High-throughput droplet-based analysis of influenza A virus genetic reassortment by single-virus RNA sequencing.

The segmented RNA genome of influenza A viruses (IAVs) enables viral evolution through genetic reassortment after multiple IAVs coinfect the same cell, leading to viruses harboring combinations of eight genomic segments from distinct parental viruses. Existing data indicate that reassortant genotypes are not equiprobable; however, the low throughput of available virology techniques does not allow quantitative analysis. Here, we have developed a high-throughput single-cell droplet microfluidic system allowing encapsulation of IAV-infected cells, each cell being infected by a single progeny virion resulting from a coinfection process. Customized barcoded primers for targeted viral RNA sequencing enabled the analysis of 18,422 viral genotypes resulting from coinfection with two circulating human H1N1pdm09 and H3N2 IAVs. Results were highly reproducible, confirmed that genetic reassortment is far from random, and allowed accurate quantification of reassortants including rare events. In total, 159 out of the 254 possible reassortant genotypes were observed but with widely varied prevalence (from 0.038 to 8.45%). In cells where eight segments were detected, all 112 possible pairwise combinations of segments were observed. The inclusion of data from single cells where less than eight segments were detected allowed analysis of pairwise cosegregation between segments with very high confidence. Direct coupling analysis accurately predicted the fraction of pairwise segments and full genotypes. Overall, our results indicate that a large proportion of reassortant genotypes can emerge upon coinfection and be detected over a wide range of frequencies, highlighting the power of our tool for systematic and exhaustive monitoring of the reassortment potential of IAVs.

p53/p21 pathway activation contributes to the ependymal fate decision downstream of GemC1.

Multiciliated ependymal cells and adult neural stem cells are components of the adult neurogenic niche, essential for brain homeostasis. These cells share a common glial cell lineage regulated by the Geminin family members Geminin and GemC1/Mcidas. Ependymal precursors require GemC1/Mcidas expression to massively amplify centrioles and become multiciliated cells. Here, we show that GemC1-dependent differentiation is initiated in actively cycling radial glial cells, in which a DNA damage response, including DNA replication-associated damage and dysfunctional telomeres, is induced, without affecting cell survival. Genotoxic stress is not sufficient by itself to induce ependymal cell differentiation, although the absence of p53 or p21 in progenitors hinders differentiation by maintaining cell division. Activation of the p53-p21 pathway downstream of GemC1 leads to cell-cycle slowdown/arrest, which permits timely onset of ependymal cell differentiation in progenitor cells.

m5C Methylation Guides Systemic Transport of Messenger RNA over Graft Junctions in Plants.

In plants, transcripts move to distant body parts to potentially act as systemic signals regulating development and growth. Thousands of messenger RNAs (mRNAs) are transported across graft junctions via the phloem to distinct plant parts. Little is known regarding features, structural motifs, and potential base modifications of transported transcripts and how these may affect their mobility. We identified Arabidopsis thaliana mRNAs harboring the modified base 5-methylcytosine (m5C) and found that these are significantly enriched in mRNAs previously described as mobile, moving over graft junctions to distinct plant parts. We confirm this finding with graft-mobile methylated mRNAs TRANSLATIONALLY CONTROLLED TUMOR PROTEIN 1 (TCTP1) and HEAT SHOCK COGNATE PROTEIN 70.1 (HSC70.1), whose mRNA transport is diminished in mutants deficient in m5C mRNA methylation. Together, our results point toward an essential role of cytosine methylation in systemic mRNA mobility in plants and that TCTP1 mRNA mobility is required for its signaling function.

ALFA: annotation landscape for aligned reads.

BACKGROUND: The last 10 years have seen the rise of countless functional genomics studies based on Next-Generation Sequencing (NGS). In the vast majority of cases, whatever the species, whatever the experiment, the two first steps of data analysis consist of a quality control of the raw reads followed by a mapping of those reads to a reference genome/transcriptome. Subsequent steps then depend on the type of study that is being made. While some tools have been proposed for investigating data quality after the mapping step, there is no commonly adopted framework that would be easy to use and broadly applicable to any NGS data type. RESULTS: We present ALFA, a simple but universal tool that can be used after the mapping step on any kind of NGS experiment data for any organism with available genomic annotations. In a single command line, ALFA can compute and display distribution of reads by categories (exon, intron, UTR, etc.) and biotypes (protein coding, miRNA, etc.) for a given aligned dataset with nucleotide precision. We present applications of ALFA to Ribo-Seq and RNA-Seq on Homo sapiens, CLIP-Seq on Mus musculus, RNA-Seq on Saccharomyces cerevisiae, Bisulfite sequencing on Arabidopsis thaliana and ChIP-Seq on Caenorhabditis elegans. CONCLUSIONS: We show that ALFA provides a powerful and broadly applicable approach for post mapping quality control and to produce a global overview using common or dedicated annotations. It is made available to the community as an easy to install command line tool and from the Galaxy Tool Shed.

RSAT 2018: regulatory sequence analysis tools 20th anniversary.

RSAT (Regulatory Sequence Analysis Tools) is a suite of modular tools for the detection and the analysis of cis-regulatory elements in genome sequences. Its main applications are (i) motif discovery, including from genome-wide datasets like ChIP-seq/ATAC-seq, (ii) motif scanning, (iii) motif analysis (quality assessment, comparisons and clustering), (iv) analysis of regulatory variations, (v) comparative genomics. Six public servers jointly support 10 000 genomes from all kingdoms. Six novel or refactored programs have been added since the 2015 NAR Web Software Issue, including updated programs to analyse regulatory variants (retrieve-variation-seq, variation-scan, convert-variations), along with tools to extract sequences from a list of coordinates (retrieve-seq-bed), to select motifs from motif collections (retrieve-matrix), and to extract orthologs based on Ensembl Compara (get-orthologs-compara). Three use cases illustrate the integration of new and refactored tools to the suite. This Anniversary update gives a 20-year perspective on the software suite. RSAT is well-documented and available through Web sites, SOAP/WSDL (Simple Object Access Protocol/Web Services Description Language) web services, virtual machines and stand-alone programs at http://www.rsat.eu/.

Discovery of Human-Similar Gene Fusions in Canine Cancers.

Canine cancers represent a tremendous natural resource due to their incidence and striking similarities to human cancers, sharing similar clinical and pathologic features as well as oncogenic events, including identical somatic mutations. Considering the importance of gene fusions as driver alterations, we explored their relevance in canine cancers. We focused on three distinct human-comparable canine cancers representing different tissues and embryonic origins. Through RNA-Seq, we discovered similar gene fusions as those found in their human counterparts: IGK-CCND3 in B-cell lymphoma, MPB-BRAF in glioma, and COL3A1-PDGFB in dermatofibrosarcoma protuberans-like. We showed not only similar partner genes but also identical breakpoints leading to oncogene overexpression. This study demonstrates similar gene fusion partners and mechanisms in human-dog corresponding tumors and allows for selection of targeted therapies in preclinical and clinical trials with pet dogs prior to human trials, within the framework of personalized medicine. Cancer Res; 77(21); 5721-7. ©2017 AACR.

Translation in astrocyte distal processes sets molecular heterogeneity at the gliovascular interface.

Astrocytes send out long processes that are terminated by endfeet at the vascular surface and regulate vascular functions as well as homeostasis at the vascular interface. To date, the astroglial mechanisms underlying these functions have been poorly addressed. Here we demonstrate that a subset of messenger RNAs is distributed in astrocyte endfeet. We identified, among this transcriptome, a pool of messenger RNAs bound to ribosomes, the endfeetome, that primarily encodes for secreted and membrane proteins. We detected nascent protein synthesis in astrocyte endfeet. Finally, we determined the presence of smooth and rough endoplasmic reticulum and the Golgi apparatus in astrocyte perivascular processes and endfeet, suggesting for local maturation of membrane and secreted proteins. These results demonstrate for the first time that protein synthesis occurs in astrocyte perivascular distal processes that may sustain their structural and functional polarization at the vascular interface.