Chromosomal evolution, environmental heterogeneity, and migration drive spatial patterns of species richness in Calochortus (Liliaceae).

We used nuclear genomic data and statistical models to evaluate the ecological and evolutionary processes shaping spatial variation in species richness in Calochortus (Liliaceae, 74 spp.). Calochortus occupies diverse habitats in the western United States and Mexico and has a center of diversity in the California Floristic Province, marked by multiple orogenies, winter rainfall, and highly divergent climates and substrates (including serpentine). We used sequences of 294 low-copy nuclear loci to produce a time-calibrated phylogeny, estimate historical biogeography, and test hypotheses regarding drivers of present-day spatial patterns in species number. Speciation and species coexistence require reproductive isolation and ecological divergence, so we examined the roles of chromosome number, environmental heterogeneity, and migration in shaping local species richness. Six major clades-inhabiting different geographic/climatic areas, and often marked by different base chromosome numbers (n = 6 to 10)-began diverging from each other ~10.3 Mya. As predicted, local species number increased significantly with local heterogeneity in chromosome number, elevation, soil characteristics, and serpentine presence. Species richness is greatest in the Transverse/Peninsular Ranges where clades with different chromosome numbers overlap, topographic complexity provides diverse conditions over short distances, and several physiographic provinces meet allowing immigration by several clades. Recently diverged sister-species pairs generally have peri-patric distributions, and maximum geographic overlap between species increases over the first million years since divergence, suggesting that chromosomal evolution, genetic divergence leading to gametic isolation or hybrid inviability/sterility, and/or ecological divergence over small spatial scales may permit species co-occurrence.

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.

psiTurk: An open-source framework for conducting replicable behavioral experiments online.

Online data collection has begun to revolutionize the behavioral sciences. However, conducting carefully controlled behavioral experiments online introduces a number of new of technical and scientific challenges. The project described in this paper, psiTurk, is an open-source platform which helps researchers develop experiment designs which can be conducted over the Internet. The tool primarily interfaces with Amazon's Mechanical Turk, a popular crowd-sourcing labor market. This paper describes the basic architecture of the system and introduces new users to the overall goals. psiTurk aims to reduce the technical hurdles for researchers developing online experiments while improving the transparency and collaborative nature of the behavioral sciences.

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.

Time to Radiation after Oncoplastic Reduction versus After Lumpectomy.

Prior studies contrasting oncoplastic reduction (OCR) to traditional lumpectomy have validated oncoplastic reduction surgery with similar survival and oncological outcomes. The purpose of this study was to evaluate if there was a significant difference in the time to initiation of radiation therapy after OCR in comparison with the standard breast-conserving therapy (lumpectomy). Methods: The patients included were from a database of breast cancer patients who all underwent postoperative adjuvant radiation after either OCR or lumpectomy at a single institution between 2003 and 2020. Patients who experienced delays in radiation for nonsurgical reasons were excluded. Comparisons were made between the groups in the time to radiation and complication rates. Results: A total of 487 patients underwent breast-conserving therapy, with 220 having undergone OCR and 267 lumpectomy patients. There was no significant difference in days to radiation between patient cohorts (60.5 OCR, 56.2 lumpectomy, P = 0.059). There was a significant difference in the number of complications between OCR and lumpectomy patients (20.4% OCR, 2.2% lumpectomy, P < 0.001). However, of patients who had complications, there was no significant difference in the number of days to radiation (74.3 OCR, 69.3 lumpectomy, P = 0.732). Conclusions: Compared with lumpectomy, OCR was not associated with an increased time to radiation but was associated with higher complications. Statistical analysis did not reveal surgical technique or complications to be independent, significant predictors of increased time to radiation. Surgeons should be aware that although complications may remain higher in OCR, this does not necessarily translate to delays in radiation.

Balancing read length and sequencing depth: Optimizing Nanopore long-read sequencing for monocots with an emphasis on the Liliales.

Premise: We present approaches used to generate long-read Nanopore sequencing reads for the Liliales and demonstrate how modifications to standard protocols directly impact read length and total output. The goal is to help those interested in generating long-read sequencing data determine which steps may be necessary for optimizing output and results. Methods: Four species of Calochortus (Liliaceae) were sequenced. Modifications made to sodium dodecyl sulfate (SDS) extractions and cleanup protocols included grinding with a mortar and pestle, using cut or wide-bore tips, chloroform cleaning, bead cleaning, eliminating short fragments, and using highly purified DNA. Results: Steps taken to maximize read length can decrease overall output. Notably, the number of pores in a flow cell is correlated with the overall output, yet we did not see an association between the pore number and the read length or the number of reads produced. Discussion: Many factors contribute to the overall success of a Nanopore sequencing run. We showed the direct impact that several modifications to the DNA extraction and cleaning steps have on the total sequencing output, read size, and number of reads generated. We show a tradeoff between read length and the number of reads and, to a lesser extent, the total sequencing output, all of which are important factors for successful de novo genome assembly.

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.