A small ribosome-associated ncRNA globally inhibits translation by restricting ribosome dynamics.

Ribosome-associated non-coding RNAs (rancRNAs) have been recognized as an emerging class of regulatory molecules capable of fine-tuning translation in all domains of life. RancRNAs are ideally suited for allowing a swift response to changing environments and are therefore considered pivotal during the first wave of stress adaptation. Previously, we identified an mRNA-derived 18 nucleotides long rancRNA (rancRNA_18) in Saccharomyces cerevisiae that rapidly downregulates protein synthesis during hyperosmotic stress. However, the molecular mechanism of action remained enigmatic. Here, we combine biochemical, genetic, transcriptome-wide and structural evidence, thus revealing rancRNA_18 as global translation inhibitor by targeting the E-site region of the large ribosomal subunit. Ribosomes carrying rancRNA_18 possess decreased affinity for A-site tRNA and impaired structural dynamics. Cumulatively, these discoveries reveal the mode of action of a rancRNA involved in modulating protein biosynthesis at a thus far unequalled precision.

The Bgee suite: integrated curated expression atlas and comparative transcriptomics in animals.

Bgee is a database to retrieve and compare gene expression patterns in multiple animal species, produced by integrating multiple data types (RNA-Seq, Affymetrix, in situ hybridization, and EST data). It is based exclusively on curated healthy wild-type expression data (e.g., no gene knock-out, no treatment, no disease), to provide a comparable reference of normal gene expression. Curation includes very large datasets such as GTEx (re-annotation of samples as 'healthy' or not) as well as many small ones. Data are integrated and made comparable between species thanks to consistent data annotation and processing, and to calls of presence/absence of expression, along with expression scores. As a result, Bgee is capable of detecting the conditions of expression of any single gene, accommodating any data type and species. Bgee provides several tools for analyses, allowing, e.g., automated comparisons of gene expression patterns within and between species, retrieval of the prefered conditions of expression of any gene, or enrichment analyses of conditions with expression of sets of genes. Bgee release 14.1 includes 29 animal species, and is available at https://bgee.org/ and through its Bioconductor R package BgeeDB.

Targeting CD70 with cusatuzumab eliminates acute myeloid leukemia stem cells in patients treated with hypomethylating agents.

Acute myeloid leukemia (AML) is driven by leukemia stem cells (LSCs) that resist conventional chemotherapy and are the major cause of relapse1,2. Hypomethylating agents (HMAs) are the standard of care in the treatment of older or unfit patients with AML, but responses are modest and not durable3-5. Here we demonstrate that LSCs upregulate the tumor necrosis factor family ligand CD70 in response to HMA treatment resulting in increased CD70/CD27 signaling. Blocking CD70/CD27 signaling and targeting CD70-expressing LSCs with cusatuzumab, a human αCD70 monoclonal antibody with enhanced antibody-dependent cellular cytotoxicity activity, eliminated LSCs in vitro and in xenotransplantation experiments. Based on these preclinical results, we performed a phase 1/2 trial in previously untreated older patients with AML with a single dose of cusatuzumab monotherapy followed by a combination therapy with the HMA azacitidine ( NCT03030612 ). We report results from the phase 1 dose escalation part of the clinical trial. Hematological responses in the 12 patients enrolled included 8 complete remission, 2 complete remission with incomplete blood count recovery and 2 partial remission with 4 patients achieving minimal residual disease negativity by flow cytometry at <10-3. Median time to response was 3.3 months. Median progression-free survival was not reached yet at the time of the data cutoff. No dose-limiting toxicities were reported and the maximum tolerated dose of cusatuzumab was not reached. Importantly, cusatuzumab treatment substantially reduced LSCs and triggered gene signatures related to myeloid differentiation and apoptosis.

rRNA expansion segment 27Lb modulates the factor recruitment capacity of the yeast ribosome and shapes the proteome.

Fine-tuned regulation of protein biosynthesis is crucial for cellular fitness and became even more vital when cellular and organismal complexity increased during the course of evolution. In order to cope with this augmented demand for translation control, eukaryal ribosomes have gained extensions both at the ribosomal protein and rRNA levels. Here we analyze the functional role of ES27L, an rRNA expansion segment in the large ribosomal subunit of Saccharomyces cerevisiae. Deletion of the b-arm of this expansion segment, called ES27Lb, did not hamper growth during optimal conditions, thus demonstrating that this 25S rRNA segment is not inherently crucial for ribosome functioning. However, reductive stress results in retarded growth and rendered unique protein sets prone to aggregation. Lack of ES27Lb negatively affects ribosome-association of known co-translational N-terminal processing enzymes which in turn contributes to the observed protein aggregation. Likely as a compensatory response to these challenges, the truncated ribosomes showed re-adjusted translation of specific sets of mRNAs and thus fine-tune the translatome in order to re-establish proteostasis. Our study gives comprehensive insight into how a highly conserved eukaryal rRNA expansion segment defines ribosomal integrity, co-translational protein maturation events and consequently cellular fitness.

Tn-sequencing of Mycoplasma hyopneumoniae and Mycoplasma hyorhinis mutant libraries reveals non-essential genes of porcine mycoplasmas differing in pathogenicity.

Mycoplasma hyopneumoniae and Mycoplasma hyorhinis are two phylogenetically related species colonizing the respiratory tract of pigs but differing in pathogenicity, the basis of which is not well resolved. We hypothesize that genes belonging to the species-specific portion of the genome and being non-essential during ideal laboratory growth conditions encode possible virulent determinants and are the driver of interspecies differences. To investigate this, transposon mutant libraries were generated for both species and a transposon sequencing (Tn-seq) method for mycoplasmas was established to identify non-essential genes. Tn-seq datasets combined with bidirectional Blastp analysis revealed that 101 out of a total 678 coding sequences (CDS) are species-specific and non-essential CDS of M. hyopneumoniae strain F7.2C, while 96 out of a total 751 CDS are species-specific and non-essential CDS in the M. hyorhinis strain JF5820. Among these species-specific and non-essential CDS were genes involved in metabolic pathways. In particular, the myo-inositol and the sialic acid pathways were found to be non-essential and therefore could be considered important to the specific pathogenicity of M. hyopneumoniae and M. hyorhinis, respectively. Such pathways could enable the use of an alternative energy source providing an advantage in their specific niche and might be interesting targets to knock out in order to generate attenuated live vaccines.

Unexpected diversity in eukaryotic transcription revealed by the retrotransposon hotspot family of Trypanosoma brucei.

The path from DNA to RNA to protein in eukaryotes is guided by a series of factors linking transcription, mRNA export and translation. Many of these are conserved from yeast to humans. Trypanosomatids, which diverged early in the eukaryotic lineage, exhibit unusual features such as polycistronic transcription and trans-splicing of all messenger RNAs. They possess basal transcription factors, but lack recognisable orthologues of many factors required for transcription elongation and mRNA export. We show that retrotransposon hotspot (RHS) proteins fulfil some of these functions and that their depletion globally impairs nascent RNA synthesis by RNA polymerase II. Three sub-families are part of a coordinated process in which RHS6 is most closely associated with chromatin, RHS4 is part of the Pol II complex and RHS2 connects transcription with the translation machinery. In summary, our results show that the components of eukaryotic transcription are far from being universal, and reveal unsuspected plasticity in the course of evolution.

Lack of evidence for selection favouring MHC haplotypes that combine high functional diversity.

High rates of gene duplication and the highest levels of functional allelic diversity in vertebrate genomes are the main hallmarks of the major histocompatibility complex (MHC), a multigene family with a primordial role in pathogen recognition. The usual tight linkage among MHC gene duplicates may provide an opportunity for the evolution of haplotypes that associate functionally divergent alleles and thus grant the transmission of optimal levels of diversity to coming generations. Even though such associations may be a crucial component of disease resistance, this hypothesis has been given little attention in wild populations. Here, we leveraged pedigree data from a barn owl (Tyto alba) population to characterize MHC haplotype structure across two MHC class I (MHC-I) and two MHC class IIB (MHC-IIB) duplicates, in order to test the hypothesis that haplotypes' genetic diversity is higher than expected from randomly associated alleles. After showing that MHC loci are tightly linked within classes, we found limited evidence for shifts towards MHC haplotypes combining high diversity. Neither amino acid nor functional within-haplotype diversity were significantly higher than in random sets of haplotypes, regardless of MHC class. Our results therefore provide no evidence for selection towards high-diversity MHC haplotypes in barn owls. Rather, high rates of concerted evolution may constrain the evolution of high-diversity haplotypes at MHC-I, while, in contrast, for MHC-IIB, fixed differences among loci may provide barn owls with already optimized functional diversity. This suggests that at the MHC-I and MHC-IIB respectively, different evolutionary dynamics may govern the evolution of within-haplotype diversity.

Sequencing and characterizing the genome of Estrella lausannensis as an undergraduate project: training students and biological insights.

With the widespread availability of high-throughput sequencing technologies, sequencing projects have become pervasive in the molecular life sciences. The huge bulk of data generated daily must be analyzed further by biologists with skills in bioinformatics and by "embedded bioinformaticians," i.e., bioinformaticians integrated in wet lab research groups. Thus, students interested in molecular life sciences must be trained in the main steps of genomics: sequencing, assembly, annotation and analysis. To reach that goal, a practical course has been set up for master students at the University of Lausanne: the "Sequence a genome" class. At the beginning of the academic year, a few bacterial species whose genome is unknown are provided to the students, who sequence and assemble the genome(s) and perform manual annotation. Here, we report the progress of the first class from September 2010 to June 2011 and the results obtained by seven master students who specifically assembled and annotated the genome of Estrella lausannensis, an obligate intracellular bacterium related to Chlamydia. The draft genome of Estrella is composed of 29 scaffolds encompassing 2,819,825 bp that encode for 2233 putative proteins. Estrella also possesses a 9136 bp plasmid that encodes for 14 genes, among which we found an integrase and a toxin/antitoxin module. Like all other members of the Chlamydiales order, Estrella possesses a highly conserved type III secretion system, considered as a key virulence factor. The annotation of the Estrella genome also allowed the characterization of the metabolic abilities of this strictly intracellular bacterium. Altogether, the students provided the scientific community with the Estrella genome sequence and a preliminary understanding of the biology of this recently-discovered bacterial genus, while learning to use cutting-edge technologies for sequencing and to perform bioinformatics analyses.

The aminoacyl-tRNA synthetases of Drosophila melanogaster.

Aminoacyl-tRNA synthetases (aaRSs) ligate amino acids to their cognate tRNAs, allowing them to decode the triplet code during translation. Through different mechanisms aaRSs also perform several non-canonical functions in transcription, translation, apoptosis, angiogenesis and inflammation. Drosophila has become a preferred system to model human diseases caused by mutations in aaRS genes, to dissect effects of reduced translation or non-canonical activities, and to study aminoacylation and translational fidelity. However, the lack of a systematic annotation of this gene family has hampered such studies. Here, we report the identification of the entire set of aaRS genes in the fly genome and we predict their roles based on experimental evidence and/or orthology. Further, we propose a new, systematic and logical nomenclature for aaRSs. We also review the research conducted on Drosophila aaRSs to date. Together, our work provides the foundation for further research in the fly aaRS field.

Comparative genome analysis of Pseudomonas knackmussii†B13, the first bacterium known to degrade chloroaromatic compounds.

Pseudomonas knackmussii B13 was the first strain to be isolated in 1974 that could degrade chlorinated aromatic hydrocarbons. This discovery was the prologue for subsequent characterization of numerous bacterial metabolic pathways, for genetic and biochemical studies, and which spurred ideas for pollutant bioremediation. In this study, we determined the complete genome sequence of B13 using next generation sequencing technologies and optical mapping. Genome annotation indicated that B13 has a variety of metabolic pathways for degrading monoaromatic hydrocarbons including chlorobenzoate, aminophenol, anthranilate and hydroxyquinol, but not polyaromatic compounds. Comparative genome analysis revealed that B13 is closest to Pseudomonas denitrificans and Pseudomonas aeruginosa. The B13 genome contains at least eight genomic islands [prophages and integrative conjugative elements (ICEs)], which were absent in closely related pseudomonads. We confirm that two ICEs are identical copies of the 103 kb self-transmissible element ICEclc that carries the genes for chlorocatechol metabolism. Comparison of ICEclc showed that it is composed of a variable and a 'core' region, which is very conserved among proteobacterial genomes, suggesting a widely distributed family of so far uncharacterized ICE. Resequencing of two spontaneous B13 mutants revealed a number of single nucleotide substitutions, as well as excision of a large 220 kb region and a prophage that drastically change the host metabolic capacity and survivability.

Selectome update: quality control and computational improvements to a database of positive selection.

Selectome (http://selectome.unil.ch/) is a database of positive selection, based on a branch-site likelihood test. This model estimates the number of nonsynonymous substitutions (dN) and synonymous substitutions (dS) to evaluate the variation in selective pressure (dN/dS ratio) over branches and over sites. Since the original release of Selectome, we have benchmarked and implemented a thorough quality control procedure on multiple sequence alignments, aiming to provide minimum false-positive results. We have also improved the computational efficiency of the branch-site test implementation, allowing larger data sets and more frequent updates. Release 6 of Selectome includes all gene trees from Ensembl for Primates and Glires, as well as a large set of vertebrate gene trees. A total of 6810 gene trees have some evidence of positive selection. Finally, the web interface has been improved to be more responsive and to facilitate searches and browsing.

The branch-site test of positive selection is surprisingly robust but lacks power under synonymous substitution saturation and variation in GC.

Positive selection is widely estimated from protein coding sequence alignments by the nonsynonymous-to-synonymous ratio ω. Increasingly elaborate codon models are used in a likelihood framework for this estimation. Although there is widespread concern about the robustness of the estimation of the ω ratio, more efforts are needed to estimate this robustness, especially in the context of complex models. Here, we focused on the branch-site codon model. We investigated its robustness on a large set of simulated data. First, we investigated the impact of sequence divergence. We found evidence of underestimation of the synonymous substitution rate for values as small as 0.5, with a slight increase in false positives for the branch-site test. When dS increases further, underestimation of dS is worse, but false positives decrease. Interestingly, the detection of true positives follows a similar distribution, with a maximum for intermediary values of dS. Thus, high dS is more of a concern for a loss of power (false negatives) than for false positives of the test. Second, we investigated the impact of GC content. We showed that there is no significant difference of false positives between high GC (up to ∼80%) and low GC (∼30%) genes. Moreover, neither shifts of GC content on a specific branch nor major shifts in GC along the gene sequence generate many false positives. Our results confirm that the branch-site is a very conservative test.

When orthologs diverge between human and mouse.

Despite the common assumption that orthologs usually share the same function, there have been various reports of divergence between orthologs, even among species as close as mammals. The comparison of mouse and human is of special interest, because mouse is often used as a model organism to understand human biology. We review the literature on evidence for divergence between human and mouse orthologous genes, and discuss it in the context of biomedical research.