tRNA Precursors Research Articles

Towards plant resistance to viruses using protein-only RNase P

Plant viruses cause massive crop yield loss worldwide. Most plant viruses are RNA viruses, many of which contain a functional tRNA-like structure. RNase P has the enzymatic activity to catalyze the 5′ maturation of precursor tRNAs. It is also able to cleave tRNA-like structures. However, RNase P enzymes only accumulate in the nucleus, mitochondria, and chloroplasts rather than cytosol where virus replication takes place. Here, we report a biotechnology strategy based on the re-localization of plant protein-only RNase P to the cytosol (CytoRP) to target plant viruses tRNA-like structures and thus hamper virus replication. We demonstrate the cytosol localization of protein-only RNase P in Arabidopsis protoplasts. In addition, we provide in vitro evidences for CytoRP to cleave turnip yellow mosaic virus and oilseed rape mosaic virus. However, we observe varied in vivo results. The possible reasons have been discussed. Overall, the results provided here show the potential of using CytoRP for combating some plant viral diseases.

Ligand-dependent tRNA processing by a rationally designed RNase P riboswitch.

We describe a synthetic riboswitch element that implements a regulatory principle which directly addresses an essential tRNA maturation step. Constructed using a rational in silico design approach, this riboswitch regulates RNase P-catalyzed tRNA 5′-processing by either sequestering or exposing the single-stranded 5′-leader region of the tRNA precursor in response to a ligand. A single base pair in the 5′-leader defines the regulatory potential of the riboswitch both in vitro and in vivo. Our data provide proof for prior postulates on the importance of the structure of the leader region for tRNA maturation. We demonstrate that computational predictions of ligand-dependent structural rearrangements can address individual maturation steps of stable non-coding RNAs, thus making them amenable as promising target for regulatory devices that can be used as functional building blocks in synthetic biology.

TRNA-derived fragments as New Hallmarks of Aging and Age-related Diseases

tRNA-derived fragments (tRFs), which are non-coding RNAs produced via tRNA cleavage with lengths of 14 to 50 nucleotides, originate from precursor tRNAs or mature tRNAs and exist in a wide range of organisms. tRFs are produced not by random fracture of tRNAs but by specific mechanisms. Considerable evidence shows that tRFs are detectable in model organisms of different ages and are associated with age-related diseases in humans, such as cancer and neurodegenerative diseases. In this literature review, the origin and classification of tRFs and the regulatory mechanisms of tRFs in aging and age-related diseases are summarized. We also describe the available tRF databases and research techniques and lay a foundation for the exploration of tRFs as biomarkers for the diagnosis and treatment of aging and age-related diseases.

Disease-associated mutations in mitochondrial precursor tRNAs affect binding, m1R9 methylation, and tRNA processing by mtRNase P.

Mitochondrial diseases linked to mutations in mitochondrial (mt) tRNA sequences are common. However, the contributions of these tRNA mutations to the development of diseases is mostly unknown. Mutations may affect interactions with (mt)tRNA maturation enzymes or protein synthesis machinery leading to mitochondrial dysfunction. In human mitochondria, in most cases the first step of tRNA processing is the removal of the 5′ leader of precursor tRNAs (pre-tRNA) catalyzed by the three-component enzyme, mtRNase P. Additionally, one component of mtRNase P, mitochondrial RNase P protein 1 (MRPP1), catalyzes methylation of the R9 base in pre-tRNAs. Despite the central role of 5′ end processing in mitochondrial tRNA maturation, the link between mtRNase P and diseases is mostly unexplored. Here, we investigate how 11 different human disease-linked mutations in (mt)pre-tRNAIle, (mt)pre-tRNALeu(UUR), and (mt)pre-tRNAMet affect the activities of mtRNase P. We find that several mutations weaken the pre-tRNA binding affinity (KDs are approximately two- to sixfold higher than that of wild-type), while the majority of mutations decrease 5′ end processing and methylation activity catalyzed by mtRNase P (up to ∼55% and 90% reduction, respectively). Furthermore, all of the investigated mutations in (mt)pre-tRNALeu(UUR) alter the tRNA fold which contributes to the partial loss of function of mtRNase P. Overall, these results reveal an etiological link between early steps of (mt)tRNA-substrate processing and mitochondrial disease.

A stress-induced tyrosine-tRNA depletion response mediates codon-based translational repression and growth suppression.

Eukaryotic transfer RNAs can become selectively fragmented upon various stresses, generating tRNA-derived small RNA fragments. Such fragmentation has been reported to impact a small fraction of the tRNA pool and thus presumed to not directly impact translation. We report that oxidative stress can rapidly generate tyrosine-tRNAGUA fragments in human cells-causing significant depletion of the precursor tRNA. Tyrosine-tRNAGUA depletion impaired translation of growth and metabolic genes enriched in cognate tyrosine codons. Depletion of tyrosine tRNAGUA or its translationally regulated targets USP3 and SCD repressed proliferation-revealing a dedicated tRNA-regulated growth-suppressive pathway for oxidative stress response. Tyrosine fragments are generated in a DIS3L2 exoribonuclease-dependent manner and inhibit hnRNPA1-mediated transcript destabilization. Moreover, tyrosine fragmentation is conserved in C. elegans. Thus, tRNA fragmentation can coordinately generate trans-acting small RNAs and functionally deplete a tRNA. Our findings reveal the existence of an underlying adaptive codon-based regulatory response inherent to the genetic code.

Splicing Endonuclease Is an Important Player in rRNA and tRNA Maturation in Archaea.

In all three domains of life, tRNA genes contain introns that must be removed to yield functional tRNA. In archaea and eukarya, the first step of this process is catalyzed by a splicing endonuclease. The consensus structure recognized by the splicing endonuclease is a bulge-helix-bulge (BHB) motif which is also found in rRNA precursors. So far, a systematic analysis to identify all biological substrates of the splicing endonuclease has not been carried out. In this study, we employed CRISPRi to repress expression of the splicing endonuclease in the archaeon Haloferax volcanii to identify all substrates of this enzyme. Expression of the splicing endonuclease was reduced to 1% of its normal level, resulting in a significant extension of lag phase in H. volcanii growth. In the repression strain, 41 genes were down-regulated and 102 were up-regulated. As an additional approach in identifying new substrates of the splicing endonuclease, we isolated and sequenced circular RNAs, which identified excised introns removed from tRNA and rRNA precursors as well as from the 5′ UTR of the gene HVO_1309. In vitro processing assays showed that the BHB sites in the 5′ UTR of HVO_1309 and in a 16S rRNA-like precursor are processed by the recombinant splicing endonuclease. The splicing endonuclease is therefore an important player in RNA maturation in archaea.

The abundance of a transfer RNA-derived RNA fragment small RNA subpopulation is enriched in cauda spermatozoa

The small RNA (sRNA) landscape of mammalian spermatozoa is considerably altered as these gametic cells migrate through the segment specific microenvironments of the epididymis. More specifically, the microRNA (miRNA) species of sRNA dominates the sRNA landscape of spermatozoa of the proximal caput segment of the epididymis. However, in sperm cells sourced from the distal cauda epididymal segment, the transfer RNA (tRNA)-derived RNA fragment (tRF) sRNA species is the most abundant. Here we show that the 5′ halves of fifteen mature tRNAs were used as processing substrates for the production of a specific subpopulation of tRF sRNAs, 30 to 33 nucleotides (30–33-nt) in length. A quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) approach was used to experimentally validate the sRNA sequencing identified trend of enriched abundance of this specific 30–33-nt tRF subpopulation in cauda spermatozoa. The length, and exclusive alignment of the cauda spermatozoa enriched tRF subpopulation to the 5′ half of each processed tRNA precursor, identified ANGIOGENIN (ANG) as the endonuclease likely responsible for tRF production in the mouse epididymis: a prediction confirmed via immunoblotting assessment of ANG abundance in spermatozoa sourced from the caput, corpus and cauda epididymal segments. When taken together with our previous profiling of miRNA and Piwi-interacting RNA (piRNA) sRNA abundance in spermatozoa sourced from the three segments of physiologically normal mouse epididymides, the tRF profile reported here adds greater depth of coverage to the global sRNA landscape of the mouse epididymis; a roadmap constructed to assist with the future molecular characterization of sRNA-directed responses to a wide range of imposed environmental stressors.

On the expanding roles of tRNA fragments in modulating cell behavior.

The fragments that derive from transfer RNAs (tRNAs) are an emerging category of regulatory RNAs. Known as tRFs, these fragments were reported for the first time only a decade ago, making them a relatively recent addition to the ever-expanding pantheon of non-coding RNAs. tRFs are short, 16–35 nucleotides (nts) in length, and produced through cleavage of mature and precursor tRNAs at various positions. Both cleavage positions and relative tRF abundance depend strongly on context, including the tissue type, tissue state, and disease, as well as the sex, population of origin, and race/ethnicity of an individual. These dependencies increase the urgency to understand the regulatory roles of tRFs. Such efforts are gaining momentum, and comprise experimental and computational approaches. System-level studies across many tissues and thousands of samples have produced strong evidence that tRFs have important and multi-faceted roles. Here, we review the relevant literature on tRF biology in higher organisms, single cell eukaryotes, and prokaryotes.

Characterization of the Methanomicrobial Archaeal RNase Zs for Processing the CCA-Containing tRNA Precursors.

RNase Z is a widely distributed and usually essential endoribonuclease involved in the 3′-end maturation of transfer RNAs (tRNAs). A CCA triplet that is needed for tRNA aminoacylation in protein translation is added by a nucleotidyl-transferase after the 3′-end processing by RNase Z. However, a considerable proportion of the archaeal pre-tRNAs genetically encode a CCA motif, while the enzymatic characteristics of the archaeal RNase (aRNase) Zs in processing CCA-containing pre-tRNAs remain unclear. This study intensively characterized two methanomicrobial aRNase Zs, the Methanolobus psychrophilus mpy-RNase Z and the Methanococcus maripaludis mmp-RNase Z, particularly focusing on the properties of processing the CCA-containing pre-tRNAs, and in parallel comparison with a bacterial bsu-RNase Z from Bacillus subtilis. Kinetic analysis found that Co2+ supplementation enhanced the cleavage efficiency of mpy-RNase Z, mmp-RNase Z, and bsu-RNase Z for 1400-, 2990-, and 34-fold, respectively, and Co2+ is even more indispensable to the aRNase Zs than to bsu-RNase Z. Mg2+ also elevated the initial cleavage velocity (V0) of bsu-RNase Z for 60.5-fold. The two aRNase Zs exhibited indiscriminate efficiencies in processing CCA-containing vs. CCA-less pre-tRNAs. However, V0 of bsu-RNase Z was markedly reduced for 1520-fold by the CCA motif present in pre-tRNAs under Mg2+ supplementation, but only 5.8-fold reduced under Co2+ supplementation, suggesting Co2+ could ameliorate the CCA motif inhibition on bsu-RNase Z. By 3′-RACE, we determined that the aRNase Zs cleaved just downstream the discriminator nucleotide and the CCA triplet in CCA-less and CCA-containing pre-tRNAs, thus exposing the 3′-end for linking CCA and the genetically encoded CCA triplet, respectively. The aRNase Zs, but not bsu-RNase Z, were also able to process the intron-embedded archaeal pre-tRNAs, and even process pre-tRNAs that lack the D, T, or anticodon arm, but strictly required the acceptor stem. In summary, the two methanomicrobial aRNase Zs use cobalt as a metal ligand and process a broad spectrum of pre-tRNAs, and the characteristics would extend our understandings on aRNase Zs.

Abstract 426: Effects of Non-syndromic Mitral Valve Prolapse on Valvular Interstitial Cell Extracellular Vesicle-associated Non-coding RNA in a Spontaneous Canine Disease Model

Non-syndromic mitral valve prolapse (MVP), or Barlow’s Disease, is a valvular heart disease with a prevalence of 2-3% in the human adult population. Consequences of MVP include mitral regurgitation that without surgical treatment can progress to congestive heart failure, arrhythmias and sudden death. Since MVP is a degenerative disease that worsens with age, surgical risk is of concern with an older population, necessitating the development of alternative strategies for early detection and effective medical intervention. Similar to humans, canine MVP is age-related with a lifetime prevalence close to 90%, providing a large population for study. Canine MVP represents a relevant spontaneous large animal model to dissect the mechanistic underpinnings of disease pathogenesis and evaluate novel treatment. We hypothesize that extracellular vesicles (EVs) and their associated non-coding RNA (ncRNA) may play a role in this disease by enabling propagation of the fibroblast-to-myofibroblast phenotypic switch in mitral valvular interstitial cells (VICs). VICs were isolated from 12 normal and 14 diseased canine mitral valves and cultured in defined chemical media for 48 hours. EVs were isolated from conditioned media using size exclusion chromatography. Total RNA was isolated from EVs with the Qiagen miReasy Plasma/Serum kit, and from VIC cells with the mirVana miRNA Isolation Kit. Sequencing libraries were created using QIAseq miRNA Library Kit, and RNAseq was performed with Illumina HiSeq 2500 sequencer with single read 100 base format. Reads were mapped to canine ncRNA databases (miRbase, RFam). VICs from diseased valves had higher expression of αSMA than cells from normal valves (p = 0.002) based on RT-qPCR. Sequence analysis showed decreased content of tRNA (p = 0.023) and miRNA precursor (p = 0.036) in EVs from diseased VICs, in addition to decreased expression of let-7f. Cells from diseased valves had lower Y-RNA content (p = 0.007). miR-8859a and miR-451 were preferentially packaged into EVs from both normal and diseased VICs. Preferential packaging of miR-155 was seen in normal VIC EVs and miR-191 in diseased VIC EVs. Functional roles of these miRs will need to be confirmed to determine their therapeutic potential for treatment of MVP.

Pentatricopeptide repeats of protein-only RNase P use a distinct mode to recognize conserved bases and structural elements of pre-tRNA.

Pentatricopeptide repeat (PPR) motifs are α-helical structures known for their modular recognition of single-stranded RNA sequences with each motif in a tandem array binding to a single nucleotide. Protein-only RNase P 1 (PRORP1) in Arabidopsis thaliana is an endoribonuclease that uses its PPR domain to recognize precursor tRNAs (pre-tRNAs) as it catalyzes removal of the 5'-leader sequence from pre-tRNAs with its NYN metallonuclease domain. To gain insight into the mechanism by which PRORP1 recognizes tRNA, we determined a crystal structure of the PPR domain in complex with yeast tRNAPhe at 2.85 Å resolution. The PPR domain of PRORP1 bound to the structurally conserved elbow of tRNA and recognized conserved structural features of tRNAs using mechanisms that are different from the established single-stranded RNA recognition mode of PPR motifs. The PRORP1 PPR domain-tRNAPhe structure revealed a conformational change of the PPR domain upon tRNA binding and moreover demonstrated the need for pronounced overall flexibility in the PRORP1 enzyme conformation for substrate recognition and catalysis. The PRORP1 PPR motifs have evolved strategies for protein-tRNA interaction analogous to tRNA recognition by the RNA component of ribonucleoprotein RNase P and other catalytic RNAs, indicating convergence on a common solution for tRNA substrate recognition.

MAF1 is a chronic repressor of RNA polymerase III transcription in the mouse

Maf1−/− mice are lean, obesity-resistant and metabolically inefficient. Their increased energy expenditure is thought to be driven by a futile RNA cycle that reprograms metabolism to meet an increased demand for nucleotides stemming from the deregulation of RNA polymerase (pol) III transcription. Metabolic changes consistent with this model have been reported in both fasted and refed mice, however the impact of the fasting-refeeding-cycle on pol III function has not been examined. Here we show that changes in pol III occupancy in the liver of fasted versus refed wild-type mice are largely confined to low and intermediate occupancy genes; high occupancy genes are unchanged. However, in Maf1−/− mice, pol III occupancy of the vast majority of active loci in liver and the levels of specific precursor tRNAs in this tissue and other organs are higher than wild-type in both fasted and refed conditions. Thus, MAF1 functions as a chronic repressor of active pol III loci and can modulate transcription under different conditions. Our findings support the futile RNA cycle hypothesis, elaborate the mechanism of pol III repression by MAF1 and demonstrate a modest effect of MAF1 on global translation via reduced mRNA levels and translation efficiencies for several ribosomal proteins.

Relationship between tRNA-derived fragments and human cancers.

tRNA-derived fragments, a class of small noncoding RNAs (sncRNAs), have been identified in numerous studies in recent years. tRNA-derived fragments are classified into two main groups, including tRNA halves (tiRNAs) and tRNA-derived small RNA fragments (tRFs), according to different cleavage positions of the precursor or mature tRNAs. Instead of random tRNA degradation debris, a growing body of evidence has shown that tRNA-derived fragments are precise products of specific tRNA modifications and play important roles in biological activities, such as regulating protein translation, affecting gene expression, and altering immune signaling. Recently, the relations between tRNA-derived fragments and the occurrence of human diseases, especially cancers, have generated wide interest. It has been demonstrated that tRNA-derived fragments are involved in cancer cell proliferation, metastasis, progression and survival. In this review, we will describe the biogenesis of tRNA-derived fragments, the distinct expression and function of tRNA-derived fragments in the development of cancers, and their emerging roles as diagnostic and prognostic biomarkers and precise targets of future treatments.

Abstract IA31: Novel nuclear and mitochondrial RNAs that are linked to key pathways and depend on sex, population origin, race, tissue, and disease

Abstract IA31: Novel nuclear and mitochondrial RNAs that are linked to key pathways and depend on sex, population origin, race, tissue, and disease

Mechanistic and In silico Characterization of Metal ion Requirements of Escherichia coli Zinc Phosphodiesterase Activity

Abstract Zinc phosphodiesterase (ZiPD) participates in the maturation of tRNA precursors. The roles of metal ions in promoting phosphoryl transfer reaction on zinc phosphodiesterase (ZiPD) activity have not been fully characterized. Therefore, this study investigated the effects of some metal ions on phosphodiesterase activity of Escherichia coli ZiPD as well as the binding site and binding affinity of the metal ions. ZiPD activity was measured by monitoring the rate of hydrolysis of bis-para-nitrophenyl phosphate (bis-pNPP) in the presence of some selected divalent metal ions (Mn2+, Co2+, Mg2+ and Zn2+). The results obtained revealed that Mn2+ at 1 mM activated ZiPD activity by 4-fold with binding affinity score of 1.795. Co2+ at 0.5 mM activated ZiPD activity by 2-fold with binding affinity score of 1.773. Mg2+ at 0.5 mM enhanced the binding affinity of ZiPD for bis-pNPP but did not increase the turnover rate of ZiPD. Zn2+ at 1.5 mM activated ZiPD activity by 2-fold via increased affinity of ZiPD for bis-pNPP. In conclusion, the findings from this study showed that Mn2+ and Zn2+ are the most effective stimulatory ions of ZiPD for bis-pNPP while Zn2+ exerted the highest binding affinity of ZiPD for bis-pNPP.

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.

TRNA: Splicing and Intron Turnover

In most eukaryotic genomes a subset of tRNA genes contains introns. Introns in tRNAs genes are short sequences located 1 nt. 3′ of the anticodon. Introns are removed from precursor tRNAs (pre‐tRNA) by a conserved heterotetrameric protein enzyme, tRNA splicing endonuclease (SEN), that recognizes pre‐tRNA structure rather than sequence motifs at splice junctions. In vertebrate cells intron removal from pre‐tRNAs occurs in the nucleoplasm; in contrast, pre‐tRNA splicing in budding yeast was surprisingly discovered to occur on the mitochondrial surface (Yoshihisa et al. 2003). We recently reported that the SEN complex also locates to the mitochondrial surface in fission yeast (Wan & Hopper 2018) and, therefore, pre‐tRNA splicing on the mitochondrial surface has been conserved for at least 500 million years. The mitochondrial outer membrane protein, Tom70, is required for efficient SEN subunit location to mitochondria in both budding and fission yeast (Wan & Hopper 2018). The freed introns are rarely detected in cells and until recently it was unknown how are they are turned over. We identified at least 5 separate, species‐specific mechanisms that function in tRNA intron destruction (Wu & Hopper, 2014; Bao, unpublished). Interestingly, under particular stress conditions, specific tRNA introns accumulate, possibly indicating their functions in stress response (Peltier, unpublished). Moreover, we discovered that tRNA introns possess long stretches of perfect complementarity to particular budding yeast mRNAs (Bao, unpublished), also indicative of their possible roles in gene regulation. Our initial experiments support the hypothesis that tRNA introns may function as a novel type of noncoding regulatory RNA (Nostramo, unpublished).Support or Funding InformationNIH GMS 122884

The Role of tRNA Derived Small RNAs in Gene Regulation in Normal Tissues and Cancer

The function of tRNAs is not merely to provide the specific amino acids for a growing peptide chain during protein synthesis. Precursor and mature tRNAs can be enzymatically cleaved into various tRNA derived small RNAs. While there are thousands of different RNA species that can be generated, the function of most are not known. I will provide a brief introduction into this field and describe the mechanism of how at least one specific tsRNA derived from the 3′end of a mature tRNA is involved in regulating ribosome biogenesis (Kim et al. Nature 2017). This 3′tsRNA is upregulated in many tumors. Reducing the tsRNA in patient derived hepatocellular carcinomas using antisense oligonucleotides induced apoptosis and tumor shrinkage in mice. This as well as other 3′tsRNAs are being studied as potential targets for cancer therapeutics. I will provide an update on current approaches we are developing to help us identify, quantify, and define the targets of the myriad of various tsRNAs present in various cell types. This will further allow investigation as to their role in maintaining health and disease states.Support or Funding InformationNational Institutes of Health ‐ National Institute of Diabetes and Digestive and Kidney Diseases R01‐DK114483

CLP1 acts as the main RNA kinase in mice

CLP1 plays an essential role in the protein complex involved in mRNA 3′-end formation and polyadenylation as well as in the tRNA splicing endonuclease (TSEN) complex involved in the splicing of precursor tRNAs. NOL9 localizes in the nucleolus of cells and plays an essential role in ribosomal RNA maturation. Both CLP1 and NOL9 are RNA kinases that phosphorylate the 5′ end of RNAs. From the evidence that phosphorylation of the 5′ end of a siRNA is essential for its efficient RNA cleavage, it was expected that CLP1 and NOL9 would be corresponding molecules. However, there had been no direct evidence that this is the case. In this study, murine NOL9 showed no apparent RNA kinase activity in cells or even in an RNA kinase assay using recombinant murine NOL9 protein. Although siRNA efficiency was decreased in CLP1 kinase-dead (Clp1K/K) cells, it was not influenced by NOL9 overexpression. These findings indicate that in mouse cells it is CLP1 that mainly acts to phosphorylate the 5′ end of RNAs in the siRNA pathway, with no apparent involvement of NOL9.

Polyethylene glycol molecular crowders enhance the catalytic ability of bimolecular bacterial RNase P ribozymes

The modular structure of bacterial ribonuclease P (RNase P) ribozymes, which recognize tertiary structures of precursor tRNAs (pre-tRNAs) to cleave their 5′ leader sequence, can be dissected physically into the two structured domain RNAs (S-domain and C-domain). Separately prepared S-domain RNA and C-domain RNA assemble to form bimolecular forms of RNase P ribozymes. We analyzed the effects of polyethylene glycols (PEGs) on pre-tRNA cleavage catalyzed by bimolecular RNase P ribozymes to examine the effects of molecular crowding on the reaction. PEG molecular crowders significantly enhanced the activities of bimolecular RNase P ribozymes, some of which were hardly active without PEGs.