Biology subjects

Moore, M. J.

Publications and source records attributed to Moore, M. J..

8 recordsLinked to original sources

bioRxiv2018

DEBrowser: Interactive Differential Expression Analysis and Visualization Tool for Count Data

BackgroundSequencing data has become a standard measure for studying diverse cellular activities. For example, gene expression is accurately measured by RNA sequencing (RNA-Seq) libraries, protein-DNA interactions are captured by chromatin immunoprecipitation sequencing (ChIP-Seq), protein-RNA interactions by crosslinking immunoprecipitation (CLIP-Seq) or RNA immunoprecipitation (RIP-Seq) sequencing, DNA accessibility by assay for transposase-accessible chromatin (ATAC-Seq), and DNase or MNase sequencing libraries. Analysis of these sequencing techniques involve library-specific approaches. However, in all cases, once the sequencing libraries are processed, the result is a count table specifying the estimated number of reads originating from a genomic locus. Differential analysis to determine which loci have different cellular activity under different conditions starts with the count table and iterates through a cycle of data assessment, preparation and analysis. Such iterative approach relies on multiple programs and is therefore a challenge for those without programming skills.\n\nResultsWe developed DEBrowser, as an R bioconductor project, to interactively visualize each step of the differential analysis of count data, without any requirement for programming expertise. The application presents a rich and interactive web based graphical user interface based on Rs shiny infrastructure. We use shinys reactive programming interface for a dynamic webpage that responds to user input and integrates its visualization widgets at each stage of the analysis. In this way, every step of the analysis can be displayed in one application that combines many approaches and multiple results. We show DEBrowsers capabilities by reproducing the analysis of two previously published data sets.\n\nConclusionsDEBrowser is a flexible, intuitive, web-based analysis platform that enables an iterative and interactive analysis of count data without any requirement of programming knowledge.

bioinformatics↗

bioRxiv2018

Evolution of Portulacineae marked by gene tree conflict and gene family expansion associated with adaptation to harsh environments

Several plant lineages have evolved adaptations that allow survival in extreme and harsh environments including many within the plant clade Portulacineae (Caryophyllales) such as the Cactaceae, Didiereaceae of Madagascar, and high altitude Montiaceae. Here, using newly generated transcriptomic data, we reconstructed the phylogeny of Portulacineae and examine potential correlates between molecular evolution within this clade and adaptation to harsh environments. Our phylogenetic results were largely congruent with previous analyses, but we identified several early diverging nodes characterized by extensive gene tree conflict. For particularly contentious nodes, we presented detailed information about the phylogenetic signal for alternative relationships. We also analyzed the frequency of gene duplications, confirmed previously identified whole genome duplications (WGD), and identified a previously unidentified WGD event within the Didiereaceae. We found that the WGD events were typically associated with shifts in climatic niche and did not find a direct association with WGDs and diversification rate shifts. Diversification shifts occurred within the Portulacaceae, Cactaceae, and Anacampserotaceae, and while these did not experience WGDs, the Cactaceae experienced extensive gene duplications. We examined gene family expansion and molecular evolutionary patterns with a focus on genes associated with environmental stress responses and found evidence for significant gene family expansion in genes with stress adaptation and clades found in extreme environments. These results provide important directions for further and deeper examination of the potential links between molecular evolutionary patterns and adaptation to harsh environments.

evolutionary biology↗

bioRxiv2018

Higher-Order Organization Principles of Pre-translational mRNPs

Compared to noncoding RNAs (ncRNAs) such as rRNAs and ribozymes, for which high resolution structures abound, little is known about the tertiary structures of mRNAs. In eukaryotic cells, newly made mRNAs are packaged with proteins in highly compacted mRNPs, but the manner of this mRNA compaction is unknown. Here we developed and implemented RIPPLiT (RNA ImmunoPrecipitation and Proximity Ligation in Tandem), a transcriptome-wide method for probing the 3D conformations of RNAs stably-associated with defined proteins, in this case exon junction complex (EJC) core factors. EJCs multimerize with other mRNP components to form megadalton sized complexes that protect large swaths of newly synthesized mRNAs from endonuclease digestion. Unlike ncRNAs, mRNAs behave more like flexible polymers without strong locus-specific interactions. Polymer analysis of proximity ligation data for hundreds of mRNA species demonstrates that pre-translational mammalian mRNPs fold as linear rod-like structures with no strong propensity for 5 and 3 end interaction.

molecular biology↗

bioRxiv2018

ZFP36 RNA-binding proteins restrain T-cell activation and anti-viral immunity

Dynamic post-transcriptional control of RNA expression by RNA-binding proteins (RBPs) is critical during immune response. ZFP36 RBPs are prominent inflammatory regulators linked to autoimmunity and cancer, but functions in adaptive immunity are less clear. We used HITS-CLIP to define ZFP36 targets in T-cells, which revealed unanticipated actions in regulating T-cell activation, proliferation, and effector functions. Transcriptome and ribosome profiling showed that ZFP36 represses mRNA target abundance and translation, notably through a novel class of AU-rich sites in coding sequence. Functional studies revealed that ZFP36 regulates early T-cell activation kinetics in a cell autonomous manner, by attenuating activation marker expression, limiting T-cell expansion, and promoting apoptosis. Strikingly, loss of ZFP36 in vivo accelerated T-cell responses to acute viral infection, and enhanced anti-viral immunity. These findings uncover a critical role for ZFP36 RBPs in restraining T-cell expansion and effector functions, and suggest ZFP36 inhibition as a novel strategy to enhance immune-based therapies.

molecular biology↗

bioRxiv2018

A master regulatory network restoring brain glutamate homeostasis is coordinately activated in stroke

Post-transcriptional regulation by miRNAs is essential for complex molecular responses to physiological insult and disease. Although many disease-associated miRNAs are known, their global targets and culminating network effects on pathophysiology remain poorly understood. We applied AGO CLIP to systematically elucidate altered miRNA-target interactions in brain following ischemia/reperfusion (I/R) injury. Among 1,190 identified, most prominent was the cumulative loss of target regulation by miR-29 family members. Integration of translational and time-course RNA profiles revealed a dynamic mode of miR-29 target de-regulation, led by acute translational activation and later increase in RNA levels, allowing rapid proteomic changes to take effect. These functional regulatory events rely on canonical and non-canonical miR-29 binding and engage glutamate reuptake signals to control local glutamate levels. These results uncover a miRNA target network that acts acutely to maintain brain homeostasis after ischemic stroke.

neuroscience↗

bioRxiv2017

Transcriptome-wide analysis of the functional intronome using spliceosome profiling

Full understanding of eukaryotic transcriptomes and how they respond to different conditions requires deep knowledge of all sites of intron excision. Although RNA-Seq provides much of this information, the low abundance of many spliced transcripts (often due to their rapid cytoplasmic decay) limits the ability of RNA-Seq alone to reveal the full repertoire of spliced species. Here we present \"spliceosome profiling\", a strategy based on deep sequencing of RNAs co-purifying with late stage spliceosomes. Spliceosome profiling allows for unambiguous mapping of intron ends to single nucleotide resolution and branchpoint identification at unprecedented depths. Our data reveal hundreds of new introns in S. pombe and numerous others that were previously misannotated. By providing a means to directly interrogate sites of spliceosome assembly and catalysis genome-wide, spliceosome profiling promises to transform our understanding of RNA processing in the nucleus much like ribosome profiling has transformed our understanding mRNA translation in the cytoplasm.

genomics↗

bioRxiv2017

Single-molecule FISH in Drosophila muscle reveals location dependent mRNA composition of megaRNPs

Single-molecule fluorescence in-situ hybridization (smFISH) provides direct access to the spatial relationship between nucleic acids and specific subcellular locations. The ability to precisely localize a messenger RNA can reveal key information about its regulation. Although smFISH is well established in cell culture or thin sections, methods for its accurate application to tissues are lacking. The utility of smFISH in thick tissue sections must overcome several challenges, including probe penetration of fixed tissue, accessibility of target mRNAs for probe hybridization, high fluorescent background, spherical aberration along the optical axis, and image segmentation of organelles. Here we describe how we overcame these obstacles to study mRNA localization in Drosophila larval muscle samples that approach 50 m thickness. We use sample-specific optimization of smFISH, particle identification based on maximum likelihood testing, and 3-dimensional multiple-organelle segmentation. The latter allows using independent thresholds for different regions of interest within an image stack. Our approach therefore facilitates accurate measurement of mRNA location in thick tissues.

cell biology↗

bioRxiv2017

Widespread paleopolyploidy, gene tree conflict, and recalcitrant relationships among the carnivorous Caryophyllales

O_LIThe carnivorous members of the large, hyperdiverse Caryophyllales (e.g. Venus flytrap, sundews and Nepenthes pitcher plants) represent perhaps the oldest and most diverse lineage of carnivorous plants. However, despite numerous studies seeking to elucidate their evolutionary relationships, the early-diverging relationships remain unresolved.\nC_LIO_LITo explore the utility of phylogenomic data sets for resolving relationships among the carnivorous Caryophyllales, we sequenced ten transcriptomes, including all the carnivorous genera except those in the rare West African liana family (Dioncophyllaceae). We used a variety of methods to infer the species tree, examine gene tree conflict and infer paleopolyploidy events.\nC_LIO_LIPhylogenomic analyses support the monophyly of the carnivorous Caryophyllales, with an origin of 68-83 mya. In contrast to previous analyses recover the remaining non-core Caryophyllales as non-monophyletic, although there are multiple reasons this result may be spurious and node supporting this relationship contains a significant amount gene tree discordance. We present evidence that the clade contains at least seven independent paleopolyploidy events, previously debated nodes from the literature have high levels of gene tree conflict, and taxon sampling influences topology even in a phylogenomic data set.\nC_LIO_LIOur data demonstrate the importance of carefully considering gene tree conflict and taxon sampling in phylogenomic analyses. Moreover, they provide a remarkable example of the propensity for paleopolyploidy in angiosperms, with at least seven such events in a clade of less than 2500 species.\nC_LI

evolutionary biology↗