tRNA Fragmentation Research Articles

The tRNA methyltransferase Dnmt2 is required for accurate polypeptide synthesis during haematopoiesis.

The Dnmt2 enzyme utilizes the catalytic mechanism of eukaryotic DNA methyltransferases to methylate several tRNAs at cytosine 38. Dnmt2 mutant mice, flies, and plants were reported to be viable and fertile, and the biological function of Dnmt2 has remained elusive. Here, we show that endochondral ossification is delayed in newborn Dnmt2-deficient mice, which is accompanied by a reduction of the haematopoietic stem and progenitor cell population and a cell-autonomous defect in their differentiation. RNA bisulfite sequencing revealed that Dnmt2 methylates C38 of tRNA AspGTC, GlyGCC, and ValAAC, thus preventing tRNA fragmentation. Proteomic analyses from primary bone marrow cells uncovered systematic differences in protein expression that are due to specific codon mistranslation by tRNAs lacking Dnmt2-dependent methylation. Our observations demonstrate that Dnmt2 plays an important role in haematopoiesis and define a novel function of C38 tRNA methylation in the discrimination of near-cognate codons, thereby ensuring accurate polypeptide synthesis.

Welcome to the new tRNA world!

Transfer RNAs (tRNAs) are one of the classical non-coding RNAs, with lengths of approximately 70–100 bases. The secondary structure of tRNAs can be represented as a cloverleaf with four stems, and the three-dimensional structure as an “L” shape. Historically, the basic function of the tRNAs as essential components of translation was established in the 1960s, when it was found that each tRNA is charged with a target amino acid by a specific aminoacyl-tRNA synthetase, and delivers it to the ribosome during protein synthesis (Crick, 1966; Normanly and Abelson, 1989; Frank, 2000). However, recent studies suggest that the roles of tRNA in cellular regulation go beyond this paradigm. Now, tRNA is recognized as a regulator of many biological processes, and several unique tRNA genes have been discovered. Our understanding of the enzymes involved in tRNA functions has also increased and many tRNA-related diseases have been reported. In response to these exciting data, I have edited this special issue of tRNA, which revisits and summarizes the molecular biology of tRNA. The topics contributed by specialists in the field cover a wide range of tRNA research. In the last decade, a number of reports have described novel aspects of tRNAs in terms of the diversity of their genes. For example, several types of disrupted tRNA genes have been reported in the Archaea and primitive Eukarya. These include multiple-intron-containing tRNA genes, split tRNA genes, and permuted tRNA genes (Fujishima and Kanai, 2014; Soma, 2014). Because these tRNAs are encoded as precursor forms (pre-tRNAs) in the genome, they must be processed to yield mature functional tRNAs. Studies of tRNA introns and their processing enzymes suggest that rather complex pathways are required to generate mature tRNAs (Yoshihisa, 2014). Metazoan mitochondrial tRNA is another example of a unique tRNA, lacking either one or two arms of the typical tRNA cloverleaf structure (Watanabe et al., 2014), and transfer messenger RNA (tmRNA) is involved in trans-translation, the major ribosome rescue system in bacterial cells (Himeno et al., 2014). Most of these tRNA genes and a huge number of tRNA sequences from metagenomic analyses are registered on the tRNA gene databases (Abe et al., 2014). The universal 3′-terminal CCA sequence of the tRNAs, which is required for amino acid attachment to the molecule, is synthesized by tRNA nucleotidyltransferase or the “CCA-adding enzyme.” The molecular mechanism of the template-independent RNA polymerization catalyzed by the CCA-adding enzyme is discussed, based on its structural features (Tomita and Yamashita, 2014). It is well-known that tRNAs contain many types of base modifications. Recent progress in our understanding of two major modifications in tRNAs, methylated nucleosides (Hori, 2014) and thionucleosides (Shigi, 2014), is reviewed and summarized. As well as the canonical role of tRNA during protein biosynthesis, recent studies have shown that tRNA performs additional functions in regulating biochemical processes (Raina and Ibba, 2014). For example, aminoacyl-tRNA is involved in cell wall formation, protein labeling for degradation, and antibiotic biosynthesis. Moreover, tRNA cleavage is a conserved part of the responses of eukaryotic cells to various stresses. Age-associated and tissue-specific tRNA fragmentation have also been observed and several studies have suggested that some of these tRNA fragments are involved in the cellular RNA interference (RNAi) system. Pathological mutations in tRNA genes and tRNA-related enzymes have been linked to human diseases (Abbott et al., 2014). Mutations in the mitochondrial tRNA genes, in particular, are responsible for many diseases, and aminoacyl-tRNA synthetase mutations are associated with neurological diseases. Finally, the evolution of the tRNA molecule is discussed based on the analysis of the tRNA structure (Caetano-Anolles and Sun, 2014) and ancestral ribozymes (Fujishima and Kanai, 2014). Please enjoy reading all these articles, which will open the door to a new tRNA world!

Conformational change in transfer RNA is an early indicator of acute cellular damage.

Tissue damage by oxidative stress is a key pathogenic mechanism in various diseases, including AKI and CKD. Thus, early detection of oxidative tissue damage is important. Using a tRNA-specific modified nucleoside 1-methyladenosine (m1A) antibody, we show that oxidative stress induces a direct conformational change in tRNA structure that promotes subsequent tRNA fragmentation and occurs much earlier than DNA damage. In various models of tissue damage (ischemic reperfusion, toxic injury, and irradiation), the levels of circulating tRNA derivatives increased rapidly. In humans, the levels of circulating tRNA derivatives also increased under conditions of acute renal ischemia, even before levels of other known tissue damage markers increased. Notably, the level of circulating free m1A correlated with mortality in the general population (n=1033) over a mean follow-up of 6.7 years. Compared with healthy controls, patients with CKD had higher levels of circulating free m1A, which were reduced by treatment with pitavastatin (2 mg/d; n=29). Therefore, tRNA damage reflects early oxidative stress damage, and detection of tRNA damage may be a useful tool for identifying organ damage and forming a clinical prognosis.

The RNA Methyltransferase Dnmt2 Is Required for Efficient Dicer-2-Dependent siRNA Pathway Activity in Drosophila

Transfer RNA (tRNA) fragmentation in response to stress conditions has been described in many organisms. tRNA fragments have been found in association with small interfering RNA (siRNA) components, but the biological role of these interactions remains unclear. We report here that the tRNA methyltransferase Dnmt2 is essential for efficient Dicer-2 (Dcr-2) function in Drosophila. Using small RNA (sRNA) sequencing, we confirmed that Dnmt2 limits the extent of tRNA fragmentation during the heat-shock response. tRNAs as well as tRNA fragments serve as Dcr-2 substrates, and Dcr-2 degrades tRNA-derived sequences, especially under heat-shock conditions. tRNA-derived RNAs are able to inhibit Dcr-2 activity on long double-stranded RNAs (dsRNAs). Consequently, heat-shocked Dnmt2 mutant animals accumulate dsRNAs, produce fewer siRNAs, and show misregulation of siRNA pathway-dependent genes. These results reveal the impact of tRNA fragmentation on siRNA pathways and implicate tRNA modifications in the regulation of sRNA homeostasis during the heat-shock response.

TRNA modifications: Necessary for correct tRNA‐derived fragments during the recovery from stress?

Endonuclease-mediated tRNA fragmentation has been observed in many species suggesting functional importance for tRNA fragments. The size distribution of tRNA-derived fragments indicates the existence of mechanisms that protect tRNAs and their fragments from total degradation by exonucleases. Could post-transcriptional modifications be important for the controlled processing of tRNAs?

Mg 2+-katalysierte, spezifische spaltung von tRNA

Mg 2+- catalyzed specific splitting of tRNA In the presence of Mg 2+ ( Mg 2+ internucleotide phosphate ⩾ 0.5 ) tRNA Phe and tRNA Phe −Y are split under alkaline conditions almost specifically between the dihydrouridine residues at positions 16 and 17. tRNA fragments can be prepared by this reaction on a preparative scale. The fact that the chain scission occurs predominantly in the dihydrouridine region and only to a small extent in the also single-stranded anticodon region is explained by the differences in base stacking between the two regions. In the presence of low concentrations of Mg 2+ ( Mg 2+ P < 0.5 ) the fragmentation pattern of tRNA Phe is drastically altered. In 7 M urea only unspecific degradation is observed. Apparently, the specificity of the tRNA fragmentation depends on the actual tRNA conformation. A specific fragmentation in the presence of Mg 2+ is also observed in tRNA Ser.