Characterising the loss-of-function impact of 5’ untranslated region variants in whole genome sequence data from 15,708 individuals, bioRxiv, 2019-02-08
AbstractUpstream open reading frames (uORFs) are important tissue-specific cis-regulators of protein translation. Although isolated case reports have shown that variants that create or disrupt uORFs can cause disease, genetic sequencing approaches typically focus on protein-coding regions and ignore these variants. Here, we describe a systematic genome-wide study of variants that create and disrupt human uORFs, and explore their role in human disease using 15,708 whole genome sequences collected by the Genome Aggregation Database (gnomAD) project. We show that 14,897 variants that create new start codons upstream of the canonical coding sequence (CDS), and 2,406 variants disrupting the stop site of existing uORFs, are under strong negative selection. Furthermore, variants creating uORFs that overlap the CDS show signals of selection equivalent to coding loss-of-function variants, and uORF-perturbing variants are under strong selection when arising upstream of known disease genes and genes intolerant to loss-of-function variants. Finally, we identify specific genes where perturbation of uORFs is likely to represent an important disease mechanism, and report a novel uORF frameshift variant upstream of NF2 in families with neurofibromatosis. Our results highlight uORF-perturbing variants as an important and under-recognised functional class that can contribute to penetrant human disease, and demonstrate the power of large-scale population sequencing data to study the deleteriousness of specific classes of non-coding variants.
biorxiv genomics 200-500-users 2019Performance of neural network basecalling tools for Oxford Nanopore sequencing, bioRxiv, 2019-02-08
AbstractBackgroundBasecalling, the computational process of translating raw electrical signal to nucleotide sequence, is of critical importance to the sequencing platforms produced by Oxford Nanopore Technologies (ONT). Here we examine the performance of different basecalling tools, looking at accuracy at the level of bases within individual reads and at majority-rules consensus basecalls in an assembly. We also investigate some additional aspects of basecalling training using a taxon-specific dataset, using a larger neural network model and improving consensus basecalls in an assembly by additional signal-level analysis with Nanopolish.ResultsTraining basecallers on taxon-specific data results in a significant boost in consensus accuracy, mostly due to the reduction of errors in methylation motifs. A larger neural network is able to improve both read and consensus accuracy, but at a cost to speed. Improving consensus sequences (‘polishing’) with Nanopolish somewhat negates the accuracy differences in basecallers, but prepolish accuracy does have an effect on post-polish accuracy.ConclusionsBasecalling accuracy has seen significant improvements over the last two years. The current version of ONT’s Guppy basecaller performs well overall, with good accuracy and fast performance. If higher accuracy is required, users should consider producing a custom model using a larger neural network andor training data from the same species.
biorxiv bioinformatics 100-200-users 2019Rotary substates of mitochondrial ATP synthase reveal the basis of flexible F1-Fo coupling, bioRxiv, 2019-02-07
F1Fo-ATP synthases play a central role in cellular metabolism, making the energy of the proton-motive force across a membrane available for a large number of energy-consuming processes. We determined the single-particle cryo-EM structure of active dimeric ATP synthase from mitochondria of Polytomella sp. at 2.7- 2.8 Å resolution. Separation of 13 well-defined rotary substates by 3D classification provides a detailed picture of the molecular motions that accompany c-ring rotation and result in ATP synthesis. Crucially, the F1 head rotates along with the central stalk and c-ring rotor for the first ~30° of each 120° primary rotary step. The joint movement facilitates flexible coupling of the stoichiometrically mismatched F1 and Fo subcomplexes. Flexibility is mediated primarily by the interdomain hinge of the conserved OSCP subunit, a well-established target of physiologically important inhibitors. Our maps provide atomic detail of the c-ringa-subunit interface in the membrane, where protonation and deprotonation of c-ring cGlu111 drives rotary catalysis. An essential histidine residue in the lumenal proton access channel binds a strong non-peptide density assigned to a metal ion that may facilitate c-ring protonation, as its coordination geometry changes with c-ring rotation. We resolve ordered water molecules in the proton access and release channels and at the gating aArg239 that is critical in all rotary ATPases. We identify the previously unknown ASA10 subunit and present complete de novo atomic models of subunits ASA1-10, which make up the two interlinked peripheral stalks that stabilize the Polytomella ATP synthase dimer.
biorxiv biochemistry 200-500-users 2019A neurodevelopmental origin of behavioral individuality, bioRxiv, 2019-02-06
The genome versus experience, or Nature versus Nurture, debate has dominated our understanding of individual behavioral variation. A third factor, namely variation in complex behavior potentially due to non-heritable developmental noise in brain development, has been largely ignored. Using the Drosophila vinegar fly we demonstrate a causal link between variation in brain wiring due to developmental noise, and behavioral individuality. A population of visual system neurons called DCNs shows non-heritable, inter-individual variation in rightleft wiring asymmetry, and control object orientation in freely walking flies. We show that DCN wiring asymmetry predicts individual object responses the greater the asymmetry, the better the individual orients. Silencing DCNs abolishes correlations between anatomy and behavior, while inducing visual asymmetry via monocular deprivation rescues object orientation in DCN-symmetric individuals.
biorxiv neuroscience 0-100-users 2019Auxin export from proximal fruits drives arrest in competent inflorescence meristems, bioRxiv, 2019-02-06
A well-defined set of regulatory pathways control entry into the reproductive phase in flowering plants. Conversely, little is known about the mechanisms that control the end of the reproductive phase (floral arrest), despite this being a critical process for optimising fruit and seed production. Complete fruit removal or lack of fertile fruit-set in male sterile mutants, for example male sterile1 (ms1), prevents timely floral arrest in the model plant Arabidopsis. These observations formed the basis for Hensel and colleagues model in which end-of-flowering was proposed to result from a cumulative fruitseed-derived signal that caused simultaneous global proliferative arrest (GPA) in all inflorescences. Recent studies have suggested that end-of-flowering involves gene expression changes at the floral meristem which are at least in part controlled by the FRUITFULL-APETELA2 pathway, however there is limited understanding of how this process is controlled and the communication needed at the whole plant level. Here, we provide new information providing a framework for the fruit-to-meristem (F-M) communication implied by the GPA model. We show that floral arrest in Arabidopsis is not global and does not occur synchronously between branches, but rather that the arrest of each inflorescence is a local process, driven by auxin export from fruit proximal to the inflorescence meristem (IM). Furthermore, we show that inflorescence meristems are only competent for floral arrest once they reach a certain developmental age. Understanding the regulation of floral arrest is of major importance for the future manipulation of flowering to extend and maximise crop yields.
biorxiv plant-biology 0-100-users 2019B cells engineered to express pathogen-specific antibodies using CRISPRCas9 protect against infection, bioRxiv, 2019-02-06
Effective vaccines inducing lifelong protection against many important infections such as respiratory syncytial virus (RSV), human immunodeficiency virus (HIV), influenza and Epstein-Barr virus (EBV) are not yet available despite decades of research. As an alternative to a protective vaccine we developed a genetic engineering strategy in which CRISPRCas9 was utilized to replace endogenously-encoded antibodies with antibodies protective against RSV, HIV, influenza or EBV in primary human or murine B cells. The engineered antibodies were expressed in up to 59% of primary B cells under the control of endogenous regulatory elements, which maintained normal antibody expression and secretion. Importantly, a single transfer of murine B cells engineered to express an antibody protective against RSV resulted in potent and durable protection against RSV infection in immunocompromised hosts. This approach offers the opportunity to achieve sterilizing immunity against pathogens for which traditional vaccination has failed to induce or maintain protective antibody responses.
biorxiv immunology 0-100-users 2019