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Mitochondria generate most cellular energy via oxidative phosphorylation. Twenty-two species of mitochondrial (mt-)tRNAs encoded in mtDNA translate essential subunits of the respiratory chain complexes. mt-tRNAs contain post-transcriptional modifications introduced by nuclear-encoded tRNA-modifying enzymes. They are required for deciphering genetic code accurately, as well as stabilizing tRNA. Loss of tRNA modifications frequently results in severe pathological consequences. Here, we perform a comprehensive analysis of post-transcriptional modifications of all human mt-tRNAs, including 14 previously-uncharacterized species. In total, we find 18 kinds of RNA modifications at 137 positions (8.7% in 1575 nucleobases) in 22 species of human mt-tRNAs. An up-to-date list of 34 genes responsible for mt-tRNA modifications are provided. We identify two genes required for queuosine (Q) formation in mt-tRNAs. Our results provide insight into the molecular mechanisms underlying the decoding system and could help to elucidate the molecular pathogenesis of human mitochondrial diseases caused by aberrant tRNA modifications. Mitochondrial tRNA modifications are important for tRNA stability and accurate decoding. By employing RNA mass spectrometry and deep sequencing, here the authors provide a comprehensive analysis of post-transcriptional modifications of 22 species of human mitochondrial tRNAs.

In plants, the vascular tissue contains the enucleated sieve tubes facilitating long-distance transport of nutrients, hormones, and proteins. In addition, several mRNAs and small interfering RNAs/microRNAs were shown to be delivered via sieve tubes whose content is embodied by the phloem sap (PS). A number of these phloem transcripts are transported from source to sink tissues and function at targeted tissues. To gain additional insights into phloem-delivered RNAs and their potential role in signaling, we isolated and characterized PS RNA molecules distinct from microRNAs/small interfering RNAs with a size ranging from 30 to 90 bases. We detected a high number of full-length and phloem-specific fragments of noncoding RNAs such as tRNAs, ribosomal RNAs, and spliceosomal RNAs in the PS of pumpkin (Cucurbita maxima). In vitro assays show that small quantities of PS RNA molecules efficiently inhibit translation in an unspecific manner. Proof of concept that PS-specific tRNA fragments may interfere with ribosomal activity was obtained with artificially produced tRNA fragments. The results are discussed in terms of a functional role for long distance delivered noncoding PS RNAs.

The discovery of small noncoding RNAs (sncRNAs) with regulatory functions is a recent breakthrough in biology. Among sncRNAs, microRNA (miRNA), derived from host or virus, has emerged as elements with high importance in control of viral replication and host responses. However, the expression pattern and functional aspects of other types of sncRNAs, following viral infection, are unexplored. In order to define expression patterns of sncRNAs, as well as to discover novel regulatory sncRNAs in response to viral infection, we applied deep sequencing to cells infected with human respiratory syncytial virus (RSV), the most common cause of bronchiolitis and pneumonia in babies. RSV infection leads to abundant production of transfer RNA (tRNA)-derived RNA Fragments (tRFs) that are ~30 nucleotides (nts) long and correspond to the 5′-half of mature tRNAs. At least one tRF, which is derived from tRNA-Glu-CTC, represses target mRNA in the cytoplasm and promotes RSV replication. This demonstrates that this tRF is not a random by-product of tRNA degradation but a functional molecule. The biogenesis of this tRF is also specific, as it is mediated by the endonuclease angiogenin (ANG), not by other nucleases. In summary, our study presents novel information on the induction of a functional tRF by viral infection.

Post-transcriptional modification of RNA is an important determinant of RNA quality control, translational efficiency, RNA-protein interactions and stress response. This is illustrated by the observation of toxicant-specific changes in the spectrum of tRNA modifications in a stress-response mechanism involving selective translation of codon-biased mRNA for crucial proteins. To facilitate systems-level studies of RNA modifications, we developed a liquid chromatography–mass spectrometry (LC-MS) technique for the quantitative analysis of modified ribonucleosides in tRNA. The protocol includes tRNA purification by HPLC, enzymatic hydrolysis, reversed-phase HPLC resolution of the ribonucleosides, and identification and quantification of individual ribonucleosides by LC-MS via dynamic multiple reaction monitoring (DMRM). In this approach, the relative proportions of modified ribonucleosides are quantified in several micrograms of tRNA in a 15-min LC-MS run. This protocol can be modified to analyze other types of RNA by modifying the steps for RNA purification as appropriate. By comparison, traditional methods for detecting modified ribonucleosides are labor- and time-intensive, they require larger RNA quantities, they are modification-specific or require radioactive labeling.

MicroRNAs (miRNAs) are approximately 22-nt small non-coding regulatory RNAs that have generally been considered to regulate gene expression at the post-transcriptional level in the cytoplasm. However, recent studies have reported that some miRNAs localize to and function in the nucleus. To determine the number of miRNAs localized to the nucleus, we systematically investigated the subcellular distribution of small RNAs (sRNAs) by independent deep sequencing sequenced of the nuclear and cytoplasmic pools of 18- to 30-nucleotide sRNAs from human cells. We identified 339 nuclear and 324 cytoplasmic known miRNAs, 300 of which overlap, suggesting that the majority of miRNAs are imported into the nucleus. With the exception of a few miRNAs evidently enriched in the nuclear pool, such as the mir-29b, the ratio of miRNA abundances in the nuclear fraction versus in the cytoplasmic fraction vary to some extent. Moreover, our results revealed that a large number of tRNA 3' trailers are exported from the nucleus and accumulate in the cytoplasm. These tRNA 3' trailers accumulate in a variety of cell types, implying that the biogenesis of tRNA 3' trailers is conserved and that they have a potential functional role in vertebrate cells. Our results provide the first comprehensive view of the subcellular distribution of diverse sRNAs and new insights into the roles of miRNAs and tRNA 3' trailers in the cell.

MS analysis of yeast transfer RNA precursors (pre-tRNAs) isolated at different stages of tRNA processing and maturation reveals the presence of 5′-terminal methylguanosine cap structures that protect pre-tRNA from 5′-exonucleolytic degradation. Efficient maturation of transfer RNAs (tRNAs) is required for rapid cell growth. However, the precise timing of tRNA processing in coordination with the order of tRNA modifications has not been thoroughly elucidated. To analyze the modification status of tRNA precursors (pre-tRNAs) during maturation, we isolated pre-tRNAs at various stages from Saccharomyces cerevisiae and subjected them to MS analysis. We detected methylated guanosine cap structures at the 5′ termini of pre-tRNAs bearing 5′ leader sequences. These capped pre-tRNAs accumulated substantially after inhibition of RNase P activity. Upon depletion of the capping enzyme Ceg1p, the steady state level of capped pre-tRNA was markedly reduced. In addition, a population of capped pre-tRNAs accumulated in strains in which 5′ exonucleases were inhibited, indicating that the 5′ cap structures protect pre-tRNAs from 5′-exonucleolytic degradation during maturation.

Bean nuclear genes for tRNA(Pro), tRNA(Thr) and tRNA(Leu) were isolated. Expression of the tRNA(Pro) genes was demonstrated in vivo and sequence analysis suggested amplification of the tRNA(Pro) gene copy number through duplication of a gene cluster at the same locus of the bean genome. The two tRNA(Thr) genes isolated were actively transcribed and their transcripts processed in a HeLa cell system. In vivo expression tests of these genes and aminoacylation assays of the corresponding in vitro transcripts showed the presence of identity determinants in the anticodon of plant tRNA(Thr). The tRNA(Leu) gene was not expressed due to deviation from the consensus in the internal B-box promoter. The same sequence deviation also prevented aminoacylation of the corresponding in vitro transcript. This tRNA(Leu) however exists in plants and is synthesized from another gene with a consensus B-box promoter. Plant mitochondria import from the cytosol a number of nucleus-encoded tRNAs, including tRNA(Leu) and tRNA(Thr). From the available sequence data, we could not identify any conserved structural motif characteristic for the nucleus-encoded tRNAs imported into plant mitochondria, either in the tRNAs, or in the gene flanking sequences. These results suggest that recognition of tRNAs for import is idiosyncratic and likely to depend on protein/RNA interactions that are specific to each tRNA or each isoacceptor group.

We developed a general method to detect cellular small molecule–RNA conjugates that does not rely on the reactivity of the small molecule. This technique revealed NAD-linked RNA in Escherichia coli and Streptomyces venezuelae . Subsequent characterization showed that NAD is a 5′ modification of RNA, cannot be installed in vitro through aberrant transcriptional initiation, is only found among smaller cellular RNAs and is present at a surprisingly high abundance of ∼3,000 copies per cell.

In this study, we first described the complete mitochondrial genome for the red crab ( Charybdis feriata ), elucidated its phylogenetic relationship among 20 species within Decapoda and estimated the population genetic diversity. The mitochondrial genome was 15,660 bp in size and encoded 13 protein-coding genes, 22 transfer RNA (tRNA) genes and two ribosomal RNA genes. The gene arrangement of the mitochondrial genome was the same as that of its sister species, C. japonica . Phylogenomic analysis suggested that genus Charybdis should be classified into subfamily Portuninae but not into subfamily Thalamitinae. Moreover, a total of 33 haplotypes of complete cytochrome c oxidase subunit I gene were defined in 70 individuals of C. feriata derived from three localities. Haplotype diversity and nucleotide diversity values among three localities indicated a high level of genetic diversity in C. feriata . AMOVA analysis suggested a low level of genetic differentiation among the three localities ( F ST  = 0.0023, P  > 0.05). Neutrality tests and mismatch analysis revealed that C. feriata might have undergone a population expansion event that possibly occurred in the last 61,498 to 43,814 years. This study should be helpful to better understand the evolutionary status and population genetic diversity of C. feriata and related species.