Biology subjects

Flynn, R.

Publications and source records attributed to Flynn, R..

11 recordsLinked to original sources

bioRxiv2024

irCLIP-RNP and Re-CLIP reveal patterns of dynamic protein associations on RNA

RNA binding proteins (RBPs) control varied processes, including RNA splicing, stability, transport, and translation1-3. Dysfunctional RNA-RBP interactions contribute to the pathogenesis of human disease1,4,5, however, characterizing the nature and dynamics of multiprotein assemblies on RNA has been challenging. To address this, non-isotopic ligation-based ultraviolet crosslinking immunoprecipitation6 was combined with mass spectrometry (irCLIP-RNP) to identify RNA-dependent associated proteins (RDAPs) co-bound to RNA with any RBP of interest. irCLIP-RNP defined landscapes of multimeric protein assemblies on RNA, uncovering previously unknown patterns of RBP-RNA associations, including cell-type-selective combinatorial relationships between RDAPs and primary RBPs. irCLIP-RNP also defined dynamic RDAP remodeling in response to epidermal growth factor (EGF), uncovering EGF-induced recruitment of UPF1 adjacent to HNRNPC to effect splicing surveillance of cell proliferation mRNAs. To identify the RNAs simultaneously co-bound by multiple studied RBPs, a sequential immunoprecipitation irCLIP (Re-CLIP) method was also developed. Re-CLIP confirmed binding relationships seen in irCLIP-RNP and detected simultaneous HNRNPC and UPF1 co-binding on RND3 and DDX3X mRNAs. irCLIP-RNP and Re-CLIP provide a framework to identify and characterize dynamic RNA-protein assemblies in living cells.

molecular biology↗

bioRxiv2024

Synthetic genomes unveil the effects of synonymous recoding

Engineering the genetic code of an organism provides the basis for (i) making any organism safely resistant to natural viruses and (ii) preventing genetic information flow into and out of genetically modified organisms while (iii) allowing the biosynthesis of genetically encoded unnatural polymers1-4. Achieving these three goals requires the reassignment of multiple of the 64 codons nature uses to encode proteins. However, synonymous codon replacement--recoding--is frequently lethal, and how recoding impacts fitness remains poorly explored. Here, we explore these effects using whole-genome synthesis, multiplexed directed evolution, and genome-transcriptome-translatome-proteome co-profiling on multiple recoded genomes. Using this information, we assemble a synthetic Escherichia coli genome in seven sections using only 57 codons to encode proteins. By discovering the rules responsible for the lethality of synonymous recoding and developing a data-driven multi-omics-based genome construction workflow that troubleshoots synthetic genomes, we overcome the lethal effects of 62,007 synonymous codon swaps and 11,108 additional genomic edits. We show that synonymous recoding induces transcriptional noise including new antisense RNAs, leading to drastic transcriptome and proteome perturbation. As the elimination of select codons from an organisms genetic code results in the widespread appearance of cryptic promoters, we show that synonymous codon choice may naturally evolve to minimize transcriptional noise. Our work provides the first genome-scale description of how synonymous codon changes influence organismal fitness and paves the way for the construction of functional genomes that provide genetic firewalls from natural ecosystems and safely produce biopolymers, drugs, and enzymes with an expanded chemistry.

synthetic biology↗

bioRxiv2023

Human-gut phages harbor sporulation genes

Spore-forming bacteria are prevalent in mammalian guts and have implications for host health and nutrition. The production of dormant spores is thought to play an important role in the colonization, persistence, and transmission of these bacteria. Spore formation also modifies interactions among microorganisms such as infection by phages. Recent studies suggest that phages may counter dormancy-mediated defense through the expression of phage-encoded sporulation genes during infection, which can alter the transitions between active and inactive states. By mining genomes and gut-derived metagenomes, we identified sporulation genes that are preferentially encoded by phages that infect spore-forming bacteria. These included genes involved in chromosome partitioning, DNA damage repair, and cell wall-associated functions. In addition, phages contained homologs of sporulation-specific transcription factors, notably spo0A, the master regulator of sporulation, which could allow phages to control the complex genetic network responsible for spore development. Our findings suggest that phages could influence the formation of bacterial spores with implications for the health of the human gut microbiome, as well as bacterial communities in other environments. SIGNIFICANCEPhages acquire bacterial genes and use them to alter host metabolism in ways that enhance their fitness. To date, most auxiliary genes replace or modulate enzymes that are used by the host for nutrition or energy production. However, phage fitness is affected by all aspects of host physiology, including decisions that reduce metabolic activity of the cell. Here we focus on endosporulation, a complex and ancient form of dormancy found among the Bacillota that involves hundreds of genes. By coupling homology searches with host classification, we identify 31 phage-encoded homologs of sporulation genes that are mostly limited to phages infecting spore-forming bacteria. Nearly one-third the homologs recovered were regulatory genes suggesting that phages may manipulate host genetic networks by tapping into their control elements. Our findings also suggest a mechanism by which phages can overcome the defensive strategy of dormancy, which may be involved in coevolutionary dynamics of spore-forming bacteria.

microbiology↗

bioRxiv2022

Swapped genetic code blocks viral infections and gene transfer

Removing cellular transfer RNAs (tRNAs), making their cognate codons unreadable, creates a genetic firewall that prevents viral replication and horizontal gene transfer. However, numerous viruses and mobile genetic elements encode parts of the translational apparatus, including tRNAs, potentially rendering a genetic-code-based firewall ineffective. In this paper, we show that such horizontally transferred tRNA genes can enable viral replication in Escherichia coli cells despite the genome-wide lack of three codons and the previously essential cognate tRNAs and release factor 1. By repurposing viral tRNAs, we then develop recoded cells bearing an amino-acid-swapped genetic code that reassigns two of the six serine codons to leucine during translation. This amino-acid-swapped genetic code renders cells completely resistant to viral infections by mistranslating viral proteomes and prevents the escape of synthetic genetic information by engineered reliance on serine codons to produce leucine-requiring proteins. Finally, we also repurpose the third free codon to biocontain this virus-resistant host via dependence on an amino acid not found in nature.

synthetic biology↗

bioRxiv2022

Evaluation of nanopore sequencing for epigenetic epidemiology: a comparison with DNA methylation microarrays

Most epigenetic epidemiology to date has utilized microarrays to identify positions in the genome where variation in DNA methylation is associated with environmental exposures or disease. However, these profile less than 3% of DNA methylation sites in the human genome, potentially missing affected loci and preventing the discovery of disrupted biological pathways. Third generation sequencing technologies, including Nanopore sequencing, have the potential to revolutionise the generation of epigenetic data, not only by providing genuine genome-wide coverage but profiling epigenetic modifications direct from native DNA. Here we assess the viability of using Nanopore sequencing for epidemiology by performing a comparison with DNA methylation quantified using the most comprehensive microarray available, the Illumina EPIC array. We implemented a CRISPR-Cas9 targeted sequencing approach in concert with Nanopore sequencing to profile DNA methylation in three genomic regions to attempt to rediscover genomic positions that existing technologies have shown are differentially methylated in tobacco smokers. Using Nanopore sequencing reads, DNA methylation was quantified at 1,779 CpGs across three regions, providing a finer resolution of DNA methylation patterns compared to the EPIC array. The correlation of estimated levels of DNA methylation between platforms was high. Furthermore, we identified 12 CpGs where hypomethylation was significantly associated with smoking status, including 10 within the AHRR gene. In summary, Nanopore sequencing is a valid option for identifying genomic loci where large differences in DNAm are associated with a phenotype and has the potential to advance our understanding of the role differential methylation plays in the aetiology of complex disease.

neuroscience↗

bioRxiv2021

Loquacious Modulates Flaviviral RNA Replication in Mosquito Cells

Arthropod-borne viruses infect both mosquito and mammalian hosts. While much is known about virus-host interactions that modulate viral gene expression in their mammalian host, much less is known about the interactions that involve inhibition, subversion or avoidance strategies in the mosquito host. A novel RNA-Protein interaction detection assay was used to detect proteins that directly or indirectly bind to dengue viral genomes in infected mosquito cells. Membrane-associated mosquito proteins SEC61A1 and Loquacious (Loqs) were found to be in complex with the viral RNA. Depletion analysis demonstrated that both SEC61A1 and Loqs have pro-viral functions in the dengue viral infectious cycle. Co-localization and pull-down assays showed that Loqs interacts with viral protein NS3 and both full-length and subgenomic viral RNAs. While Loqs coats the entire positive-stranded viral RNA, it binds selectively to the 3 end of the negative-strand of the viral genome. In-depth analyses showed that the absence of Loqs did not affect translation or turnover of the viral RNA but modulated viral replication. Loqs also displayed pro-viral functions for several flaviviruses in infected mosquito cells, suggesting a conserved role for Loqs in flavivirus-infected mosquito cells. Author SummaryThere is a wealth of information that dictates virus-host interactions in flavivirus-infected mammalian cells, yet there is only sparse information on the mechanisms that modulate viral gene expression in the mosquito host. Using a novel RNA-protein detection assay, the interactions of SEC61A1 and Loqs with the dengue viral genome were found to have proviral functions in infected mosquito cells. In particular, Loqs forms complexes with the positive-strand of the viral RNA and the very 3 end of the negative-strand viral RNA. Further analyses showed that Loqs modulates viral RNA replication of dengue virus and gene amplification of several other flaviviral genomes. These findings argue that Loqs is an essential proviral host factor in mosquitos.

microbiology↗

bioRxiv2020

Novel epigenetic clock for fetal brain development predicts fetal epigenetic age for iPSCs and iPSC-derived neurons.

Induced pluripotent stem cells (iPSCs) and their differentiated neurons (iPSC-neurons) are a widely used cellular model in the research of the central nervous system. However, it is unknown how well they capture age-associated processes, particularly given that pluripotent cells are only present during the earliest stages of mammalian development. Epigenetic clocks utilize coordinated age-associated changes in DNA methylation to make predictions that correlate strongly with chronological age. It has been shown that the induction of pluripotency rejuvenates predicted epigenetic age. As existing clocks are not optimized for the study of brain development, we developed the fetal brain clock (FBC), a bespoke epigenetic clock trained in human prenatal brain samples in order to investigate more precisely the epigenetic age of iPSCs and iPSC-neurons. The FBC was tested in two independent validation cohorts across a total of 194 samples, confirming that the FBC outperforms other established epigenetic clocks in fetal brain cohorts. We applied the FBC to DNA methylation data from iPSCs and iPSC-derived neuronal precursor cells and neurons, finding that these cell types are epigenetically characterized as having an early fetal age. Furthermore, while differentiation from iPSCs to neurons significantly increases epigenetic age, iPSC-neurons are still predicted as being fetal. Together our findings reiterate the need to better understand the limitations of existing epigenetic clocks for answering biological research questions and highlight a limitation of iPSC-neurons as a cellular model of age-related diseases.

neuroscience↗

bioRxiv2020

Transcriptome-Wide Combinatorial RNA Structure Probing in Living Cells

RNA molecules can fold into complex structures and interact with trans-acting factors to control their biology. Recent methods have been focused on developing novel tools to measure RNA structure transcriptome-wide, but their utility to study and predict RNA-protein interactions or RNA processing has been limited thus far. Here, we extend these studies with the first transcriptomewide mapping method for cataloging RNA solvent accessibility, icLASER. By combining solvent accessibility (icLASER) with RNA flexibility (icSHAPE) data, we efficiently predict RNA-protein interactions transcriptome-wide and catalog RNA polyadenylation sites by RNA structure alone. These studies showcase the power of designing novel chemical approaches to studying RNA biology. Further, our study exemplifies merging complementary methods to measure RNA structure inside cells and its utility for predicting transcriptome-wide interactions that are critical for control of and regulation by RNA structure. We envision such approaches can be applied to studying different cell types or cells under varying conditions, using RNA structure and footprinting to characterize cellular interactions and processing involving RNA.

biochemistry↗

bioRxiv2019

Correction of amyotrophic lateral sclerosis related phenotypes in induced pluripotent stem cell-derived motor neurons carrying a hexanucleotide expansion mutation in C9orf72 by CRISPR/Cas9 genome editing using homology-directed repair

The G4C2 hexanucleotide repeat expansion (HRE) in C9orf72 is the commonest cause of familial amyotrophic lateral sclerosis (ALS). A number of different methods have been used to generate isogenic control lines using CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 and non-homologous end-joining (NHEJ) by deleting the repeat region with the risk of creating indels and genomic instability. In this study we demonstrate complete correction of an induced pluripotent stem cell (iPSC) line derived from a C9orf72-HRE positive ALS/FTD patient using CRISPR/Cas9 genome editing and homology directed repair (HDR), resulting in replacement of the excised region with a donor template carrying the wild-type repeat size to maintain the genetic architecture of the locus. The isogenic correction of the C9orf72 HRE restored normal expression and methylation at the C9orf72 locus, reduced intron retention in the edited lines, and abolished pathological phenotypes associated with the C9orf72 HRE expansion in iPSC derived motor neurons (iPSMNs). RNA sequencing of the mutant line identified 2220 differentially expressed genes compared to its isogenic control. Enrichment analysis demonstrated an over-representation of ALS relevant pathways, including calcium ion dependent exocytosis, synaptic transport and the KEGG ALS pathway, as well as new targets of potential relevance to ALS pathophysiology. Complete correction of the C9orf72 HRE in iPSMNs by CRISPR/Cas9 mediated HDR provides an ideal model to study the earliest effects of the hexanucleotide expansion on cellular homeostasis and the key pathways implicated in ALS pathophysiology.

neuroscience↗

bioRxiv2019

LRRK2 is recruited to phagosomes and co-recruits Rab8 and Rab10 in human pluripotent stem cell-derived macrophages

The Parkinsons disease-associated gene, LRRK2, is also associated with immune disorders and infectious disease, and is expressed in immune subsets. Here, we characterise a platform for interrogating the expression and function of endogenous LRRK2 in authentic human phagocytes, using human induced Pluripotent Stem Cell-derived macrophages and microglia. Endogenous LRRK2 is expressed and upregulated by interferon-{gamma} in these cells, including a 187kD cleavage product. Using LRRK2 knockout and G2019S isogenic repair lines, we find that LRRK2 is not involved in initial phagocytic uptake of bioparticles, but is recruited to LAMP1(+)/Rab9(+) maturing phagosomes, and LRRK2 kinase inhibition enhances its residency at the phagosome. Importantly, LRRK2 is required for Rab8a and Rab10 recruitment to phagosomes, implying that LRRK2 operates at the intersection between phagosome maturation and recycling pathways in these professional phagocytes.

immunology↗

bioRxiv2018

TRPV1 regulates opioid analgesia during inflammation

Acute inflammation in humans or mice enhances the analgesic properties of opioids. However, the inflammatory transducers that prime opioid receptor signaling in nociceptors are unknown. We found that TRPV1-/- mice are insensitive to peripheral opioid analgesia in an inflammatory pain model. We report that TRPV1 channel activation drives a MAPK signaling pathway accompanied by the shuttling of {beta}-arrestin2 to the nucleus. This shuttling in turn prevents: {beta}-arrestin2-receptor recruitment, subsequent internalization of agonist-bound mu opioid receptor (MOR), and suppression of DAMGO-induced inhibition of N-type calcium current observed upon desensitization. Consequently, inflammation-induced activation of TRPV1 preserves opioid analgesic potency in a mouse model of opioid receptor desensitization. Overall, our work reveals a TRPV1-mediated signaling mechanism, involving {beta}-arrestin2 nuclear translocation, that underlies the peripheral opioid control of inflammatory pain. Our data single out TRPV1 channels as modulators of opioid analgesia.

neuroscience↗