Mistranslating the genetic code with leucine in yeast and mammalian cells.

Translation fidelity relies on accurate aminoacylation of transfer RNAs (tRNAs) by aminoacyl-tRNA synthetases (AARSs). AARSs specific for alanine (Ala), leucine (Leu), serine, and pyrrolysine do not recognize the anticodon bases. Single nucleotide anticodon variants in their cognate tRNAs can lead to mistranslation. Human genomes include both rare and more common mistranslating tRNA variants. We investigated three rare human tRNALeu variants that mis-incorporate Leu at phenylalanine or tryptophan codons. Expression of each tRNALeu anticodon variant in neuroblastoma cells caused defects in fluorescent protein production without significantly increased cytotoxicity under normal conditions or in the context of proteasome inhibition. Using tRNA sequencing and mass spectrometry we confirmed that each tRNALeu variant was expressed and generated mistranslation with Leu. To probe the flexibility of the entire genetic code towards Leu mis-incorporation, we created 64 yeast strains to express all possible tRNALeu anticodon variants in a doxycycline-inducible system. While some variants showed mild or no growth defects, many anticodon variants, enriched with G/C at positions 35 and 36, including those replacing Leu for proline, arginine, alanine, or glycine, caused dramatic reductions in growth. Differential phenotypic defects were observed for tRNALeu mutants with synonymous anticodons and for different tRNALeu isoacceptors with the same anticodon. A comparison to tRNAAla anticodon variants demonstrates that Ala mis-incorporation is more tolerable than Leu at nearly every codon. The data show that the nature of the amino acid substitution, the tRNA gene, and the anticodon are each important factors that influence the ability of cells to tolerate mistranslating tRNAs.

Eukaryotic tRNA sequences present conserved and amino acid-specific structural signatures.

Metazoan organisms have many tRNA genes responsible for decoding amino acids. The set of all tRNA genes can be grouped in sets of common amino acids and isoacceptor tRNAs that are aminoacylated by corresponding aminoacyl-tRNA synthetases. Analysis of tRNA alignments shows that, despite the high number of tRNA genes, specific tRNA sequence motifs are highly conserved across multicellular eukaryotes. The conservation often extends throughout the isoacceptors and isodecoders with, in some cases, two sets of conserved isodecoders. This study is focused on non-Watson-Crick base pairs in the helical stems, especially GoU pairs. Each of the four helical stems may contain one or more conserved GoU pairs. Some are amino acid specific and could represent identity elements for the cognate aminoacyl tRNA synthetases. Other GoU pairs are found in more than a single amino acid and could be critical for native folding of the tRNAs. Interestingly, some GoU pairs are anticodon-specific, and others are found in phylogenetically-specific clades. Although the distribution of conservation likely reflects a balance between accommodating isotype-specific functions as well as those shared by all tRNAs essential for ribosomal translation, such conservations may indicate the existence of specialized tRNAs for specific translation targets, cellular conditions, or alternative functions.

tRNAscan-SE 2.0: improved detection and functional classification of transfer RNA genes.

tRNAscan-SE has been widely used for transfer RNA (tRNA) gene prediction for over twenty years, developed just as the first genomes were decoded. With the massive increase in quantity and phylogenetic diversity of genomes, the accurate detection and functional prediction of tRNAs has become more challenging. Utilizing a vastly larger training set, we created nearly one hundred specialized isotype- and clade-specific models, greatly improving tRNAscan-SE's ability to identify and classify both typical and atypical tRNAs. We employ a new comparative multi-model strategy where predicted tRNAs are scored against a full set of isotype-specific covariance models, allowing functional prediction based on both the anticodon and the highest-scoring isotype model. Comparative model scoring has also enhanced the program's ability to detect tRNA-derived SINEs and other likely pseudogenes. For the first time, tRNAscan-SE also includes fast and highly accurate detection of mitochondrial tRNAs using newly developed models. Overall, tRNA detection sensitivity and specificity is improved for all isotypes, particularly those utilizing specialized models for selenocysteine and the three subtypes of tRNA genes encoding a CAU anticodon. These enhancements will provide researchers with more accurate and detailed tRNA annotation for a wider variety of tRNAs, and may direct attention to tRNAs with novel traits.

tRNAviz: explore and visualize tRNA sequence features.

Transfer RNAs (tRNAs) are ubiquitous across the tree of life. Although tRNA structure is highly conserved, there is still significant variation in sequence features between clades, isotypes and even isodecoders. This variation not only impacts translation, but as shown by a variety of recent studies, nontranslation-associated functions are also sensitive to small changes in tRNA sequence. Despite the rapidly growing number of sequenced genomes, there is a lack of tools for both small- and large-scale comparative genomics analysis of tRNA sequence features. Here, we have integrated over 150 000 tRNAs spanning all domains of life into tRNAviz, a web application for exploring and visualizing tRNA sequence features. tRNAviz implements a framework for determining consensus sequence features and can generate sequence feature distributions by isotypes, clades and anticodons, among other tRNA properties such as score. All visualizations are interactive and exportable. The web server is publicly available at http://trna.ucsc.edu/tRNAviz/.

RNAcentral: a comprehensive database of non-coding RNA sequences.

RNAcentral is a database of non-coding RNA (ncRNA) sequences that aggregates data from specialised ncRNA resources and provides a single entry point for accessing ncRNA sequences of all ncRNA types from all organisms. Since its launch in 2014, RNAcentral has integrated twelve new resources, taking the total number of collaborating database to 22, and began importing new types of data, such as modified nucleotides from MODOMICS and PDB. We created new species-specific identifiers that refer to unique RNA sequences within a context of single species. The website has been subject to continuous improvements focusing on text and sequence similarity searches as well as genome browsing functionality. All RNAcentral data is provided for free and is available for browsing, bulk downloads, and programmatic access at http://rnacentral.org/.

tRNAscan-SE On-line: integrating search and context for analysis of transfer RNA genes.

High-throughput genome sequencing continues to grow the need for rapid, accurate genome annotation and tRNA genes constitute the largest family of essential, ever-present non-coding RNA genes. Newly developed tRNAscan-SE 2.0 has advanced the state-of-the-art methodology in tRNA gene detection and functional prediction, captured by rich new content of the companion Genomic tRNA Database. Previously, web-server tRNA detection was isolated from knowledge of existing tRNAs and their annotation. In this update of the tRNAscan-SE On-line resource, we tie together improvements in tRNA classification with greatly enhanced biological context via dynamically generated links between web server search results, the most relevant genes in the GtRNAdb and interactive, rich genome context provided by UCSC genome browsers. The tRNAscan-SE On-line web server can be accessed at http://trna.ucsc.edu/tRNAscan-SE/.

GtRNAdb 2.0: an expanded database of transfer RNA genes identified in complete and draft genomes.

Transfer RNAs represent the largest, most ubiquitous class of non-protein coding RNA genes found in all living organisms. The tRNAscan-SE search tool has become the de facto standard for annotating tRNA genes in genomes, and the Genomic tRNA Database (GtRNAdb) was created as a portal for interactive exploration of these gene predictions. Since its published description in 2009, the GtRNAdb has steadily grown in content, and remains the most commonly cited web-based source of tRNA gene information. In this update, we describe not only a major increase in the number of tRNA predictions (>367000) and genomes analyzed (>4370), but more importantly, the integration of new analytic and functional data to improve the quality and biological context of tRNA gene predictions. New information drawn from other sources includes tRNA modification data, epigenetic data, single nucleotide polymorphisms, gene expression and evolutionary conservation. A richer set of analytic data is also presented, including better tRNA functional prediction, non-canonical features, predicted structural impacts from sequence variants and minimum free energy structural predictions. Views of tRNA genes in genomic context are provided via direct links to the UCSC genome browsers. The database can be searched by sequence or gene features, and is available at http://gtrnadb.ucsc.edu/.

RNAcentral: an international database of ncRNA sequences.

The field of non-coding RNA biology has been hampered by the lack of availability of a comprehensive, up-to-date collection of accessioned RNA sequences. Here we present the first release of RNAcentral, a database that collates and integrates information from an international consortium of established RNA sequence databases. The initial release contains over 8.1 million sequences, including representatives of all major functional classes. A web portal (http://rnacentral.org) provides free access to data, search functionality, cross-references, source code and an integrated genome browser for selected species.

IscR is essential for yersinia pseudotuberculosis type III secretion and virulence.

Type III secretion systems (T3SS) are essential for virulence in dozens of pathogens, but are not required for growth outside the host. Therefore, the T3SS of many bacterial species are under tight regulatory control. To increase our understanding of the molecular mechanisms behind T3SS regulation, we performed a transposon screen to identify genes important for T3SS function in the food-borne pathogen Yersinia pseudotuberculosis. We identified two unique transposon insertions in YPTB2860, a gene that displays 79% identity with the E. coli iron-sulfur cluster regulator, IscR. A Y. pseudotuberculosis iscR in-frame deletion mutant (ΔiscR) was deficient in secretion of Ysc T3SS effector proteins and in targeting macrophages through the T3SS. To determine the mechanism behind IscR control of the Ysc T3SS, we carried out transcriptome and bioinformatic analysis to identify Y. pseudotuberculosis genes regulated by IscR. We discovered a putative IscR binding motif upstream of the Y. pseudotuberculosis yscW-lcrF operon. As LcrF controls transcription of a number of critical T3SS genes in Yersinia, we hypothesized that Yersinia IscR may control the Ysc T3SS through LcrF. Indeed, purified IscR bound to the identified yscW-lcrF promoter motif and mRNA levels of lcrF and 24 other T3SS genes were reduced in Y. pseudotuberculosis in the absence of IscR. Importantly, mice orally infected with the Y. pseudotuberculosis ΔiscR mutant displayed decreased bacterial burden in Peyer's patches, mesenteric lymph nodes, spleens, and livers, indicating an essential role for IscR in Y. pseudotuberculosis virulence. This study presents the first characterization of Yersinia IscR and provides evidence that IscR is critical for virulence and type III secretion through direct regulation of the T3SS master regulator, LcrF.

The UCSC Archaeal Genome Browser: 2012 update.

The UCSC Archaeal Genome Browser (http://archaea.ucsc.edu) offers a graphical web-based resource for exploration and discovery within archaeal and other selected microbial genomes. By bringing together existing gene annotations, gene expression data, multiple-genome alignments, pre-computed sequence comparisons and other specialized analysis tracks, the genome browser is a powerful aggregator of varied genomic information. The genome browser environment maintains the current look-and-feel of the vertebrate UCSC Genome Browser, but also integrates archaeal and bacterial-specific tracks with a few graphic display enhancements. The browser currently contains 115 archaeal genomes, plus 31 genomes of viruses known to infect archaea. Some of the recently developed or enhanced tracks visualize data from published high-throughput RNA-sequencing studies, the NCBI Conserved Domain Database, sequences from pre-genome sequencing studies, predicted gene boundaries from three different protein gene prediction algorithms, tRNAscan-SE gene predictions with RNA secondary structures and CRISPR locus predictions. We have also developed a companion resource, the Archaeal COG Browser, to provide better search and display of arCOG gene function classifications, including their phylogenetic distribution among available archaeal genomes.

Reclassification of Thermoproteus neutrophilus Stetter and Zillig 1989 as Pyrobaculum neutrophilum comb. nov. based on phylogenetic analysis.

The hyperthermophilic crenarchaeon Thermoproteus neutrophilus V24Sta(T) was originally classified before sequence-based phylogenetic analysis became standard for bacterial taxonomy. Subsequent phylogenetic analyses by various groups have shown that strain V24Sta(T) groups more closely with strains of the genus Pyrobaculum than with those in the genus Thermoproteus. Based on phylogenetic comparison of rRNA gene sequences and ribosomal proteins, we propose that strain V24Sta(T) be reclassified as Pyrobaculum neutrophilum comb. nov., with the type strain V24Sta(T) ( = DSM 2338(T) = JCM 9278(T)). An emended description of the genus Pyrobaculum is also presented.

Discovery of a minimal form of RNase P in Pyrobaculum.

RNase P RNA is an ancient, nearly universal feature of life. As part of the ribonucleoprotein RNase P complex, the RNA component catalyzes essential removal of 5' leaders in pre-tRNAs. In 2004, Li and Altman computationally identified the RNase P RNA gene in all but three sequenced microbes: Nanoarchaeum equitans, Pyrobaculum aerophilum, and Aquifex aeolicus (all hyperthermophiles) [Li Y, Altman S (2004) RNA 10:1533-1540]. A recent study concluded that N. equitans does not have or require RNase P activity because it lacks 5' tRNA leaders. The "missing" RNase P RNAs in the other two species is perplexing given evidence or predictions that tRNAs are trimmed in both, prompting speculation that they may have developed novel alternatives to 5' pre-tRNA processing. Using comparative genomics and improved computational methods, we have now identified a radically minimized form of the RNase P RNA in five Pyrobaculum species and the related crenarchaea Caldivirga maquilingensis and Vulcanisaeta distributa, all retaining a conventional catalytic domain, but lacking a recognizable specificity domain. We confirmed 5' tRNA processing activity by high-throughput RNA sequencing and in vitro biochemical assays. The Pyrobaculum and Caldivirga RNase P RNAs are the smallest naturally occurring form yet discovered to function as trans-acting precursor tRNA-processing ribozymes. Loss of the specificity domain in these RNAs suggests altered substrate specificity and could be a useful model for finding other potential roles of RNase P. This study illustrates an effective combination of next-generation RNA sequencing, computational genomics, and biochemistry to identify a divergent, formerly undetectable variant of an essential noncoding RNA gene.

Complete genome sequence of Desulfurococcus fermentans, a hyperthermophilic cellulolytic crenarchaeon isolated from a freshwater hot spring in Kamchatka, Russia.

Desulfurococcus fermentans is the first known cellulolytic archaeon. This hyperthermophilic and strictly anaerobic crenarchaeon produces hydrogen from fermentation of various carbohydrates and peptides without inhibition by accumulating hydrogen. The complete genome sequence reported here suggested that D. fermentans employs membrane-bound hydrogenases and novel glycohydrolases for hydrogen production from cellulose.

Modeling the Thermoproteaceae RNase P RNA.

The RNA component of the RNase P complex is found throughout most branches of the tree of life and is principally responsible for removing the 5' leader sequence from pre-tRNA transcripts during tRNA maturation. RNase P RNA has a number of universal core features, however variations in sequence and structure found in homologs across the tree of life require multiple Rfam covariance search models to detect accurately. We describe a new Rfam search model to enable efficient detection of the diminutive archaeal Type T RNase P RNAs, which are missed by existing Rfam models. Using the new model, we establish effective score detection thresholds, and detect four new RNase P RNA genes in recently completed genomes from the crenarchaeal family Thermoproteaceae.

Systematic identification of tRNA genes in Drosophila melanogaster.

Transfer RNAs (tRNAs) are ubiquitous adapter molecules that link specific codons in messenger RNA (mRNA) with their corresponding amino acids during protein synthesis. The tRNA genes of Drosophila have been investigated for over half a century but have lacked systematic identification and nomenclature. Here, we review and integrate data within FlyBase and the Genomic tRNA Database (GtRNAdb) to identify the full complement of tRNA genes in the D. melanogaster nuclear and mitochondrial genomes. We apply a logical and informative nomenclature to all tRNA genes, and provide an overview of their characteristics and genomic features.

Distinct Stress-Dependent Signatures of Cellular and Extracellular tRNA-Derived Small RNAs.

The cellular response to stress is an important determinant of disease pathogenesis. Uncovering the molecular fingerprints of distinct stress responses may identify novel biomarkers and key signaling pathways for different diseases. Emerging evidence shows that transfer RNA-derived small RNAs (tDRs) play pivotal roles in stress responses. However, RNA modifications present on tDRs are barriers to accurately quantifying tDRs using traditional small RNA sequencing. Here, AlkB-facilitated methylation sequencing is used to generate a comprehensive landscape of cellular and extracellular tDR abundances in various cell types during different stress responses. Extracellular tDRs are found to have distinct fragmentation signatures from intracellular tDRs and these tDR signatures are better indicators of different stress responses than miRNAs. These distinct extracellular tDR fragmentation patterns and signatures are also observed in plasma from patients on cardiopulmonary bypass. It is additionally demonstrated that angiogenin and RNASE1 are themselves regulated by stressors and contribute to the stress-modulated abundance of sub-populations of cellular and extracellular tDRs. Finally, a sub-population of extracellular tDRs is identified for which AGO2 appears to be required for their expression. Together, these findings provide a detailed profile of stress-responsive tDRs and provide insight about tDR biogenesis and stability in response to cellular stressors.

Functional curation of the Sulfolobus solfataricus P2 and S. acidocaldarius 98-3 complete genome sequences.

The thermoacidophiles Sulfolobus solfataricus P2 and S. acidocaldarius 98-3 are considered key model organisms representing a major phylum of the Crenarchaeota. Because maintaining current, accurate genome information is indispensable for modern biology, we have updated gene function annotation using the arCOGs database, plus other available functional, structural and phylogenetic information. The goal of this initiative is continuous improvement of genome annotation with the support of the Sulfolobus research community.

GtRNAdb: a database of transfer RNA genes detected in genomic sequence.

Transfer RNAs (tRNAs) represent the single largest, best-understood class of non-protein coding RNA genes found in all living organisms. By far, the major source of new tRNAs is computational identification of genes within newly sequenced genomes. To organize the rapidly growing collection and enable systematic analyses, we created the Genomic tRNA Database (GtRNAdb), currently including over 74,000 tRNA genes predicted from 740 species. The web resource provides overview statistics of tRNA genes within each analyzed genome, including information by isotype and genetic locus, easily downloadable primary sequences, graphical secondary structures and multiple sequence alignments. Direct links for each gene to UCSC eukaryotic and microbial genome browsers provide graphical display of tRNA genes in the context of all other local genetic information. The database can be searched by primary sequence similarity, tRNA characteristics or phylogenetic group. The database is publicly available at http://gtrnadb.ucsc.edu.