Structural basis for human mitochondrial tRNA maturation.

The human mitochondrial genome is transcribed into two RNAs, containing mRNAs, rRNAs and tRNAs, all dedicated to produce essential proteins of the respiratory chain. The precise excision of tRNAs by the mitochondrial endoribonucleases (mt-RNase), P and Z, releases all RNA species from the two RNA transcripts. The tRNAs then undergo 3'-CCA addition. In metazoan mitochondria, RNase P is a multi-enzyme assembly that comprises the endoribonuclease PRORP and a tRNA methyltransferase subcomplex. The requirement for this tRNA methyltransferase subcomplex for mt-RNase P cleavage activity, as well as the mechanisms of pre-tRNA 3'-cleavage and 3'-CCA addition, are still poorly understood. Here, we report cryo-EM structures that visualise four steps of mitochondrial tRNA maturation: 5' and 3' tRNA-end processing, methylation and 3'-CCA addition, and explain the defined sequential order of the tRNA processing steps. The methyltransferase subcomplex recognises the pre-tRNA in a distinct mode that can support tRNA-end processing and 3'-CCA addition, likely resulting from an evolutionary adaptation of mitochondrial tRNA maturation complexes to the structurally-fragile mitochondrial tRNAs. This subcomplex can also ensure a tRNA-folding quality-control checkpoint before the sequential docking of the maturation enzymes. Altogether, our study provides detailed molecular insight into RNA-transcript processing and tRNA maturation in human mitochondria.

RNase P: Beyond Precursor tRNA Processing.

Ribonuclease P (RNase P) was first described in the 1970's as an endoribonuclease acting in the maturation of precursor transfer RNAs (tRNAs). More recent studies, however, have uncovered non-canonical roles for RNase P and its components. Here, we review the recent progress of its involvement in chromatin assembly, DNA damage response, and maintenance of genome stability with implications in tumorigenesis. The possibility of RNase P as a therapeutic target in cancer is also discussed.

Rapid Ribonuclease P Kinetics Measured by Stopped-Flow Fluorescence and Fluorescence Anisotropy.

Stopped-flow fluorescence spectroscopy is a highly sensitive method for measuring rapid enzyme kinetics. A wide range of fluorophores can be employed, and fluorescence and fluorescence polarization can be measured. Thus, binding, conformational changes, and catalysis can, in principle, be measured, making it helpful in probing the entire kinetic landscape of a reaction. In this chapter, we use the bacterial RNA processing enzyme ribonuclease P (RNase P) as a model system to illustrate the determination of the kinetic constants for substrate binding and cleavage, thus allowing mechanistic questions regarding the effects of reaction conditions, mutations, or drug binding to be answered.

In Vitro Amplification and Selection of Engineered RNase P Ribozyme for Gene Targeting Applications.

Ribozymes engineered from the RNase P catalytic RNA (M1 RNA) represent promising gene-targeting agents for clinical applications. We describe in this report an in vitro amplification and selection procedure for generating active RNase P ribozyme variants with improved catalytic efficiency. Using the amplification and selection procedure, we have previously generated ribozyme variants that were highly active in cleaving a herpes simplex virus 1-encoded mRNA in vitro and inhibiting its expression in virally infected human cells. In this chapter, we use an overlapping region of the mRNAs for the IE1 and IE2 proteins of human cytomegalovirus (HCMV) as a target substrate. We provide detailed protocols and include methods for establishing the procedure for the amplification and selection of active mRNA-cleaving RNase P ribozymes. The in vitro amplification and selection system represents an excellent approach for engineering highly active RNase P ribozymes that can be used in both basic research and clinical applications.

A tribute to Sidney Altman, one of the architects of modern RNA biology.

This special issue of JBC pays tribute to Sidney Altman, whose discovery of a catalytic role for RNA, a breakthrough made independently by Thomas Cech, overturned the long-held dogma that only proteins can serve as catalysts in biological systems. The discovery of RNA catalysis galvanized biologists to think expansively in new directions and has given rise to a remarkable RNAissance in science and medicine. The collection of articles begins with the story of the discovery of RNase P and builds up to the emerging picture of an unexpectedly vast repertoire of RNase P variants in the three domains of life, including insights derived from recent high-resolution structures on how RNAs, ribonucleoproteins, or protein scaffolds can be used variably to generate an active site for catalyzing the same RNA processing reaction. The series of articles ends with a discussion of more recently discovered endonucleases (Argonautes, Cas), whose parallels with RNase P underscore recurring themes in diverse biological contexts.

External Guide Sequence Effectively Suppresses the Gene Expression and Replication of Herpes Simplex Virus 2.

Ribonuclease P (RNase P) complexed with an external guide sequence (EGS) represents a promising nucleic acid-based gene targeting approach for gene expression knock-down and modulation. The RNase P-EGS strategy is unique as an EGS can be designed to basepair any mRNA sequence and recruit intracellular RNase P for hydrolysis of the target mRNA. In this study, we provide the first direct evidence that the RNase P-based approach effectively blocks the gene expression and replication of herpes simplex virus 2 (HSV-2), the causative agent of genital herpes. We constructed EGSs to target the mRNA encoding HSV-2 single-stranded DNA binding protein ICP8, which is essential for viral DNA genome replication and growth. In HSV-2 infected cells expressing a functional EGS, ICP8 levels were reduced by 85%, and viral growth decreased by 3000 folds. On the contrary, ICP8 expression and viral growth exhibited no substantial differences between cells expressing no EGS and those expressing a disabled EGS with mutations precluding RNase P recognition. The anti-ICP8 EGS is specific in targeting ICP8 because it only affects ICP8 expression but does not affect the expression of the other viral immediate-early and early genes examined. This study shows the effective and specific anti-HSV-2 activity of the RNase P-EGS approach and demonstrates the potential of EGS RNAs for anti-HSV-2 applications.

The discovery of a catalytic RNA within RNase P and its legacy.

Sidney Altman's discovery of the processing of one RNA by another RNA that acts like an enzyme was revolutionary in biology and the basis for his sharing the 1989 Nobel Prize in Chemistry with Thomas Cech. These breakthrough findings support the key role of RNA in molecular evolution, where replicating RNAs (and similar chemical derivatives) either with or without peptides functioned in protocells during the early stages of life on Earth, an era referred to as the RNA world. Here, we cover the historical background highlighting the work of Altman and his colleagues and the subsequent efforts of other researchers to understand the biological function of RNase P and its catalytic RNA subunit and to employ it as a tool to downregulate gene expression. We primarily discuss bacterial RNase P-related studies but acknowledge that many groups have significantly contributed to our understanding of archaeal and eukaryotic RNase P, as reviewed in this special issue and elsewhere.

The protein-only RNase Ps, endonucleases that cleave pre-tRNA: Biological relevance, molecular architectures, substrate recognition and specificity, and protein interactomes.

Protein-only RNase P (PRORP) is an essential enzyme responsible for the 5' maturation of precursor tRNAs (pre-tRNAs). PRORPs are classified into three categories with unique molecular architectures, although all three classes of PRORPs share a mechanism and have similar active sites. Single subunit PRORPs, like those found in plants, have multiple isoforms with different localizations, substrate specificities, and temperature sensitivities. Most recently, Arabidopsis thaliana PRORP2 was shown to interact with TRM1A and B, highlighting a new potential role between these enzymes. Work with At PRORPs led to the development of a ribonuclease that is being used to protect against plant viruses. The mitochondrial RNase P complex, found in metazoans, consists of PRORP, TRMT10C, and SDR5C1, and has also been shown to have substrate specificity, although the cause is unknown. Mutations in mitochondrial tRNA and mitochondrial RNase P have been linked to human disease, highlighting the need to continue understanding this complex. The last class of PRORPs, homologs of Aquifex RNase P (HARPs), is found in thermophilic archaea and bacteria. This most recently discovered type of PRORP forms a large homo-oligomer complex. Although numerous structures of HARPs have been published, it is still unclear how HARPs bind pre-tRNAs and in what ratio. There is also little investigation into the substrate specificity and ideal conditions for HARPs. Moving forward, further work is required to fully characterize each of the three classes of PRORP, the pre-tRNA binding recognition mechanism, the rules of substrate specificity, and how these three distinct classes of PRORP evolved. This article is categorized under: RNA Structure and Dynamics > RNA Structure, Dynamics and Chemistry RNA Structure and Dynamics > Influence of RNA Structure in Biological Systems.

Multiple structural flavors of RNase P in precursor tRNA processing.

The precursor transfer RNAs (pre-tRNAs) require extensive processing to generate mature tRNAs possessing proper fold, structural stability, and functionality required to sustain cellular viability. The road to tRNA maturation follows an ordered process: 5'-processing, 3'-processing, modifications at specific sites, if any, and 3'-CCA addition before aminoacylation and recruitment to the cellular protein synthesis machinery. Ribonuclease P (RNase P) is a universally conserved endonuclease in all domains of life, performing the hydrolysis of pre-tRNA sequences at the 5' end by the removal of phosphodiester linkages between nucleotides at position -1 and +1. Except for an archaeal species: Nanoarchaeum equitans where tRNAs are transcribed from leaderless-position +1, RNase P is indispensable for life and displays fundamental variations in terms of enzyme subunit composition, mechanism of substrate recognition and active site architecture, utilizing in all cases a two metal ion-mediated conserved catalytic reaction. While the canonical RNA-based ribonucleoprotein RNase P has been well-known to occur in bacteria, archaea, and eukaryotes, the occurrence of RNA-free protein-only RNase P in eukaryotes and RNA-free homologs of Aquifex RNase P in prokaryotes has been discovered more recently. This review aims to provide a comprehensive overview of structural diversity displayed by various RNA-based and RNA-free RNase P holoenzymes towards harnessing critical RNA-protein and protein-protein interactions in achieving conserved pre-tRNA processing functionality. Furthermore, alternate roles and functional interchangeability of RNase P are discussed in the context of its employability in several clinical and biotechnological applications. This article is categorized under: RNA Processing > tRNA Processing RNA Evolution and Genomics > RNA and Ribonucleoprotein Evolution RNA Interactions with Proteins and Other Molecules > RNA-Protein Complexes.

Coevolution of RNA and protein subunits in RNase P and RNase MRP, two RNA processing enzymes.

RNase P and RNase mitochondrial RNA processing (MRP) are ribonucleoproteins (RNPs) that consist of a catalytic RNA and a varying number of protein cofactors. RNase P is responsible for precursor tRNA maturation in all three domains of life, while RNase MRP, exclusive to eukaryotes, primarily functions in rRNA biogenesis. While eukaryotic RNase P is associated with more protein cofactors and has an RNA subunit with fewer auxiliary structural elements compared to its bacterial cousin, the double-anchor precursor tRNA recognition mechanism has remarkably been preserved during evolution. RNase MRP shares evolutionary and structural similarities with RNase P, preserving the catalytic core within the RNA moiety inherited from their common ancestor. By incorporating new protein cofactors and RNA elements, RNase MRP has established itself as a distinct RNP capable of processing ssRNA substrates. The structural information on RNase P and MRP helps build an evolutionary trajectory, depicting how emerging protein cofactors harmonize with the evolution of RNA to shape different functions for RNase P and MRP. Here, we outline the structural and functional relationship between RNase P and MRP to illustrate the coevolution of RNA and protein cofactors, a key driver for the extant, diverse RNP world.

Discovery, structure, mechanisms, and evolution of protein-only RNase P enzymes.

The endoribonuclease RNase P is responsible for tRNA 5' maturation in all domains of life. A unique feature of RNase P is the variety of enzyme architectures, ranging from dual- to multi-subunit ribonucleoprotein forms with catalytic RNA subunits to protein-only enzymes, the latter occurring as single- or multi-subunit forms or homo-oligomeric assemblies. The protein-only enzymes evolved twice: a eukaryal protein-only RNase P termed PRORP and a bacterial/archaeal variant termed homolog of Aquifex RNase P (HARP); the latter replaced the RNA-based enzyme in a small group of thermophilic bacteria but otherwise coexists with the ribonucleoprotein enzyme in a few other bacteria as well as in those archaea that also encode a HARP. Here we summarize the history of the discovery of protein-only RNase P enzymes and review the state of knowledge on structure and function of bacterial HARPs and eukaryal PRORPs, including human mitochondrial RNase P as a paradigm of multi-subunit PRORPs. We also describe the phylogenetic distribution and evolution of PRORPs, as well as possible reasons for the spread of PRORPs in the eukaryal tree and for the recruitment of two additional protein subunits to metazoan mitochondrial PRORP. We outline potential applications of PRORPs in plant biotechnology and address diseases associated with mutations in human mitochondrial RNase P genes. Finally, we consider possible causes underlying the displacement of the ancient RNA enzyme by a protein-only enzyme in a small group of bacteria.

The specificity landscape of bacterial ribonuclease P.

Developing quantitative models of substrate specificity for RNA processing enzymes is a key step toward understanding their biology and guiding applications in biotechnology and biomedicine. Optimally, models to predict relative rate constants for alternative substrates should integrate an understanding of structures of the enzyme bound to "fast" and "slow" substrates, large datasets of rate constants for alternative substrates, and transcriptomic data identifying in vivo processing sites. Such data are either available or emerging for bacterial ribonucleoprotein RNase P a widespread and essential tRNA 5' processing endonuclease, thus making it a valuable model system for investigating principles of biological specificity. Indeed, the well-established structure and kinetics of bacterial RNase P enabled the development of high throughput measurements of rate constants for tRNA variants and provided the necessary framework for quantitative specificity modeling. Several studies document the importance of conformational changes in the precursor tRNA substrate as well as the RNA and protein subunits of bacterial RNase P during binding, although the functional roles and dynamics are still being resolved. Recently, results from cryo-EM studies of E. coli RNase P with alternative precursor tRNAs are revealing prospective mechanistic relationships between conformational changes and substrate specificity. Yet, extensive uncharted territory remains, including leveraging these advances for drug discovery, achieving a complete accounting of RNase P substrates, and understanding how the cellular context contributes to RNA processing specificity in vivo.

The previously uncharacterized RnpM (YlxR) protein modulates the activity of ribonuclease P in Bacillus subtilis in vitro.

Even though Bacillus subtilis is one of the most studied organisms, no function has been identified for about 20% of its proteins. Among these unknown proteins are several RNA- and ribosome-binding proteins suggesting that they exert functions in cellular information processing. In this work, we have investigated the RNA-binding protein YlxR. This protein is widely conserved in bacteria and strongly constitutively expressed in B. subtilis suggesting an important function. We have identified the RNA subunit of the essential RNase P as the binding partner of YlxR. The main activity of RNase P is the processing of 5' ends of pre-tRNAs. In vitro processing assays demonstrated that the presence of YlxR results in reduced RNase P activity. Chemical cross-linking studies followed by in silico docking analysis and experiments with site-directed mutant proteins suggest that YlxR binds to the region of the RNase P RNA that is important for binding and cleavage of the pre-tRNA substrate. We conclude that the YlxR protein is a novel interaction partner of the RNA subunit of RNase P that serves to finetune RNase P activity to ensure appropriate amounts of mature tRNAs for translation. We rename the YlxR protein RnpM for RNase P modulator.

Raising NAD+ Level Stimulates Short-Chain Dehydrogenase/Reductase Proteins to Alleviate Heart Failure Independent of Mitochondrial Protein Deacetylation.

BACKGROUND: Strategies to increase cellular NAD+ (oxidized nicotinamide adenine dinucleotide) level have prevented cardiac dysfunction in multiple models of heart failure, but molecular mechanisms remain unclear. Little is known about the benefits of NAD+-based therapies in failing hearts after the symptoms of heart failure have appeared. Most pretreatment regimens suggested mechanisms involving activation of sirtuin, especially Sirt3 (sirtuin 3), and mitochondrial protein acetylation. METHODS: We induced cardiac dysfunction by pressure overload in SIRT3-deficient (knockout) mice and compared their response with nicotinamide riboside chloride treatment with wild-type mice. To model a therapeutic approach, we initiated the treatment in mice with established cardiac dysfunction. RESULTS: We found nicotinamide riboside chloride improved mitochondrial function and blunted heart failure progression. Similar benefits were observed in wild-type and knockout mice. Boosting NAD+ level improved the function of NAD(H) redox-sensitive SDR (short-chain dehydrogenase/reductase) family proteins. Upregulation of Mrpp2 (mitochondrial ribonuclease P protein 2), a multifunctional SDR protein and a subunit of mitochondrial ribonuclease P, improves mitochondrial DNA transcripts processing and electron transport chain function. Activation of SDRs in the retinol metabolism pathway stimulates RXRα (retinoid X receptor α)/PPARα (proliferator-activated receptor α) signaling and restores mitochondrial oxidative metabolism. Downregulation of Mrpp2 and impaired mitochondrial ribonuclease P were found in human failing hearts, suggesting a shared mechanism of defective mitochondrial biogenesis in mouse and human heart failure. CONCLUSIONS: These findings identify SDR proteins as important regulators of mitochondrial function and molecular targets of NAD+-based therapy. Furthermore, the benefit is observed regardless of Sirt3-mediated mitochondrial protein deacetylation, a widely held mechanism for NAD+-based therapy for heart failure. The data also show that NAD+-based therapy can be useful in pre-existing heart failure.

A tRNA-modifying enzyme facilitates RNase P activity in Arabidopsis nuclei.

RNase P is the essential activity that performs the 5' maturation of transfer RNA (tRNA) precursors. Beyond the ancestral form of RNase P containing a ribozyme, protein-only RNase P enzymes termed PRORP were identified in eukaryotes. In human mitochondria, PRORP forms a complex with two protein partners to become functional. In plants, although PRORP enzymes are active alone, we investigate their interaction network to identify potential tRNA maturation complexes. Here we investigate functional interactions involving the Arabidopsis nuclear RNase P PRORP2. We show, using an immuno-affinity strategy, that PRORP2 occurs in a complex with the tRNA methyl transferases TRM1A and TRM1B in vivo. Beyond RNase P, these enzymes can also interact with RNase Z. We show that TRM1A/TRM1B localize in the nucleus and find that their double knockout mutation results in a severe macroscopic phenotype. Using a combination of immuno-detections, mass spectrometry and a transcriptome-wide tRNA sequencing approach, we observe that TRM1A/TRM1B are responsible for the m22G26 modification of 70% of cytosolic tRNAs in vivo. We use the transcriptome wide tRNAseq approach as well as RNA blot hybridizations to show that RNase P activity is impaired in TRM1A/TRM1B mutants for specific tRNAs, in particular, tRNAs containing a m22G modification at position 26 that are strongly downregulated in TRM1A/TRM1B mutants. Altogether, results indicate that the m22G-adding enzymes TRM1A/TRM1B functionally cooperate with nuclear RNase P in vivo for the early steps of cytosolic tRNA biogenesis.

Transcriptome analysis reveals a lncRNA-miRNA-mRNA regulatory network in OsRpp30-mediated disease resistance in rice.

BACKGROUND: Long non-coding RNAs (lncRNAs) play critical roles in various biological processes in plants. Extensive studies utilizing high-throughput RNA sequencing have revealed that many lncRNAs are involved in plant disease resistance. Oryza sativa RNase P protein 30 (OsRpp30) has been identified as a positive regulator of rice immunity against fungal and bacterial pathogens. Nevertheless, the specific functions of lncRNAs in relation to OsRpp30-mediated disease resistance in rice remain elusive. RESULTS: We conducted a comprehensive analysis of lncRNAs, miRNAs, and mRNAs expression patterns in wild type (WT), OsRpp30 overexpression (OsRpp30-OE), and OsRpp30 knockout (OsRpp30-KO) rice plants. In total, we identified 91 differentially expressed lncRNAs (DElncRNAs), 1671 differentially expressed mRNAs (DEmRNAs), and 41 differentially expressed miRNAs (DEmiRNAs) across the different rice lines. To gain further insights, we investigated the interaction between DElncRNAs and DEmRNAs, leading to the discovery of 10 trans- and 27 cis-targeting pairs specific to the OsRpp30-OE and OsRpp30-KO samples. In addition, we constructed a competing endogenous RNA (ceRNA) network comprising differentially expressed lncRNAs, miRNAs, and mRNAs to elucidate their intricate interplay in rice disease resistance. The ceRNA network analysis uncovered a set of gene targets regulated by lncRNAs and miRNAs, which were found to be involved in pathogen recognition, hormone pathways, transcription factor activation, and other biological processes related to plant immunity. CONCLUSIONS: Our study provides a comprehensive expression profiling of lncRNAs, miRNAs, and mRNAs in a collection of defense mutants in rice. To decipher the putative functional significance of lncRNAs, we constructed trans- and cis-targeting networks involving differentially expressed lncRNAs and mRNAs, as well as a ceRNA network incorporating differentially expressed lncRNAs, miRNAs, and mRNAs. Together, the findings from this study provide compelling evidence supporting the pivotal roles of lncRNAs in OsRpp30-mediated disease resistance in rice.

Cleavage kinetics of human mitochondrial RNase P and contribution of its non-nuclease subunits.

RNase P is the endonuclease responsible for the 5' processing of precursor tRNAs (pre-tRNAs). Unlike the single-subunit protein-only RNase P (PRORP) found in plants or protists, human mitochondrial RNase P is a multi-enzyme assembly that in addition to the homologous PRORP subunit comprises a methyltransferase (TRMT10C) and a dehydrogenase (SDR5C1) subunit; these proteins, but not their enzymatic activities, are required for efficient pre-tRNA cleavage. Here we report a kinetic analysis of the cleavage reaction by human PRORP and its interplay with TRMT10C-SDR5C1 including 12 different mitochondrial pre-tRNAs. Surprisingly, we found that PRORP alone binds pre-tRNAs with nanomolar affinity and can even cleave some of them at reduced efficiency without the other subunits. Thus, the ancient binding mode, involving the tRNA elbow and PRORP's PPR domain, appears basically retained by human PRORP, and its metallonuclease domain is in principle correctly folded and functional. Our findings support a model according to which the main function of TRMT10C-SDR5C1 is to direct PRORP's nuclease domain to the cleavage site, thereby increasing the rate and accuracy of cleavage. This functional dependence of human PRORP on an extra tRNA-binding protein complex likely reflects an evolutionary adaptation to the erosion of canonical structural features in mitochondrial tRNAs.

Association of SARS-CoV-2 viral load with abnormal laboratory characteristics and clinical outcomes in hospitalised COVID-19 patients.

We conducted a retrospective, analytical cross-sectional and single-centre study that included 190 hospitalised COVID-19 patients in the Fujian Provincial Hospital South Branch between December 2022 and January 2023 to analyse the correlation of viral loads of throat swabs with clinical progression and outcomes. To normalise the Ct value as quantification of viral loads, we used RNase P gene as internal control gene and subtracted the Ct value of SARS-CoV-2 N gene from the Ct value of RNase P gene, termed △Ct. Most patients were discharged (84.2%), and only 10 (5.6%) individuals who had a lower △Ct value died. The initial △Ct value of participants was also significantly correlated with some abnormal laboratory characteristics, and the duration time of SARS-CoV-2 was longer in patients with severe symptoms and a lower △Ct value at admission. Our study suggested that the △Ct value may be used as a predictor of disease progression and outcomes in hospitalised COVID-19 patients.

Human RNase P exhibits and controls distinct ribonucleolytic activities required for ordered maturation of tRNA.

Precursor tRNAs are transcribed with flanking and intervening sequences known to be processed by specific ribonucleases. Here, we show that transcription complexes of RNA polymerase III assembled on tRNA genes comprise RNase P that cleaves precursor tRNA and subsequently degrades the excised 5' leader. Degradation is based on a 3'-5' exoribonucleolytic activity carried out by the protein subunit Rpp14, as determined by biochemical and reverse genetic analyses. Neither reconstituted nor purified RNase P displays this magnesium ion-dependent, processive exoribonucleolytic activity. Markedly, knockdown of Rpp14 by RNA interference leads to a wide-ranging inhibition of cleavage of flanking and intervening sequences of various precursor tRNAs in extracts and cells. This study reveals that RNase P controls tRNA splicing complex and RNase Z for ordered maturation of nascent precursor tRNAs by transcription complexes.

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.