General amino acid control in fission yeast is regulated by a nonconserved transcription factor, with functions analogous to Gcn4/Atf4

Eukaryotes respond to amino acid starvation by enhancing the translation of mRNAs encoding b-ZIP family transcription factors (GCN4 in Saccharomyces cerevisiae and ATF4 in mammals), which launch transcriptional programs to counter this stress. This pathway involves phosphorylation of the eIF2 translation factor by Gcn2-protein kinases and is regulated by upstream ORFs (uORFs) in the GCN4/ATF4 5′ leaders. Here, we present evidence that the transcription factors that mediate this response are not evolutionarily conserved. Although cells of the fission yeast Schizosaccharomyces pombe respond transcriptionally to amino acid starvation, they lack clear Gcn4 and Atf4 orthologs. We used ribosome profiling to identify mediators of this response in S. pombe, looking for transcription factors that behave like GCN4. We discovered a transcription factor (Fil1) translationally induced by amino acid starvation in a 5′ leader and Gcn2-dependent manner. Like Gcn4, Fil1 is required for the transcriptional response to amino acid starvation, and Gcn4 and Fil1 regulate similar genes. Despite their similarities in regulation, function, and targets, Fil1 and Gcn4 belong to different transcription factor families (GATA and b-ZIP, respectively). Thus, the same functions are performed by nonorthologous proteins under similar regulation. These results highlight the plasticity of transcriptional networks, which maintain conserved principles with nonconserved regulators

The Scots at the Battle of Neville's Cross, 17 October 1346

This is an analysis of the loyalties and political rivalries of the army of David II of Scotland at the battle of Neville's Cross in 1346 where that king was apparently deserted by some of his subjects and led off to eleven years' captivity in England

Quantitative principles of cis-translational control by general mRNA sequence features in eukaryotes.

BackgroundGeneral translational cis-elements are present in the mRNAs of all genes and affect the recruitment, assembly, and progress of preinitiation complexes and the ribosome under many physiological states. These elements include mRNA folding, upstream open reading frames, specific nucleotides flanking the initiating AUG codon, protein coding sequence length, and codon usage. The quantitative contributions of these sequence features and how and why they coordinate to control translation rates are not well understood.ResultsHere, we show that these sequence features specify 42-81% of the variance in translation rates in Saccharomyces cerevisiae, Schizosaccharomyces pombe, Arabidopsis thaliana, Mus musculus, and Homo sapiens. We establish that control by RNA secondary structure is chiefly mediated by highly folded 25-60 nucleotide segments within mRNA 5' regions, that changes in tri-nucleotide frequencies between highly and poorly translated 5' regions are correlated between all species, and that control by distinct biochemical processes is extensively correlated as is regulation by a single process acting in different parts of the same mRNA.ConclusionsOur work shows that general features control a much larger fraction of the variance in translation rates than previously realized. We provide a more detailed and accurate understanding of the aspects of RNA structure that directs translation in diverse eukaryotes. In addition, we note that the strongly correlated regulation between and within cis-control features will cause more even densities of translational complexes along each mRNA and therefore more efficient use of the translation machinery by the cell

Large-scale profiling of noncoding RNA function in yeast

Noncoding RNAs (ncRNAs) are emerging as key regulators of cellular function. We have exploited the recently developed barcoded ncRNA gene deletion strain collections in the yeast Saccharomyces cerevisiae to investigate the numerous ncRNAs in yeast with no known function. The ncRNA deletion collection contains deletions of tRNAs, snoRNAs, snRNAs, stable unannotated transcripts (SUTs), cryptic unstable transcripts (CUTs) and other annotated ncRNAs encompassing 532 different individual ncRNA deletions. We have profiled the fitness of the diploid heterozygous ncRNA deletion strain collection in six conditions using batch and continuous liquid culture, as well as the haploid ncRNA deletion strain collections arrayed individually onto solid rich media. These analyses revealed many novel environmental-specific haplo-insufficient and haplo-proficient phenotypes providing key information on the importance of each specific ncRNA in every condition. Co-fitness analysis using fitness data from the heterozygous ncRNA deletion strain collection identified two ncRNA groups required for growth during heat stress and nutrient deprivation. The extensive fitness data for each ncRNA deletion strain has been compiled into an easy to navigate database called Yeast ncRNA Analysis (YNCA). By expanding the original ncRNA deletion strain collection we identified four novel essential ncRNAs; SUT527, SUT075, SUT367 and SUT259/691. We defined the effects of each new essential ncRNA on adjacent gene expression in the heterozygote background identifying both repression and induction of nearby genes. Additionally, we discovered a function for SUT527 in the expression, 3’ end formation and localization of SEC4, an essential protein coding mRNA. Finally, using plasmid complementation we rescued the SUT075 lethal phenotype revealing that this ncRNA acts in trans. Overall, our findings provide important new insights into the function of ncRNAs

Time-resolved RNA SHAPE chemistry: quantitative RNA structure analysis in one-second snapshots and at single-nucleotide resolution

RNA SHAPE chemistry exploits the discovery that conformationally dynamic nucleotides preferentially adopt conformations that facilitate reaction between the 2′-OH group and a hydroxyl-selective electrophile, such as benzoyl cyanide (BzCN), to form a 2′-O-adduct. BzCN is ideally suited for quantitative, time-resolved analysis of RNA folding and RNP assembly mechanisms because this reagent both reacts with flexible RNA nucleotides and also undergoes auto-inactivating hydrolysis with a half-life of 0.25 s at 37 °C. RNA folding is initiated by addition of Mg(2+) or protein, or other change in solution conditions, and nucleotide resolution structural images are obtained by adding aliquots of the evolving reaction to BzCN and then “waiting” for 1 sec. Sites of 2′-O-adduct formation are subsequently scored as stops to primer extension using reverse transcriptase. This time resolved SHAPE protocol makes it possible to obtain 1 sec snapshots in time-resolved kinetic studies for RNAs of arbitrary length and complexity in a straightforward and concise experiment

Impact of uORFs in mediating regulation of translation in stress conditions

Background: A large fraction of genes contains upstream ORFs (uORFs) in the 5' untranslated region (5'UTR). The translation of uORFs can inhibit the translation of the main coding sequence, for example by causing premature dissociation of the two ribosomal units or ribosome stalling. However, it is currently unknown if most uORFs are inhibitory or if this activity is restricted to specific cases. Here we interrogate ribosome profiling data from three different stress experiments in yeast to gain novel insights into this question. Results: By comparing ribosome occupancies in different conditions and experiments we obtain strong evidence that, in comparison to primary coding sequences (CDS), which undergo translational arrest during stress, the translation of uORFs is mostly unaffected by changes in the environment. As a result, the relative abundance of uORF-encoded peptides increases during stress. In general, the changes in the translational efficiency of regions containing uORFs do not seem to affect downstream translation. The exception are uORFs found in a subset of genes that are significantly up-regulated at the level of translation during stress; these uORFs tend to be translated at lower levels in stress conditions than in optimal growth conditions, facilitating the translation of the CDS during stress. We find new examples of uORF-mediated regulation of translation, including the Gcn4 functional homologue fil1 and ubi4 genes in S. pombe. Conclusion: We find evidence that the relative amount of uORF-encoded peptides increases during stress. The increased translation of uORFs is however uncoupled from the general CDS translational repression observed during stress. In a subset of genes that encode proteins that need to be rapidly synthesized upon stress uORFs act as translational switches.1. Ministerio de Ciencia e Innovación (MCI), Agencia Estatal de Investigación (AEI) grants BFU2015–65235-P and PGC2018–094091-B-I00, co-funded by Fondo Europeo de Desarrollo Regional (FEDER); 2. Agència de Gestió d’Ajuts Universitaris i de Recerca, Generalitat de Catalunya, grant 2017SGR01020; 3. Spanish National Bioinformatics Institute (INB), funded by ISCIII and FEDER, grant PT17/0009/0014 of the PE I + D + i 2013–2016

Selective 2′-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP) for direct, versatile and accurate RNA structure analysis

SHAPE chemistries exploit small electrophilic reagents that react with the 2′-hydroxyl group to interrogate RNA structure at single-nucleotide resolution. Mutational profiling (MaP) identifies modified residues based on the ability of reverse transcriptase to misread a SHAPE-modified nucleotide and then counting the resulting mutations by massively parallel sequencing. The SHAPE-MaP approach measures the structure of large and transcriptome-wide systems as accurately as for simple model RNAs. This protocol describes the experimental steps, implemented over three days, required to perform SHAPE probing and construct multiplexed SHAPE-MaP libraries suitable for deep sequencing. These steps include RNA folding and SHAPE structure probing, mutational profiling by reverse transcription, library construction, and sequencing. Automated processing of MaP sequencing data is accomplished using two software packages. ShapeMapper converts raw sequencing files into mutational profiles, creates SHAPE reactivity plots, and provides useful troubleshooting information, often within an hour. SuperFold uses these data to model RNA secondary structures, identify regions with well-defined structures, and visualize probable and alternative helices, often in under a day. We illustrate these algorithms with the E. coli thiamine pyrophosphate riboswitch, E. coli 16S rRNA, and HIV-1 genomic RNAs. SHAPE-MaP can be used to make nucleotide-resolution biophysical measurements of individual RNA motifs, rare components of complex RNA ensembles, and entire transcriptomes. The straightforward MaP strategy greatly expands the number, length, and complexity of analyzable RNA structures

Three-dimensional RNA structure refinement by hydroxyl radical probing

Molecular modeling guided by experimentally-derived structural information is an attractive approach for three-dimensional structure determination of complex RNAs that are not amenable to study by high-resolution methods. Hydroxyl radical probing (HRP), performed routinely in many laboratories, provides a measure of solvent accessibility at individual nucleotides. HRP measurements have, to date, only been used to evaluate RNA models qualitatively. Here, we report development of a quantitative structure refinement approach using HRP measurements to drive discrete molecular dynamics simulations for RNAs ranging in size from 80 to 230 nucleotides. HRP reactivities were first used to identify RNAs that form extensive helical packing interactions. For these RNAs, we achieved highly significant structure predictions, given inputs of RNA sequence and base pairing. This HRP-directed tertiary structure refinement approach generates robust structural hypotheses useful for guiding explorations of structure-function interrelationships in RNA