DYRK3 enables secretory trafficking by maintaining the liquid-like state of ER exit sites.

The dual-specificity kinase DYRK3 controls the formation and dissolution of multiple biomolecular condensates, regulating processes including stress recovery and mitotic progression. Here, we report that DYRK3 functionally interacts with proteins associated with endoplasmic reticulum (ER) exit sites (ERESs) and that inhibition of DYRK3 perturbs the organization of the ERES-Golgi interface and secretory trafficking. DYRK3-mediated regulation of ERES depends on the N-terminal intrinsically disordered region (IDR) of the peripheral membrane protein SEC16A, which co-phase separates with ERES components to form liquid-like condensates on the surface of the ER. By modulating the liquid-like properties of ERES, we show that their physical state is essential for functional cargo trafficking through the early secretory pathway. Our findings support a mechanism whereby phosphorylation by DYRK3 and its reversal by serine-threonine phosphatases regulate the material properties of ERES to create a favorable physicochemical environment for directional membrane traffic in eukaryotic cells.

Post-transcriptional regulation of antiviral gene expression by N6-methyladenosine.

Type I interferons (IFNs) induce hundreds of IFN-stimulated genes (ISGs) in response to viral infection. Induction of these ISGs must be regulated for an efficient and controlled antiviral response, but post-transcriptional controls of these genes have not been well defined. Here, we identify a role for the RNA base modification N6-methyladenosine (m6A) in the regulation of ISGs. Using ribosome profiling and quantitative mass spectrometry, coupled with m6A-immunoprecipitation and sequencing, we identify a subset of ISGs, including IFITM1, whose translation is enhanced by m6A and the m6A methyltransferase proteins METTL3 and METTL14. We further determine that the m6A reader YTHDF1 increases the expression of IFITM1 in an m6A-binding-dependent manner. Importantly, we find that the m6A methyltransferase complex promotes the antiviral activity of type I IFN. Thus, these studies identify m6A as having a role in post-transcriptional control of ISG translation during the type I IFN response for antiviral restriction.

Direct RNA sequencing reveals m6A modifications on adenovirus RNA are necessary for efficient splicing.

Adenovirus is a nuclear replicating DNA virus reliant on host RNA processing machinery. Processing and metabolism of cellular RNAs can be regulated by METTL3, which catalyzes the addition of N6-methyladenosine (m6A) to mRNAs. While m6A-modified adenoviral RNAs have been previously detected, the location and function of this mark within the infectious cycle is unknown. Since the complex adenovirus transcriptome includes overlapping spliced units that would impede accurate m6A mapping using short-read sequencing, here we profile m6A within the adenovirus transcriptome using a combination of meRIP-seq and direct RNA long-read sequencing to yield both nucleotide and transcript-resolved m6A detection. Although both early and late viral transcripts contain m6A, depletion of m6A writer METTL3 specifically impacts viral late transcripts by reducing their splicing efficiency. These data showcase a new technique for m6A discovery within individual transcripts at nucleotide resolution, and highlight the role of m6A in regulating splicing of a viral pathogen.

Limits in the detection of m6A changes using MeRIP/m6A-seq.

Many cellular mRNAs contain the modified base m6A, and recent studies have suggested that various stimuli can lead to changes in m6A. The most common method to map m6A and to predict changes in m6A between conditions is methylated RNA immunoprecipitation sequencing (MeRIP-seq), through which methylated regions are detected as peaks in transcript coverage from immunoprecipitated RNA relative to input RNA. Here, we generated replicate controls and reanalyzed published MeRIP-seq data to estimate reproducibility across experiments. We found that m6A peak overlap in mRNAs varies from ~30 to 60% between studies, even in the same cell type. We then assessed statistical methods to detect changes in m6A peaks as distinct from changes in gene expression. However, from these published data sets, we detected few changes under most conditions and were unable to detect consistent changes across studies of similar stimuli. Overall, our work identifies limits to MeRIP-seq reproducibility in the detection both of peaks and of peak changes and proposes improved approaches for analysis of peak changes.

Evolutionary dynamics of microRNA target sites across vertebrate evolution.

MicroRNAs (miRNAs) control the abundance of the majority of the vertebrate transcriptome. The recognition sequences, or target sites, for bilaterian miRNAs are found predominantly in the 3' untranslated regions (3'UTRs) of mRNAs, and are amongst the most highly conserved motifs within 3'UTRs. However, little is known regarding the evolutionary pressures that lead to loss and gain of such target sites. Here, we quantify the selective pressures that act upon miRNA target sites. Notably, selective pressure extends beyond deeply conserved binding sites to those that have undergone recent substitutions. Our approach reveals that even amongst ancient animal miRNAs, which exert the strongest selective pressures on 3'UTR sequences, there are striking differences in patterns of target site evolution between miRNAs. Considering only ancient animal miRNAs, we find three distinct miRNA groups, each exhibiting characteristic rates of target site gain and loss during mammalian evolution. The first group both loses and gains sites rarely. The second group shows selection only against site loss, with site gains occurring at a neutral rate, whereas the third loses and gains sites at neutral or above expected rates. Furthermore, mutations that alter the strength of existing target sites are disfavored. Applying our approach to individual transcripts reveals variation in the distribution of selective pressure across the transcriptome and between miRNAs, ranging from strong selection acting on a small subset of targets of some miRNAs, to weak selection on many targets for other miRNAs. miR-20 and miR-30, and many other miRNAs, exhibit broad, deeply conserved targeting, while several other comparably ancient miRNAs show a lack of selective constraint, and a small number, including mir-146, exhibit evidence of rapidly evolving target sites. Our approach adds valuable perspective on the evolution of miRNAs and their targets, and can also be applied to characterize other 3'UTR regulatory motifs.

Altered m6A Modification of Specific Cellular Transcripts Affects Flaviviridae Infection.

The RNA modification N6-methyladenosine (m6A) modulates mRNA fate and thus affects many biological processes. We analyzed m6A across the transcriptome following infection by dengue virus (DENV), Zika virus (ZIKV), West Nile virus (WNV), and hepatitis C virus (HCV). We found that infection by these viruses in the Flaviviridae family alters m6A modification of specific cellular transcripts, including RIOK3 and CIRBP. During viral infection, the addition of m6A to RIOK3 promotes its translation, while loss of m6A in CIRBP promotes alternative splicing. Importantly, viral activation of innate immune sensing or the endoplasmic reticulum (ER) stress response contributes to the changes in m6A in RIOK3 or CIRBP, respectively. Further, several transcripts with infection-altered m6A profiles, including RIOK3 and CIRBP, encode proteins that influence DENV, ZIKV, and HCV infection. Overall, this work reveals that cellular signaling pathways activated during viral infection lead to alterations in m6A modification of host mRNAs to regulate infection.

Correction to: Comprehensive benchmarking and ensemble approaches for metagenomic classifiers.

Following publication of the original article [1], the authors would like to highlight the following two corrections.

Single-molecule sequencing detection of N6-methyladenine in microbial reference materials.

The DNA base modification N6-methyladenine (m6A) is involved in many pathways related to the survival of bacteria and their interactions with hosts. Nanopore sequencing offers a new, portable method to detect base modifications. Here, we show that a neural network can improve m6A detection at trained sequence contexts compared to previously published methods using deviations between measured and expected current values as each adenine travels through a pore. The model, implemented as the mCaller software package, can be extended to detect known or confirm suspected methyltransferase target motifs based on predictions of methylation at untrained contexts. We use PacBio, Oxford Nanopore, methylated DNA immunoprecipitation sequencing (MeDIP-seq), and whole-genome bisulfite sequencing data to generate and orthogonally validate methylomes for eight microbial reference species. These well-characterized microbial references can serve as controls in the development and evaluation of future methods for the identification of base modifications from single-molecule sequencing data.

N6-methyladenosine modification of hepatitis B virus RNA differentially regulates the viral life cycle.

N6-methyladenosine (m6A) RNA methylation is the most abundant epitranscriptomic modification of eukaryotic messenger RNAs (mRNAs). Previous reports have found m6A on both cellular and viral transcripts and defined its role in regulating numerous biological processes, including viral infection. Here, we show that m6A and its associated machinery regulate the life cycle of hepatitis B virus (HBV). HBV is a DNA virus that completes its life cycle via an RNA intermediate, termed pregenomic RNA (pgRNA). Silencing of enzymes that catalyze the addition of m6A to RNA resulted in increased HBV protein expression, but overall reduced reverse transcription of the pgRNA. We mapped the m6A site in the HBV RNA and found that a conserved m6A consensus motif situated within the epsilon stem loop structure, is the site for m6A modification. The epsilon stem loop is located in the 3' terminus of all HBV mRNAs and at both the 5' and 3' termini of the pgRNA. Mutational analysis of the identified m6A site in the 5' epsilon stem loop of pgRNA revealed that m6A at this site is required for efficient reverse transcription of pgRNA, while m6A methylation of the 3' epsilon stem loop results in destabilization of all HBV transcripts, suggesting that m6A has dual regulatory function for HBV RNA. Overall, this study reveals molecular insights into how m6A regulates HBV gene expression and reverse transcription, leading to an increased level of understanding of the HBV life cycle.

Nanopore DNA Sequencing and Genome Assembly on the International Space Station.

We evaluated the performance of the MinION DNA sequencer in-flight on the International Space Station (ISS), and benchmarked its performance off-Earth against the MinION, Illumina MiSeq, and PacBio RS II sequencing platforms in terrestrial laboratories. Samples contained equimolar mixtures of genomic DNA from lambda bacteriophage, Escherichia coli (strain K12, MG1655) and Mus musculus (female BALB/c mouse). Nine sequencing runs were performed aboard the ISS over a 6-month period, yielding a total of 276,882 reads with no apparent decrease in performance over time. From sequence data collected aboard the ISS, we constructed directed assemblies of the ~4.6 Mb E. coli genome, ~48.5 kb lambda genome, and a representative M. musculus sequence (the ~16.3 kb mitochondrial genome), at 100%, 100%, and 96.7% consensus pairwise identity, respectively; de novo assembly of the E. coli genome from raw reads yielded a single contig comprising 99.9% of the genome at 98.6% consensus pairwise identity. Simulated real-time analyses of in-flight sequence data using an automated bioinformatic pipeline and laptop-based genomic assembly demonstrated the feasibility of sequencing analysis and microbial identification aboard the ISS. These findings illustrate the potential for sequencing applications including disease diagnosis, environmental monitoring, and elucidating the molecular basis for how organisms respond to spaceflight.

Comprehensive benchmarking and ensemble approaches for metagenomic classifiers.

BACKGROUND: One of the main challenges in metagenomics is the identification of microorganisms in clinical and environmental samples. While an extensive and heterogeneous set of computational tools is available to classify microorganisms using whole-genome shotgun sequencing data, comprehensive comparisons of these methods are limited. RESULTS: In this study, we use the largest-to-date set of laboratory-generated and simulated controls across 846 species to evaluate the performance of 11 metagenomic classifiers. Tools were characterized on the basis of their ability to identify taxa at the genus, species, and strain levels, quantify relative abundances of taxa, and classify individual reads to the species level. Strikingly, the number of species identified by the 11 tools can differ by over three orders of magnitude on the same datasets. Various strategies can ameliorate taxonomic misclassification, including abundance filtering, ensemble approaches, and tool intersection. Nevertheless, these strategies were often insufficient to completely eliminate false positives from environmental samples, which are especially important where they concern medically relevant species. Overall, pairing tools with different classification strategies (k-mer, alignment, marker) can combine their respective advantages. CONCLUSIONS: This study provides positive and negative controls, titrated standards, and a guide for selecting tools for metagenomic analyses by comparing ranges of precision, accuracy, and recall. We show that proper experimental design and analysis parameters can reduce false positives, provide greater resolution of species in complex metagenomic samples, and improve the interpretation of results.

N6-Methyladenosine in Flaviviridae Viral RNA Genomes Regulates Infection.

The RNA modification N6-methyladenosine (m6A) post-transcriptionally regulates RNA function. The cellular machinery that controls m6A includes methyltransferases and demethylases that add or remove this modification, as well as m6A-binding YTHDF proteins that promote the translation or degradation of m6A-modified mRNA. We demonstrate that m6A modulates infection by hepatitis C virus (HCV). Depletion of m6A methyltransferases or an m6A demethylase, respectively, increases or decreases infectious HCV particle production. During HCV infection, YTHDF proteins relocalize to lipid droplets, sites of viral assembly, and their depletion increases infectious viral particles. We further mapped m6A sites across the HCV genome and determined that inactivating m6A in one viral genomic region increases viral titer without affecting RNA replication. Additional mapping of m6A on the RNA genomes of other Flaviviridae, including dengue, Zika, yellow fever, and West Nile virus, identifies conserved regions modified by m6A. Altogether, this work identifies m6A as a conserved regulatory mark across Flaviviridae genomes.

Clinical Genomics: Challenges and Opportunities.

Next-generation sequencing (NGS) approaches are highly applicable to clinical studies. We review recent advances in sequencing technologies, as well as their benefits and tradeoffs, to provide an overview of clinical genomics from study design to computational analysis. Sequencing technologies enable genomic, transcriptomic, and epigenomic evaluations. Studies that use a combination of whole genome, exome, mRNA, and bisulfite sequencing are now feasible due to decreasing sequencing costs. Single-molecule sequencing increases read length, with the MinIONTM nanopore sequencer, which offers a uniquely portable option at a lower cost. Many of the published comparisons we review here address the challenges associated with different sequencing methods. Overall, NGS techniques, coupled with continually improving analysis algorithms, are useful for clinical studies in many realms, including cancer, chronic illness, and neurobiology. We, and others in the field, anticipate the clinical use of NGS approaches will continue to grow, especially as we shift into an era of precision medicine.

Extensive sequencing of seven human genomes to characterize benchmark reference materials.

The Genome in a Bottle Consortium, hosted by the National Institute of Standards and Technology (NIST) is creating reference materials and data for human genome sequencing, as well as methods for genome comparison and benchmarking. Here, we describe a large, diverse set of sequencing data for seven human genomes; five are current or candidate NIST Reference Materials. The pilot genome, NA12878, has been released as NIST RM 8398. We also describe data from two Personal Genome Project trios, one of Ashkenazim Jewish ancestry and one of Chinese ancestry. The data come from 12 technologies: BioNano Genomics, Complete Genomics paired-end and LFR, Ion Proton exome, Oxford Nanopore, Pacific Biosciences, SOLiD, 10X Genomics GemCode WGS, and Illumina exome and WGS paired-end, mate-pair, and synthetic long reads. Cell lines, DNA, and data from these individuals are publicly available. Therefore, we expect these data to be useful for revealing novel information about the human genome and improving sequencing technologies, SNP, indel, and structural variant calling, and de novo assembly.

Biophysical constraints on lateral inhibition in the olfactory bulb.

The mitral cells (MCs) of the mammalian olfactory bulb (OB) constitute one of two populations of principal neurons (along with middle/deep tufted cells) that integrate afferent olfactory information with top-down inputs and intrinsic learning and deliver output to downstream olfactory areas. MC activity is regulated in part by inhibition from granule cells, which form reciprocal synapses with MCs along the extents of their lateral dendrites. However, with MC lateral dendrites reaching over 1.5 mm in length in rats, the roles of distal inhibitory synapses pose a quandary. Here, we systematically vary the properties of a MC model to assess the capacity of inhibitory synaptic inputs on lateral dendrites to influence afferent information flow through MCs. Simulations using passivized models with varying dendritic morphologies and synaptic properties demonstrated that, even with unrealistically favorable parameters, passive propagation fails to convey effective inhibitory signals to the soma from distal sources. Additional simulations using an active model exhibiting action potentials, subthreshold oscillations, and a dendritic morphology closely matched to experimental values further confirmed that distal synaptic inputs along the lateral dendrite could not exert physiologically relevant effects on MC spike timing at the soma. Larger synaptic conductances representative of multiple simultaneous inputs were not sufficient to compensate for the decline in signal with distance. Reciprocal synapses on distal MC lateral dendrites may instead serve to maintain a common fast oscillatory clock across the OB by delaying spike propagation within the lateral dendrites themselves.

Nanopore sequencing in microgravity.

Rapid DNA sequencing and analysis has been a long-sought goal in remote research and point-of-care medicine. In microgravity, DNA sequencing can facilitate novel astrobiological research and close monitoring of crew health, but spaceflight places stringent restrictions on the mass and volume of instruments, crew operation time, and instrument functionality. The recent emergence of portable, nanopore-based tools with streamlined sample preparation protocols finally enables DNA sequencing on missions in microgravity. As a first step toward sequencing in space and aboard the International Space Station (ISS), we tested the Oxford Nanopore Technologies MinION during a parabolic flight to understand the effects of variable gravity on the instrument and data. In a successful proof-of-principle experiment, we found that the instrument generated DNA reads over the course of the flight, including the first ever sequenced in microgravity, and additional reads measured after the flight concluded its parabolas. Here we detail modifications to the sample-loading procedures to facilitate nanopore sequencing aboard the ISS and in other microgravity environments. We also evaluate existing analysis methods and outline two new approaches, the first based on a wave-fingerprint method and the second on entropy signal mapping. Computationally light analysis methods offer the potential for in situ species identification, but are limited by the error profiles (stays, skips, and mismatches) of older nanopore data. Higher accuracies attainable with modified sample processing methods and the latest version of flow cells will further enable the use of nanopore sequencers for diagnostics and research in space.