Genome-wide Identification and Quantitative Analysis of Cleaved tRNA Fragments Induced by Cellular Stress

Certain stress conditions can induce cleavage of tRNAs around the anticodon loop via the use of the ribonuclease angiogenin. The cellular factors that regulate tRNA cleavage are not well known. In this study we used normal and eIF2α phosphorylation-deficient mouse embryonic fibroblasts and applied a microarray-based methodology to identify and compare tRNA cleavage patterns in response to hypertonic stress, oxidative stress (arsenite), and treatment with recombinant angiogenin. In all three scenarios mouse embryonic fibroblasts deficient in eIF2α phosphorylation showed a higher accumulation of tRNA fragments including those derived from initiator-tRNA^Met. We have shown that tRNA cleavage is regulated by the availability of angiogenin, its substrate (tRNA), the levels of the angiogenin inhibitor RNH1, and the rates of protein synthesis. These conclusions are supported by the following findings: (i) exogenous treatment with angiogenin or knockdown of RNH1 increased tRNA cleavage; (ii) tRNA fragment accumulation was higher during oxidative stress than hypertonic stress, in agreement with a dramatic decrease of RNH1 levels during oxidative stress; and (iii) a positive correlation was observed between angiogenin-mediated tRNA cleavage and global protein synthesis rates. Identification of the stress-specific tRNA cleavage mechanisms and patterns will provide insights into the role of tRNA fragments in signaling pathways and stress-related disorders.     

血管生成素与tRNA片段化

tRNA主要功能是转运氨基酸参与蛋白质合成,在蛋白质生物合成过程中起着关键性的作用．近年来发现,tRNA是细胞内小RNA分子的重要来源,具有其它重要的生物学功能．来源于成熟tRNA分子的tRNA片段根据切割位置及生成机制的不同,主要分为两类：一类是tRNA半分子（tRNA halves）;另一类是较小的tRNA片段,称为tRFs( tRNA fragments)．在哺乳动物细胞中,tRNA半分子由血管生成素在tRNA分子反密码环处切割生成．本文主要针对tRNA半分子的加工机制、功能及在临床上的潜在应用进行综述．     

tRNA cleavage is a conserved response to oxidative stress in eukaryotes

Recent results have identified a diversity of small RNAs in a wide range of organisms. In this work, we demonstrate that Saccharomyces cerevisiae contains a small RNA population consisting primarily of tRNA halves and rRNA fragments. Both 5′ and 3′ fragments of tRNAs are detectable by Northern blot analysis, suggesting a process of endonucleolytic cleavage. tRNA and rRNA fragment production in yeast is most pronounced during oxidative stress conditions, especially during entry into stationary phase. Similar tRNA fragments are also observed in human cell lines and in plants during oxidative stress. These results demonstrate that tRNA cleavage is a conserved aspect of the response to oxidative stress.     

Stress‐induced tRNA cleavage and tiRNA generation in rat neuronal PC12 cells

Transfer RNA (tRNA) plays a role in stress response programs involved in various pathological conditions including neurological diseases. Under cell stress conditions, intracellular tRNA is cleaved by a specific ribonuclease, angiogenin, generating tRNA‐derived fragments or tRNA‐derived stress‐induced RNA (tiRNA). Generated tiRNA contributes to the cell stress response and has potential cell protective effects. However, tiRNA generation under stress conditions in neuronal cells has not been fully elucidated. To examine angiogenin‐mediated tiRNA generation in neuronal cells, we used the rat neuronal cell line, PC12, in combination with analysis of SYBR staining and immuno‐northern blotting using anti‐1‐methyladenosine antibody, which specifically and sensitively detects tiRNA. Oxidative stress induced by arsenite and hydrogen peroxide caused tRNA cleavage and tiRNA generation in PC12 cells. We also demonstrated that oxygen‐glucose deprivation, which is an in vitro model of ischemic–reperfusion injury, induced tRNA cleavage and tiRNA generation. In these stress conditions, the amount of generated tiRNA was associated with the degree of morphological cell damage. Time course analysis indicated that generation of tiRNA was prior to severe cell damage and cell death. Angiogenin over‐expression did not influence the amount of tiRNA in normal culture conditions; however, it significantly increased tiRNA generation induced by cell stress conditions. Our findings show that angiogenin‐mediated tiRNA generation can be induced in neuronal cells by different cell stressors, including ischemia–reperfusion. Additionally, detection of tiRNA could be used as a potential cell damage marker in neuronal cells.

Cover Image for this issue: doi: 10.1111/jnc.14191 .

     

来源于tRNA的小分子RNA——降解碎片还是新的调控分子

具有经典三叶草结构的tRNA作为细胞蛋白质合成机器的重要元件,已经拥有几十年深入细致的研究历史.但是,对于其功能的认识远没有止境,尤其在其作为潜在的基因表达调控分子前体的功能目前正逐渐被人们认识.最新的多项研究结果表明,在多种细胞系中通过高通量测序发现某种来源于tRNA的小片段RNA,这些剪切产物被认为与多种microRNA加工体系关键分子(如Dicer、Ago家族中的蛋白质)具有相互作用的能力.同时,报告基因检测系统的研究结果也暗示,这些小片段RNA具有类似microRNA的潜在调控功能,可能在细胞应对外界环境刺激时发挥重要的调节作用.如其具体的作用机制能够被更多的实验结果阐明,将极大地扩展我们对于非编码RNA调控功能的认识.     

The RNase Rny1p cleaves tRNAs and promotes cell death during oxidative stress in Saccharomyces cerevisiae

The cellular response to stress conditions involves a decision between survival or cell death when damage is severe. A conserved stress response in eukaryotes involves endonucleolytic cleavage of transfer RNAs (tRNAs). The mechanism and significance of such tRNA cleavage is unknown. We show that in yeast, tRNAs are cleaved by the RNase T2 family member Rny1p, which is released from the vacuole into the cytosol during oxidative stress. Rny1p modulates yeast cell survival during oxidative stress independently of its catalytic ability. This suggests that upon release to the cytosol, Rny1p promotes cell death by direct interactions with downstream components. Thus, detection of Rny1p, and possibly its orthologues, in the cytosol may be a conserved mechanism for assessing cellular damage and determining cell survival, analogous to the role of cytochrome c as a marker for mitochondrial damage.     

In vitro hyperprocessing of Drosophila tRNAs by the catalytic RNA of RNase P the cloverleaf structure of tRNA is not always stable?

We have previously reported that the catalytic RNA subunit of RNase P of Escherichia coli (M1 RNA) cleaves Drosophila initiator methionine tRNA (tRNA(Met)i) within the mature tRNA sequence to produce specific fragments. This cleavage was dependent on the occurrence of an altered conformation of the tRNA substrate. We call this further cleavage hyperprocessing. In the present paper, to search for another tRNA that can be hyperprocessed in vitro, we used total mature tRNAs from Drosophila as substrates for the in vitro M1 RNA reaction. We found that some tRNAs can be hyperprocessed by M1 RNA and that two such tRNAs are an alanine tRNA and a histidine tRNA. Using mutant substrates of these tRNAs, we also show that the hyperprocessing by M1 RNA is dependent on the occurrence of altered conformations of these tRNAs. The altered conformations were very similar to that of tRNA(Met)i. We show here that M1 RNA can be used as a powerful tool to detect the alternative conformation of tRNAs. The relationship between these hyperprocessing reactions and stability of the tRNA structure will also be discussed.     

Reversible and Rapid Transfer-RNA Deactivation as a Mechanism of Translational Repression in Stress

Stress-induced changes of gene expression are crucial for survival of eukaryotic cells. Regulation at the level of translation provides the necessary plasticity for immediate changes of cellular activities and protein levels. In this study, we demonstrate that exposure to oxidative stress results in a quick repression of translation by deactivation of the aminoacyl-ends of all transfer-RNA (tRNA). An oxidative-stress activated nuclease, angiogenin, cleaves first within the conserved single-stranded 3′-CCA termini of all tRNAs, thereby blocking their use in translation. This CCA deactivation is reversible and quickly repairable by the CCA-adding enzyme [ATP(CTP):tRNA nucleotidyltransferase]. Through this mechanism the eukaryotic cell dynamically represses and reactivates translation at low metabolic costs.     

An emerging role of the 5′ termini of mature tRNAs in human diseases: Current situation and prospects

The fundamental biological roles of a class of small noncoding RNAs (sncRNAs), derived from mature tRNAs or pre-tRNAs, in human diseases have received increasing attention in recent years. These ncRNAs are called tRNA-derived fragments (tRFs) or tRNA-derived small RNAs (tsRNAs). tRFs mainly include tRF-1, tRF-5, tRF-3 and tRNA halves (tiRNAs or tRHs), which are produced by enzyme-specific cleavage of tRNAs. Here, we classify tRF-5 and 5′ tiRNAs into the same category: 5′-tRFs and review the biological functions and regulatory mechanisms of 5′-tRFs in cancer and other diseases (metabolic diseases, neurodegenerative diseases, pathological stress injury and virus infection) to provide a new theoretical basis for the diagnosis and treatment of diseases.     

Identification of an entire set of tRNA molecules and characterization of cleavage sites of the intron-containing tRNA precursors in acidothermophilic crenarchaeon Sulfolobus tokodaii strain7

The acidothermophilic crenarchaeon, Sulfolobus tokodaii strain7, was isolated from a hot spring in Beppu, Kyushu, Japan. Whole genomic data of this microorganism indicated that among 46 putative tRNA genes identified, 24 were interrupted tRNA genes containing an intron. A sequence comparison between the cDNA sequences for unspliced and spliced tRNAs indicated that all predicted tRNAs were expressed and all intron portions were spliced in this microorganism. However, the actual cleavage site in the splicing process was not determined for 13 interrupted tRNAs because of the presence of the same nucleotides at both 5′ and 3′ border regions of each intron. The cleavage sites for all the introns, which were determined by an in vitro cleavage experiment with recombinant splicing endonuclease as well as cDNA sequencing of the spliced tRNAs, indicated that non-canonical BHB structure motifs were also recognized and processed by the splicing machinery in this organism. This is the first report to empirically determine the actual cleavage and splice sites of introns in the whole set of archaeal tRNA genes, and reassigns the exon-intron borders with a novel and more plausible non-canonical BHB structure.     

Angiogenin is a cytotoxic, tRNA-specific ribonuclease in the RNase A superfamily.

Angiogenin is a 14.4-kDa human plasma protein with 65% homology to RNase A that retains the key active site residues and three of the four RNase A disulfide bonds. We demonstrate that recombinant angiogenin functions as a cytotoxic tRNA-specific RNase in cell-free lysates and when injected into Xenopus oocytes. Inhibition of protein synthesis by angiogenin correlates with degradation of endogenous oocyte tRNA. Exogenous, radiolabeled tRNA is also hydrolyzed by angiogenin, whereas oocyte rRNA and mRNA are not detectably degraded by angiogenin. Protein synthesis was restored to angiogenin-injected oocytes by injecting the RNase inhibitor RNasin plus total Xenopus or calf liver tRNAs, thereby demonstrating that the tRNA degradation induced by angiogenin was the sole cause of cytotoxicity. A similar tRNA-reversible inhibition of protein synthesis was seen in rabbit reticulocyte lysates. Angiogenin therefore appears to be a specific cellular tRNase, whereas five homologues in the RNase A superfamily lack angiogenin's specificity for tRNA. One of these homologues purified from human eosinophils, eosinophil-derived neurotoxin, nonspecifically degrades oocyte RNA similar to RNase A and is also cytotoxic at very low concentrations.     

tRNA fragments in human health and disease

Transfer RNA (tRNA) is traditionally considered to be an adaptor molecule that helps ribosomes to decode messenger RNA (mRNA) and synthesize protein. Recent studies have demonstrated that tRNAs also serve as a major source of small non-coding RNAs that possess distinct and varied functions. These tRNA fragments are heterogeneous in size, nucleotide composition, biogenesis and function. Here we describe multiple roles that tRNA fragments play in cell physiology and discuss their relevance to human health and disease.     

tRNA is a new target for cleavage by a MazF toxin

Toxin-antitoxin (TA) systems play key roles in bacterial persistence, biofilm formation and stress responses. The MazF toxin from the Escherichia coli mazEF TA system is a sequence- and single-strand-specific endoribonuclease, and many studies have led to the proposal that MazF family members exclusively target mRNA. However, recent data indicate some MazF toxins can cleave specific sites within rRNA in concert with mRNA. In this report, we identified the repertoire of RNAs cleaved by Mycobacterium tuberculosis toxin MazF-mt9 using an RNA-seq-based approach. This analysis revealed that two tRNAs were the principal targets of MazF-mt9, and each was cleaved at a single site in either the tRNA^Pro14 D-loop or within the tRNA^Lys43 anticodon. This highly selective target discrimination occurs through recognition of not only sequence but also structural determinants. Thus, MazF-mt9 represents the only MazF family member known to target tRNA and to require RNA structure for recognition and cleavage. Interestingly, the tRNase activity of MazF-mt9 mirrors basic features of eukaryotic tRNases that also generate stable tRNA-derived fragments that can inhibit translation in response to stress. Our data also suggest a role for tRNA distinct from its canonical adapter function in translation, as cleavage of tRNAs by MazF-mt9 downregulates bacterial growth.     

Recombinant photoreactive tRNA molecules as probes for cross-linking studies

Photoreactive tRNA derivatives have been used extensively for investigating the interaction of tRNA molecules with their ligands and substrates. Recombinant RNA technology facilitates the construction of such tRNA probes through site-specific incorporation of photoreactive nucleosides. The general strategy involves preparation of suitable tRNA fragments and their ligation either to a photoreactive nucleotide or to each other. tRNA fragments can be prepared by site-specific cleavage of native tRNAs, or synthesized by enzymatic and chemical means. A number of photoreactive nucleosides suitable for incorporation into tRNA are presently available. Joining of tRNA fragments is accomplished either by RNA ligase or by DNA ligase in the presence of a DNA splint. The application of this methodology to the study of tRNA binding sites on the ribosome is discussed, and a model of the tRNA-ribosome complex is presented.     

血管生成素:RNA代谢过程中重要的核糖核酸酶

血管生成素是核糖核酸酶A超家族成员之一,具有较弱的核糖核酸酶活性.最新研究发现,血管生成素参与细胞内多种RNA的代谢过程.在生长条件下,血管生成素可以发生核转位聚集于细胞核中,促进rRNA转录,并可参与其剪切加工,同时它也调控一系列mRNA基因的转录,最终促进细胞的生长和增殖;在应激条件下,血管生成素能降解tRNA形成tiRNA,抑制细胞内整体蛋白质的翻译水平,并促进应激小体的形成,激活细胞内应激保护机制,从而促进细胞存活.此外,血管生成素还可参与非编码小RNA等RNA代谢过程.本文概述了血管生成素在RNA代谢中的作用与分子机制等方面的进展,并探讨了其在疾病发生和发展中的作用,以期开拓血管生成素的研究新思路.     

Several RNase T2 enzymes function in induced tRNA and rRNA turnover in the ciliate Tetrahymena

RNase T2 enzymes are produced by a wide range of organisms and have been implicated to function in diverse cellular processes, including stress-induced anticodon loop cleavage of mature tRNAs to generate tRNA halves. Here we describe a family of eight RNase T2 genes (RNT2A-RNT2H) in the ciliate Tetrahymena thermophila. We constructed strains lacking individual or combinations of these RNT2 genes that were viable but had distinct cellular and molecular phenotypes. In strains lacking only one Rnt2 protein or lacking a subfamily of three catalytically inactive Rnt2 proteins, starvation-induced tRNA fragments continued to accumulate, with only a minor change in fragment profile in one strain. We therefore generated strains lacking pairwise combinations of the top three candidates for Rnt2 tRNases. Each of these strains showed a distinct starvation-specific profile of tRNA and rRNA fragment accumulation. These results, the delineation of a broadened range of conditions that induce the accumulation of tRNA halves, and the demonstration of a predominantly ribonucleoprotein-free state of tRNA halves in cell extract suggest that ciliate tRNA halves are degradation intermediates in an autophagy pathway induced by growth arrest that functions to recycle idle protein synthesis machinery.     

Characterization of ribonucleolytic activity of angiogenin towards tRNA

Yeast tRNA is a convenient substrate for the assay of the ribonucleolytic activity of human angiogenin. The optimal pH, [NaCl], and temperature for tRNA cleavage by angiogenin are approximately 6.8, 15-30 mM, and approximately 55 degrees C, respectively, as compared with approximately 8.0, 100-200 mM, and approximately 65 degrees C, respectively, for RNase A. Polyanions and metals both inhibit angiogenin and RNase A but to different extents.