-handed one-turn superhelical structure. The structure also revealed that the active site of Aq880 is analogous to that of eukaryotic PRORPs. The pre-tRNA docking model demonstrated that 5' processing of pre-tRNAs is achieved by two adjacent dimers within the dodecamer. One dimer is responsible for catalysis, and the PrH domains of the other dimer are responsible for pre-tRNA elbow recognition. Our study suggests that HARPs measure an invariant distance from the pre-tRNA elbow to cleave the 5' leader sequence, which is analogous to the mechanism of eukaryotic PRORPs and the ribonucleoprotein RNase P. Collectively, these findings shed light on how different types of RNase P enzymes utilize the same pre-tRNA processing.

RNA polymerase II (RNAP II)-associated factors are recruited to tRNA loci, revealing that RNAP II- and RNAP III-mediated transcriptions overlap in yeast.

In yeast (Saccharomyces cerevisiae), the synthesis of tRNAs by RNA polymerase III (RNAP III) down-regulates the transcription of the nearby RNAP II-transcribed genes by a mechanism that is poorly understood. To clarify the basis of this tRNA gene-mediated (TGM) silencing, here, conducting a bioinformatics analysis of available ChIP-chip and ChIP-sequencing genomic data from yeast, we investigated whether the RNAP III transcriptional machinery can recruit protein factors required for RNAP II transcription. An analysis of 46 genome-wide protein-density profiles revealed that 12 factors normally implicated in RNAP II-mediated gene transcription are more enriched at tRNA than at mRNA loci. These 12 factors typically have RNA-binding properties, participate in the termination stage of the RNAP II transcription, and preferentially localize to the tRNA loci by a mechanism that apparently is based on the RNAP III transcription level. The factors included two kinases of RNAP II (Bur1 and Ctk1), a histone demethylase (Jhd2), and a mutated form of a nucleosome-remodeling factor (Spt6) that have never been reported to be recruited to tRNA loci. Moreover, we show that the expression levels of RNAP II-transcribed genes downstream of tRNA loci correlate with the distance from the tRNA gene by a mechanism that depends on their orientation. These results are consistent with the notion that pre-tRNAs recruit RNAP II-associated factors, thereby reducing the availability of these factors for RNAP II transcription and contributing, at least in part, to the TGM-silencing mechanism.

An interdomain bridge influences RNA binding of the human La protein.

La proteins are RNA chaperones that perform various functions depending on distinct RNA-binding modes and their subcellular localization. In the nucleus, they help process UUU-3'OH-tailed nascent RNA polymerase III transcripts, such as pre-tRNAs, whereas in the cytoplasm they contribute to translation of poly(A)-tailed mRNAs. La accumulation in the nucleus and cytoplasm is controlled by several trafficking elements, including a canonical nuclear localization signal in the extreme C terminus and a nuclear retention element (NRE) in the RNA recognition motif 2 (RRM2) domain. Previous findings indicate that cytoplasmic export of La due to mutation of the NRE can be suppressed by mutations in RRM1, but the mechanism by which the RRM1 and RRM2 domains functionally cooperate is poorly understood. In this work, we use electromobility shift assays (EMSA) to show that mutations in the NRE and RRM1 affect binding of human La to pre-tRNAs but not UUU-3'OH or poly(A) sequences, and we present compensatory mutagenesis data supporting a direct interaction between the RRM1 and RRM2 domains. Moreover, we use collision-induced unfolding and time-resolved hydrogen-deuterium exchange MS analyses to study the conformational dynamics that occur when this interaction is intact or disrupted. Our results suggest that the intracellular distribution of La may be linked to its RNA-binding modes and provide the first evidence for a direct protein-protein interdomain interaction in La proteins.

Biophysical analysis of Arabidopsis protein-only RNase P alone and in complex with tRNA provides a refined model of tRNA binding.

RNase P is a universal enzyme that removes 5' leader sequences from tRNA precursors. The enzyme is therefore essential for maturation of functional tRNAs and mRNA translation. RNase P represents a unique example of an enzyme that can occur either as ribonucleoprotein or as protein alone. The latter form of the enzyme, called protein-only RNase P (PRORP), is widespread in eukaryotes in which it can provide organellar or nuclear RNase P activities. Here, we have focused on Arabidopsis nuclear PRORP2 and its interaction with tRNA substrates. Affinity measurements helped assess the respective importance of individual pentatricopeptide repeat motifs in PRORP2 for RNA binding. We characterized the PRORP2 structure by X-ray crystallography and by small-angle X-ray scattering in solution as well as that of its complex with a tRNA precursor by small-angle X-ray scattering. Of note, our study reports the first structural data of a PRORP-tRNA complex. Combined with complementary biochemical and biophysical analyses, our structural data suggest that PRORP2 undergoes conformational changes to accommodate its substrate. In particular, the catalytic domain and the RNA-binding domain can move around a central hinge. Altogether, this work provides a refined model of the PRORP-tRNA complex that illustrates how protein-only RNase P enzymes specifically bind tRNA and highlights the contribution of protein dynamics to achieve this specific interaction.

Mechanistic Studies Reveal Similar Catalytic Strategies for Phosphodiester Bond Hydrolysis by Protein-only and RNA-dependent Ribonuclease P.

Ribonuclease P (RNase P) is an endonuclease that catalyzes the essential removal of the 5' end of tRNA precursors. Until recently, all identified RNase P enzymes were a ribonucleoprotein with a conserved catalytic RNA component. However, the discovery of protein-only RNase P (PRORP) shifted this paradigm, affording a unique opportunity to compare mechanistic strategies used by naturally evolved protein and RNA-based enzymes that catalyze the same reaction. Here we investigate the enzymatic mechanism of pre-tRNA hydrolysis catalyzed by the NYN (Nedd4-BP1, YacP nuclease) metallonuclease of Arabidopsis thaliana, PRORP1. Multiple and single turnover kinetic data support a mechanism where a step at or before chemistry is rate-limiting and provide a kinetic framework to interpret the results of metal alteration, mutations, and pH dependence. Catalytic activity has a cooperative dependence on the magnesium concentration (nH = 2) under kcat/Km conditions, suggesting that PRORP1 catalysis is optimal with at least two active site metal ions, consistent with the crystal structure. Metal rescue of Asp-to-Ala mutations identified two aspartates important for enhancing metal ion affinity. The single turnover pH dependence of pre-tRNA cleavage revealed a single ionization (pKa ∼ 8.7) important for catalysis, consistent with deprotonation of a metal-bound water nucleophile. The pH and metal dependence mirrors that observed for the RNA-based RNase P, suggesting similar catalytic mechanisms. Thus, despite different macromolecular composition, the RNA and protein-based RNase P act as dynamic scaffolds for the binding and positioning of magnesium ions to catalyze phosphodiester bond hydrolysis.