Biology subjects

Luscombe, N.

Publications and source records attributed to Luscombe, N..

12 recordsLinked to original sources

bioRxiv2024

Integrative analysis of higher-order transcriptome organisation and RNA condensation principles

Complex RNA-protein networks play a pivotal role in the formation of many types of biomolecular condensates. How intrinsic RNA features contribute to condensate formation however remains unclear. Here, we integrate tailored transcriptomics assays to identify a distinct class of developmental condensation-prone RNAs termed smOOPs (semi-extractable, orthogonal organic phase separation-enriched RNAs). These transcripts are localised to larger intracellular foci, form denser RNA-RNA interaction subnetworks than expected and are heavily bound by RNA binding proteins (RBPs). Using an explainable deep learning framework, we reveal that smOOPs harbor characteristic sequence composition with lower sequence complexity, increased intramolecular folding and specific RBP binding patterns. Intriguingly, these RNAs encode proteins bearing extensive intrinsically disordered regions and are markedly predicted to be involved in biomolecular condensates, indicating an interplay between RNA- and protein-based features in phase separation. This work advances our understanding of condensation-prone RNAs and provides a versatile resource to further investigate RNA-driven condensation principles.

molecular biology↗

bioRxiv2024

The complete mitogenome of an unidentified Oikopleura species

1.Appendicularians are planktonic tunicates abundant all over the world. Currently, only two complete annotated mitochondrial genome assemblies are available for appendicularians, both for cryptic species of Oikopleura dioica. This underrepresentation of available appendicularian mitochondrial genomes limits environmental DNA sequencing (eDNA) studies that rely on mitochondrial markers as a taxonomic barcode. We report the complete mitochondrial genome assembly and annotation of an unknown appendicularian species isolated from the Amami Oshima island, Kagoshima prefecture, Japan, that has significant sequence difference with other currently available assemblies and will serve as a useful resource for ecological studies and further mitochondrial studies of appendicularians.

genomics↗

bioRxiv2023

Human Integrator provides a quality checkpoint during elongation to facilitate RNA polymerase II processivity

Integrator is a multi-subunit complex that directly interacts with the C-terminal domain (CTD) of RNA polymerase II (RNAPII). Through its RNA endonuclease activity, Integrator is required for 3'-end processing of both non-coding and coding transcripts. Here we demonstrate that depleting Integrator subunit 11 (INTS11), the main catalytic subunit of the Integrator complex, leads to a global elongation defect as a result of decreased polymerase processivity. We observe this defect in the region approximately 12 to 35 kb downstream of the transcription start site (TSS), where RNAPII normally transitions to its maximum processivity. We also identify an important role for INTS11, possibly in association with RNAPII CTD phospho-Tyr1, in repressing antisense transcription upstream of active promoters, as well as repressing transcription of genic regions near AsiSI-induced double-strand breaks. Altogether, this study points toward a novel function of Integrator in promoting termination of incompetent RNAPII molecules while facilitating the transition to fully processive polymerase in order to enable efficient elongation.

molecular biology↗

bioRxiv2022

A single-cell atlas of bobtail squid visual and nervous system highlights molecular principles of convergent evolution

Although the camera-type eyes of cephalopods and vertebrates are a canonical example of convergent morphological evolution, the cellular and molecular mechanisms underlying this convergence remain obscure. We used genomics and single cell transcriptomics to study these mechanisms in the visual system of the bobtail squid Euprymna berryi, an emerging cephalopod model. Analysis of 98,537 cellular transcriptomes from the squid visual and nervous system identified dozens of cell types that cannot be placed in simple correspondence with those of vertebrate or fly visual systems, as proposed by Ramon y Cajal and J.Z. Young. Instead, we find an unexpected diversity of neural types, dominated by dopamine, and previously uncharacterized glial cells. Surprisingly, we observe changes in cell populations and neurotransmitter usage during maturation and growth of the visual systems from hatchling to adult. Together these genomic and cellular findings shed new light on the parallel evolution of visual system complexity in cephalopods and vertebrates.

evolutionary biology↗

bioRxiv2022

A gradient border model for cell fate decisions at the neural plate border

The vertebrate neural plate border is a transient territory located at the edge of the neural plate containing precursors for all ectodermal derivatives: the neural plate; neural crest; placodes; and epidermis. Elegant functional experiments in a range of vertebrate models have provided an in-depth understanding of gene regulatory interactions within the ectoderm. However, these experiments conducted at tissue level raise seemingly contradictory models for fate allocation of individual cells. Here, we carry out single cell RNA sequencing of chick ectoderm from primitive streak to neurulation stage, to explore cell state diversity and heterogeneity. We characterise the dynamics of gene modules containing key factors known to regulate ectodermal cell fates, allowing us to model the order in which these fates are specified. Furthermore, we find that genes previously classified as neural plate border specifiers typically exhibit dynamic expression patterns and are biased towards either placodal or neural crest fates, revealing that the neural plate border should be seen as an anatomical region of the ectoderm and not a discrete transcriptional state. Through co-expression of placodal and neural crest markers, we identify a population of border located unstable progenitors (BLUPs) which gradually reduces in size as fate segregation occurs. Considering our findings, we propose a gradient border model for cell fate choice at the neural plate border, with the probability of cell fate allocation closely tied to the spatiotemporal positioning of cells.

developmental biology↗

bioRxiv2022

Annelid functional genomics reveal the origins of bilaterian life cycles

Indirect development with an intermediate larva exists in all major animal lineages1, making larvae central to most scenarios of animal evolution2-12. Yet how larvae evolved remains disputed. Here we show that temporal shifts (i.e., heterochronies) in trunk formation underpin the diversification of larvae and bilaterian life cycles. Combining chromosome-scale genome sequencing in the slow-evolving annelid Owenia fusiformis13 with transcriptomic and epigenomic profiling during the life cycles of this and two other annelids, we found that trunk development is deferred to pre-metamorphic stages in the feeding larva of O. fusiformis, but starts after gastrulation in the non-feeding larva with gradual metamorphosis of Capitella teleta and the direct developing embryo of Dimorphilus gyrociliatus. Accordingly, the embryos of O. fusiformis develop first into an enlarged anterior domain that forms larval tissues and the adult head. Notably, this also occurs in the so-called "head larvae" of other bilaterians14,15, with whom O. fusiformis larva shows extensive transcriptomic similarities. Together, our findings suggest that the temporal decoupling of head and trunk formation, as maximally observed in "head larvae", allowed larval evolution in Bilateria, thus diverging from prevailing scenarios that propose either co-option10,11 or innovation12 of gene regulatory programmes to explain larva and adult origins.

genomics↗

bioRxiv2021

clipplotr - a comparative visualisation and analysis tool for CLIP data

CLIP technologies are now widely used to study RNA-protein interactions and many datasets are now publicly available. An important first step in CLIP data exploration is the visual inspection and assessment of processed genomic data on selected genes or regions and performing comparisons: either across conditions within a particular project, or incorporating publicly available data. However, the output files produced by data processing pipelines or preprocessed files available to download from data repositories are often not suitable for direct comparison and usually need further processing. Furthermore, to derive biological insight it is usually necessary to visualise CLIP signal alongside other data such as annotations, or orthogonal functional genomic data (e.g. RNA-seq). We have developed a simple, but powerful, command-line tool: clipplotr, which facilitates these visual comparative and integrative analyses with normalisation and smoothing options for CLIP data and the ability to show these alongside reference annotation tracks and functional genomic data. These data can be supplied as input to clipplotr in a range of file formats, which will output a publication quality figure. It is written in R and can both run on a laptop computer independently, or be integrated into computational workflows on a high-performance cluster. Releases, source code and documentation are freely available at: https://github.com/ulelab/clipplotr.

bioinformatics↗

bioRxiv2020

CD147 (BSG) but not ACE2 expression is detectable in vascular endothelial cells within single cell RNA sequencing datasets derived from multiple tissues in healthy individuals

Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and is associated with a wide range of systemic manifestations. Several observations support a role for vascular endothelial dysfunction in the pathogenesis including an increased incidence of thrombotic events and coagulopathy and the presence of vascular risk factors as an independent predictor of poor prognosis. It has recently been reported that endothelitis is associated with viral inclusion bodies suggesting a direct role for SARS-CoV-2 in the pathogenesis. The ACE2 receptor has been shown to mediate SARS-CoV-2 uptake and it has been proposed that CD147 (BSG) can function as an alternative cell surface receptor. To define the endothelial cell populations that are susceptible to infection with SARS-CoV-2, we investigated the expression of ACE2 as well as other genes implicated in the cellular entry of SARS-Cov-2 in the vascular endothelium through the analysis of single cell sequencing data derived from multiple human tissues (skin, liver, kidney, lung and intestine). We found that CD147 (BSG) but not ACE2 is detectable in vascular endothelial cells within single cell sequencing datasets derived from multiple tissues in healthy individuals. This implies that either ACE2 is not expressed in healthy tissue but is instead induced in response to SARS-Cov2 or that SARS-Cov2 enters endothelial cells via an alternative receptor such as CD147.

genomics↗

bioRxiv2019

Transcription levels of a long noncoding RNA orchestrate opposing regulatory and cell fate outcomes in yeast

Many long noncoding RNAs (lncRNAs) act in cis through transcription-coupled chromatin alterations that drive changes in local gene expression. How some cis-acting lncRNAs promote and others repress gene expression remains poorly understood. Here we report that in S. cerevisiae transcription levels of the lncRNA IRT2, located upstream in the promoter of the inducer of meiosis gene, regulate opposing chromatin and transcription states. Low IRT2 transcription displays enhancer RNA-like features. At these levels, IRT2 promotes histone exchange delivering acetylated histone H3 lysine 56 to chromatin thereby facilitating recruitment of a transcription factor and consequently activating transcription. Conversely, increasing IRT2 transcription enhances chromatin assembly and transcriptional repression. The opposing functions of IRT2 direct a regulatory circuit, which ensures that cells expressing opposite, but not one of either, mating-type loci enter meiosis. Our data demonstrate that the transcription levels of an lncRNA are key to controlling gene expression and cell fate outcomes.

biochemistry↗

bioRxiv2018

Genome-Wide Reconstitution of Chromatin Transactions: RSC Preferentially disrupts H2A.Z-Containing Nucleosomes

Chromatin transactions are typically studied in vivo, or in vitro using artificial chromatin lacking the epigenetic complexity of the natural material. Attempting to bridge the gap between these approaches, we established a system for isolating the yeast genome as a library of mono-nucleosomes harboring the natural epigenetic signature, suitable for biochemical manipulation. Combined with deep sequencing, this library was used to investigate the intrinsic stability of individual nucleosomes, and - as proof of principle - the nucleosome preference of the chromatin remodeling complex, RSC. Our data indicate that the natural stability of nucleosomes differs greatly, with nucleosomes on tRNA genes and on promoters of protein-coding genes standing out as intrinsically unstable. Interestingly, RSC shows a distinct preference for nucleosomes derived from regions with a high density of histone variant H2A.Z, and this preference is indeed markedly diminished using nucleosomes from cells lacking H2A.Z. Importantly, the preference for H2A.Z remodeling/nucleosome ejection can also be reconstituted with recombinant nucleosome arrays. Together, our data indicate that, despite being separated from their genomic context, individual nucleosomes can retain their original identity as promoter- or TSS-nucleosomes. Besides shedding new light on nucleosome stability and the chromatin remodeler RSC, the simple experimental system outlined here should be generally applicable to the study of chromatin transactions.

biochemistry↗

bioRxiv2018

Transcriptome-wide profiling of mammalian spliceosome and branchpoints with iCLIP

Studies of spliceosomal interactions are challenging due to their dynamic nature. Here we employed spliceosome iCLIP, which immunoprecipitates SmB along with snRNPs and auxiliary RNA binding proteins (RBPs), to map human spliceosome engagement with snRNAs and pre-mRNAs. This identified over 50,000 branchpoints (BPs) that have canonical sequence and structural features. Moreover, it revealed 7 binding peaks around BPs and splice sites, each precisely overlapping with binding profiles of specific splicing factors. We show how the binding patterns of these RBPs are affected by the position and strength of BPs. For example, strong or proximally located BPs preferentially bind SF3 rather than U2AF complex. Notably, these effects are partly neutralized during spliceosomal assembly in a way that depends on the core spliceosomal protein PRPF8. These insights exemplify spliceosome iCLIP as a broadly applicable method for transcriptomic studies of splicing mechanisms.

genomics↗

bioRxiv2017

Genomic landscape of oxidative DNA damage and repair reveals regioselective protection from mutagenesis

DNA is subject to constant chemical modification and damage, which eventually results in variable mutation rates throughout the genome. Although detailed molecular mechanisms of DNA damage and repair are well-understood, damage impact and execution of repair across a genome remains poorly defined. To bridge the gap between our understanding of DNA repair and mutation distributions we developed a novel method, AP-seq, capable of mapping apurinic sitesand 8-oxo-7,8-dihydroguanine bases at [~]300bp resolution on a genome-wide scale. We directly demonstrate that the accumulation rate of oxidative damage varies widely across the genome, with hot spots acquiring many times more damage than cold spots. Unlike SNVs in cancers, damage burden correlates with marks for open chromatin notably H3K9ac and H3K4me2. Oxidative damage is also highly enriched in transposable elements and other repetitive sequences. In contrast, we observe decreased damage at promoters, exons and termination sites, but not introns, in a seemingly transcription-independent manner. Leveraging cancer genomic data, we also find locally reduced SNV rates in promoters, genes and other functional elements. Taken together, our study reveals that oxidative DNA damage accumulation and repair differ strongly across the genome, but culminate in a previously unappreciated mechanism that safe-guards the regulatory sequences and the coding regions of genes from mutations.

genomics↗