Biology subjects

Green, R.

Publications and source records attributed to Green, R..

At least 55 records · Page 3Linked to original sources

bioRxiv2021

Genetic screens identify connections between ribosome recycling and nonsense mediated decay

The decay of messenger RNA with a premature termination codon (PTC) by nonsense mediated decay (NMD) is an important regulatory pathway for eukaryotes and an essential pathway in mammals. NMD is typically triggered by the ribosome terminating at a stop codon that is aberrantly distant from the poly-A tail. Here, we use a fluorescence screen to identify factors involved in NMD in S. cerevisiae. In addition to the known NMD factors, including the entire UPF family (UPF1, UPF2 and UPF3), as well as NMD4 and EBS1, we identify factors known to function in post-termination recycling and characterize their contribution to NMD. We then use a series of modified reporter constructs that block both elongating and scanning ribosomes downstream of stop codons and demonstrate that a deficiency in recycling of 80S ribosomes or 40S subunits stabilizes NMD substrates. These observations in S. cerevisiae expand on recently reported data in mammals indicating that the 60S recycling factor ABCE1 is important for NMD (1, 2) by showing that increased activities of both elongating and scanning ribosomes (80S or 40S) in the 3UTR correlate with a loss of NMD. Author SummaryIn this work, we aim to understand the mechanism of targeting mRNAs for decay via the long-studied nonsense mediated decay (NMD) pathway. We demonstrate that efficient large and small subunit ribosome recycling are necessary components of NMD. We go on to provide evidence that either scanning or actively translating ribosomes in the 3UTR disrupt the decay of NMD targets. Our work highlights the importance of the composition of the 3UTR in NMD signaling and emphasizes the need for this region to indeed be untranslated for NMD to occur. Exon junction complexes (EJCs) in the 3UTR are known to induce NMD, however, in the budding yeast system used here, the NMD targets are EJC-free. Therefore, our data support a model in which factors other than EJCs may accumulate in the 3UTR and provide a signal for NMD.

molecular biology↗

bioRxiv2021

Mechanisms that ensure speed and fidelity in eukaryotic translation termination

Translation termination, which liberates a nascent polypeptide from the ribosome specifically at stop codons, must occur accurately and rapidly. We established single-molecule fluorescence assays to track the dynamics of ribosomes and two requisite release factors (eRF1 and eRF3) throughout termination using an in vitro-reconstituted yeast translation system. We found that the two eukaryotic release factors bind together to recognize stop codons rapidly and elicit termination via a tightly regulated, multi-step process that resembles tRNA selection during translation elongation. Because the release factors are conserved from yeast to humans, the molecular events that underlie yeast translation termination are likely broadly fundamental to eukaryotic protein synthesis. One Sentence SummaryDirect visualization of eukaryotic translation reveals the dynamics of termination at stop codons.

biochemistry↗

bioRxiv2021

Female immunity protects from cutaneous squamous cell carcinoma

PurposeCancer susceptibility and mortality are higher in males, and the mutational and transcriptomic landscape of cancer differs by sex. The current assumption is that men are at higher risk of epithelial cancers as they expose more to carcinogens and accumulate more damage than women. We present data showing women are more protected from aggressive cutaneous squamous cell carcinoma (cSCC) due to strong immune activation. MethodsWe explored clinical and molecular sexual disparity in immunocompetent and immunosuppressed patients (N= 738, N=160) with carcinoma cSCC, in FVB/N mice exposed to equal doses of DMBA, and in human keratinocytes by whole exome sequencing, bulk and single cell RNA sequencing. ResultsWe show cSCC is more aggressive in men, and immunocompetent women develop mild cSCC, later in life. To test if sex drives disparity, we exposed male and female mice to equal doses of carcinogen, and found males present more aggressive, metastatic cSCC than females. Critically, females activate cancer immune-related expression pathways and CD4 and CD8 T cell infiltration independently of mutations. In contrast, males increase the rate of mitoses and proliferation in response to carcinogen. Human female skin and keratinocytes also activate immune-cancer fighting pathways and immune cells at ultraviolet radiation-damaged sites. Critically, a compromised immune system leads to high-risk, aggressive cSCC specifically in women. ConclusionsThis work shows the immune response is sex biased in cSCC, and highlights female immunity offers greater protection than male immunity.

cancer biology↗

bioRxiv2020

Multi-omic Single-cell Atlas Reveals Broad Ex Vivo Effects on Human Immunobiology

Multi-omic profiling of human peripheral blood is increasingly utilized to identify biomarkers and pathophysiologic mechanisms of disease. The importance of these platforms in clinical and translational studies led us to investigate the impact of delayed blood processing on the numbers and state of peripheral blood mononuclear cells (PBMC) and on the plasma proteome. Similar to previous studies, we show minimal effects of delayed processing on the numbers and general phenotype of PBMCs up to 18 hours. In contrast, profound changes in the single-cell transcriptome and composition of the plasma proteome become evident as early as 6 hours after blood draw. These reflect patterns of cellular activation across diverse cell types that lead to progressive distancing of the gene expression state and plasma proteome from native in vivo biology. Differences accumulating during an overnight rest (18 hours) could confound relevant biologic variance related to many underlying disease states.

immunology↗

bioRxiv2020

Steric Interactions at Gln154 in ZEITLUPE Induce Reorganization of the LOV Domain Dimer Interface

Plants measure light, quality, intensity, and duration to coordinate growth and development with daily and seasonal changes in environmental conditions, however, the molecular details linking photochemistry to signal transduction remain incomplete. Two closely related Light, Oxygen, or Voltage (LOV) domain containing photoreceptor proteins ZEITLUPE (ZTL) and FLAVIN-BINDING, KELCH REPEAT, F-BOX 1 (FKF1) divergently regulate the protein stability of circadian clock and photoperiodic flowering components to mediate daily and seasonal development. Using structural approaches, we identified that mutations at the Gly46 position led to global rearrangements of the ZTL dimer interface. Specifically, introduction of G46S and G46A variants that mimic equivalent residues found in FKF1 induce a 180{degrees} rotation about the dimer interface that is coupled to ordering of N- and C-terminal signaling elements. These conformational changes hinge upon rotation of a C-terminal Gln residue analogous to that present in light-state structures of ZTL. The results presented herein, confirm a divergent signaling mechanism within ZTL that deviates from other members of the LOV superfamily and suggests that mechanisms of signal transduction in LOV proteins may be fluid across the LOV protein family.

biophysics↗

bioRxiv2020

Integrated single cell analysis of chromatin accessibility and cell surface markers

Single-cell measurements of cellular characteristics have been instrumental in understanding the heterogeneous pathways that drive differentiation, cellular responses to extracellular signals, and human disease states. scATAC-seq has been particularly challenging due to the large size of the human genome and processing artefacts resulting from DNA damage that are an inherent source of background signal. Downstream analysis and integration of scATAC-seq with other single-cell assays is complicated by the lack of clear phenotypic information linking chromatin state and cell type. Using the heterogeneous mixture of cells in human peripheral blood as a test case, we developed a novel scATAC-seq workflow that increases the signal-to-noise ratio and allows simultaneous measurement of cell surface markers: Integrated Cellular Indexing of Chromatin Landscape and Epitopes (ICICLE-seq). We extended this approach using a droplet-based multiomics platform to develop a trimodal assay to simultaneously measure Transcriptomic state (scRNA-seq), cell surface Epitopes, and chromatin Accessibility (scATAC-seq) from thousands of single cells, which we term TEA-seq. Together, these multimodal single-cell assays provide a novel toolkit to identify type-specific gene regulation and expression grounded in phenotypically defined cell types.

immunology↗

bioRxiv2020

eEF3 promotes late stages of tRNA translocation on the ribosome

SummaryIn addition to the conserved translation elongation factors eEF1A and eEF2, fungi require a third essential elongation factor, eEF3. While eEF3 has been implicated in tRNA binding and release at the A and E sites, its exact mechanism of action is unclear. Here we show that eEF3 acts at the mRNA–tRNA translocation step by promoting the dissociation of the tRNA from the E site, but independent of aminoacyl-tRNA recruitment to the A site. Depletion of eEF3 in vivo leads to a general slow-down in translation elongation due to accumulation of ribosomes with an occupied A site. Cryo-EM analysis of ex vivo eEF3-ribosome complexes shows that eEF3 facilitates late steps of translocation by favoring non-rotated ribosomal states as well as by opening the L1 stalk to release the E-site tRNA. Additionally, our analysis provides structural insights into novel translation elongation states, enabling presentation of a revised yeast translation elongation cycle.Competing Interest StatementThe authors have declared no competing interest.View Full Text

molecular biology↗

bioRxiv2020

Puromycin reactivity does not accurately localize translation at the subcellular level

Puromycin is a tyrosyl-tRNA mimic that blocks translation by labeling and releasing elongating polypeptide chains from translating ribosomes. Puromycin has been used in molecular biology research for decades as a translation inhibitor. The development of puromycin antibodies and derivatized puromycin analogs has enabled the quantification of active translation in bulk and single-cell assays. More recently, in vivo puromycylation assays have become popular tools for localizing translating ribosomes in cells. These assays often use elongation inhibitors to purportedly inhibit the release of puromycin-labeled nascent peptides from ribosomes. Here, using in vitro and in vivo experiments, we demonstrate that, even in the presence of elongation inhibitors, puromycylated peptides are released and diffuse away from ribosomes. Puromycylation assays reveal subcellular sites, such as nuclei, where puromycylated peptides accumulate post-release and which do not necessarily coincide with sites of active translation. Our findings urge caution when interpreting puromycylation assays in the in vivo context.

biochemistry↗

bioRxiv2020

Dynamic regulation of translation quality control associated with ribosome stalling

Translation of problematic mRNA sequences induces ribosome stalling. Collided ribosomes at the stall site are recognized by cellular quality control machinery, resulting in dissociation of the ribosome from the mRNA and subsequent degradation of the nascent polypeptide and in some organisms, decay of the mRNA. However, the timing and regulation of these processes are unclear. We developed a SunTag-based reporter to monitor translation in real time on single mRNAs harboring difficult-to-translate poly(A) stretches. This reporter recapitulates previous findings in human cells that an internal poly(A) stretch reduces protein output [~]10-fold, while mRNA levels are relatively unaffected. Long-term imaging of translation indicates that poly(A)-containing mRNAs are robustly translated in the absence of detectable mRNA cleavage. However, quantification of ribosome density reveals a [~]3-fold increase in the number of ribosomes on poly(A)-containing mRNAs compared to a control mRNA, consistent with queues of many stalled ribosomes. Using single-molecule harringtonine runoff experiments, we observe the resolution of these queues in real-time by the cellular quality control machinery, and find that rescue is very slow compared to both elongation and termination. We propose that the very slow clearance of stalled ribosomes provides the basis for the cell to distinguish between transient and deleterious stalls, and that the human quality control apparatus predominantly targets the nascent protein rather than the mRNA.

biochemistry↗

bioRxiv2020

Expression changes in immune and epigenetic gene pathways associated with nutritional metabolites in maternal blood from pregnancies resulting in autism and atypical neurodevelopment

BackgroundThe prenatal period is a critical window to study factors involved in the development of autism spectrum disorder (ASD). Environmental factors, especially in utero nutrition, can interact with genetic risk for ASD, but how specific prenatal nutrients in mothers of children later diagnosed with ASD or non-typical development (Non-TD) associate with gestational gene expression is poorly understood. Maternal blood collected prospectively during pregnancy provides a new opportunity to gain insights into nutrition, particularly one-carbon metabolites, on gene pathways and neurodevelopment. MethodsGenome-wide transcriptomes were measured using microarrays in 300 maternal blood samples from all three trimesters in the Markers of Autism Risk in Babies - Learning Early Signs (MARBLES) study. Sixteen different one-carbon metabolites, including folic acid, betaine, 5-methyltretrahydrofolate (5-MeTHF), and dimethylglycine (DMG) were measured. Differential expression analysis and weighted gene correlation network analysis (WGCNA) were used to compare gene expression between children later diagnosed as typical development (TD), Non-TD and ASD, and to nutrient metabolites. ResultsUsing differential gene expression analysis, six transcripts associated with four genes (TGR-AS1, SQSTM1, HLA-C and RFESD) showed genome-wide significance (FDR q < 0.05) with child outcomes. Genes nominally differentially expressed compared to TD specifically in ASD, but not Non-TD, significantly overlapped with seven high confidence ASD genes. 218 transcripts in common to ASD and Non-TD differential expression compared to TD were significantly enriched for functions in immune response to interferon-gamma, apoptosis, and metal ion transport. WGCNA identified co-expressed gene modules significantly correlated with 5-MeTHF, folic acid, DMG, and betaine. A module enriched in DNA methylation functions showed a protective association with folic acid/5-MeTHF concentrations and ASD risk. Independent of child outcome, maternal plasma betaine and DMG concentrations associated with a block of co-expressed genes enriched for adaptive immune, histone modification, and RNA processing functions. LimitationsBlood contains a heterogeneous mixture of cell types, and many WGCNA modules correlated with cell type and/or nutrient concentrations, but not child outcome. Gestational age correlated with some co-expressed gene modules in addition to nutrients. ConclusionsThese results support the premise that the prenatal maternal blood transcriptome is a sensitive indicator of gestational nutrition and childrens later neurodevelopmental outcomes.

bioinformatics↗

bioRxiv2020

Nuclease-mediated depletion biases in ribosome footprint profiling libraries

Ribosome footprint profiling is a high throughput sequencing based technique that provides detailed and global views of translation in living cells. An essential part of this technology is removal of unwanted, normally very abundant, ribosomal RNA sequences that dominate libraries and increase sequencing costs. The most effective commercial solution (Ribo-Zero) has been discontinued and a number of new, experimentally distinct commercial applications have emerged on the market. Here we evaluated several commercially available alternatives designed for RNA-seq of human samples and find them unsuitable for ribosome footprint profiling. We instead recommend the use of custom-designed biotinylated oligos, which were widely used in early ribosome profiling studies. Importantly, we warn that depletion solutions based on targeted nuclease cleavage significantly perturb the high-resolution information that can be derived from the data, and thus do not recommend their use for any applications that require precise determination of the ends of RNA fragments.

molecular biology↗

bioRxiv2020

Integrated lipidomics and proteomics network analysis highlights lipid and immunity pathways associated with Alzheimer's disease

INTRODUCTIONThere is an urgent need to understand the molecular mechanisms underlying Alzheimers Disease (AD) to enable early diagnosis and develop effective treatments. Here we aim to investigate Alzheimers dementia using an unsupervised lipid, protein and gene multi-omic integrative approach. METHODSA lipidomics dataset (185 AD, 40 MCI and 185 controls) and a proteomics dataset (201 AD patients, 104 MCI individuals and 97 controls) were utilised for weighted gene co-expression network analyses (WGCNA). An additional proteomics dataset (94 AD, 55 MCI and 100 controls) was included for external proteomics validation. Modules created within each modality were correlated with clinical AD diagnosis, brain atrophy measures and disease progression, as well as with each other. Gene Ontology (GO) enrichment analysis was employed to examine the biological processes and molecular and cellular functions for protein modules associated with AD phenotypes. Lipid species were annotated in the lipid modules associated with AD phenotypes. Associations between established AD risk loci and lipid/protein modules that showed high correlation with AD phenotypes were also explored. RESULTSFive of the 20 identified lipid modules and five of the 17 identified protein modules were correlated with AD phenotypes. Lipid modules comprising of phospholipids, triglycerides, sphingolipids and cholesterol esters, correlated with AD risk loci involved in immune response and lipid metabolism. Five protein modules involved in positive regulation of cytokine production, neutrophil mediated immunity, humoral immune responses were correlated with AD risk loci involved in immune and complement systems. DISCUSSIONWe have shown the first multi-omic study linking genes, proteins and lipids to study pathway dysregulation in AD. Results identified modules of tightly regulated lipids and proteins that were strongly associated with AD phenotypes and could be pathology drivers in lipid homeostasis and innate immunity. Research in ContextO_LILipid and protein modules were preserved amongst Alzheimers disease (AD) patients, participants with mild cognitive impairment (MCI) and controls. Protein modules were also externally validated. C_LIO_LIFive lipid and five protein modules out of a total of thirty-seven correlated with clinical AD diagnosis, brain atrophy measurements and the rate of cognitive decline in AD. C_LIO_LILipid and protein modules associated with AD phenotypes showed associations with established AD risk loci involved in lipid and immune pathways. C_LI

neuroscience↗

bioRxiv2019

The early local and systemic Type I interferon responses to ultraviolet B light exposure are cGAS dependent.

Most systemic lupus erythematosus (SLE) patients are photosensitive and ultraviolet B light (UVB) exposure worsens cutaneous disease and precipitates systemic flares of disease. The pathogenic link between skin disease and systemic exacerbations in SLE remains elusive. In an acute model of UVB-triggered inflammation, we observed that a single UV exposure triggered a striking IFN-I signature not only in the skin, but also in the blood and kidneys. The early IFN-I signature was significantly higher in female compared to male mice. The early IFN-I response in the skin was almost entirely, and in the blood partly, dependent on the presence of cGAS, as was skin inflammatory cell infiltration. Inhibition of cGAMP hydrolysis augmented the UVB-triggered IFN-I response. UVB skin exposure leads to cGAS-activation and both local and systemic IFN-I signature and could contribute to acute flares of disease in susceptible subjects such as patients with SLE.

immunology↗

bioRxiv2019

Insights from two decades of the Student Conference on Conservation Science

Conservation science is a crisis-oriented discipline focused on delivering robust answers to reducing human impacts on nature. To explore how the field might have changed during the past two decades, we analyzed 3,245 applications for oral presentations submitted to the Student Conference on Conservation Science (SCCS) in Cambridge, UK. SCCS has been running every year since 2000, aims for global representation by providing bursaries to early-career conservationists from lower-income countries, and has never had a thematic focus, beyond conservation in the broadest sense. We found that the majority of submissions to SCCS were based on primary biological data collection from local scale field studies in the tropics, contrary to established literature which highlights gaps in tropical research. Our results showed a small increase over time in submissions framed around how nature benefits people as well as a small increase in submissions integrating social science. Our findings also suggest that students and early-career conservationists could provide pathways to increased availability of data from the tropics and for addressing well-known biases in the published literature towards wealthier countries. We hope this research will motivate efforts to support student projects, ensuring data and results are published and made publicly available.

ecology↗

bioRxiv2019

Stop Codon Context Influences Genome-Wide Stimulation of Termination Codon Readthrough by Aminoglycosides

Stop codon readthrough (SCR) occurs when the ribosome miscodes at a stop codon. Such readthrough events can be therapeutically desirable when a premature termination codon (PTC) is found in a critical gene. To study SCR in vivo in a genome-wide manner, we treated mammalian cells with aminoglycosides and performed ribosome profiling. We find that in addition to stimulating readthrough of PTCs, aminoglycosides stimulate readthrough of normal termination codons (NTCs) genome-wide. Stop codon identity, the nucleotide following the stop codon, and the surrounding mRNA sequence context all influence the likelihood of SCR. In comparison to NTCs, downstream stop codons in 3'UTRs are recognized less efficiently by ribosomes, suggesting that targeting of critical stop codons for readthrough may be achievable without general disruption of translation termination. Finally, we find that G418 treatment globally alters gene expression with substantial effects on translation of histone genes, selenoprotein genes, and S-adenosylmethionine decarboxylase (AMD1).

molecular biology↗

bioRxiv2019

GIGYF2 and 4EHP Inhibit Translation Initiation of Defective Messenger RNAs to Assist Ribosome-Associated Quality Control

Ribosome-associated Quality Control (RQC) pathways protect cells from toxicity caused by incomplete protein products resulting from translation of damaged or problematic mRNAs. Extensive work in yeast has identified highly conserved mechanisms that lead to the degradation of the faulty mRNA and partially synthesized polypeptide. Here, we used CRISPR-Cas9-based screening to search for additional RQC strategies in mammals. We found that failed translation leads to specific silencing of translation initiation on that message. This negative feedback loop is mediated by two translation inhibitors, GIGYF2 and 4EHP. Their recruitment to defective messages can be mediated by different factors, including potentially the collision sensor ZNF598. Both model substrates and growth-based assays established that inhibition of additional rounds of translation acts in concert with known RQC pathways to prevent buildup of toxic proteins. Inability to block translation of faulty mRNAs, and subsequent accumulation of partially synthesized polypeptides, could explain the neurodevelopmental and neuropsychiatric disorders observed in mice and humans with compromised GIGYF2 function.

genetics↗

bioRxiv2019

Molecular mechanism of translational stalling by inhibitory codon combinations and poly(A) tracts

Inhibitory codon pairs and poly(A) tracts within the translated mRNA cause ribosome stalling and reduce protein output. The molecular mechanisms that drive these stalling events, however, are still unknown. Here, we use a combination of in vitro biochemistry, ribosome profiling, and cryo-EM to define molecular mechanisms that lead to these ribosome stalls. First, we use an in vitro reconstituted yeast translation system to demonstrate that inhibitory codon pairs slow elongation rates which are partially rescued by increased tRNA concentration or by an artificial tRNA not dependent on wobble base pairing. Ribosome profiling data extend these observations by revealing that paused ribosomes with empty A sites are enriched on these sequences. Cryo-EM structures of stalled ribosomes provide a structural explanation for the observed effects by showing decoding-incompatible conformations of mRNA in the A sites of all studied stall-inducing sequences. Interestingly, in the case of poly(A) tracts, the inhibitory conformation of the mRNA in the A site involves a nucleotide stacking array. Together, these data demonstrate novel mRNA-induced mechanisms of translational stalling in eukaryotic ribosomes.

biochemistry↗

bioRxiv2019

The endonuclease Cue2 cleaves mRNAs at stalled ribosomes during No Go Decay.

Translation of problematic sequences in mRNAs leads to ribosome collisions that trigger a sequence of quality control events including ribosome rescue, degradation of the stalled nascent polypeptide via the Ribosome-mediated Quality control Complex (RQC), and targeting of the mRNA for decay (No Go Decay or NGD). Previous studies provide strong evidence for the existence of an endonuclease involved in the process of NGD though the identity of the endonuclease and the extent to which it contributes to mRNA decay remain unknown. Using a reverse genetic screen in yeast, we identify Cue2 as the conserved endonuclease that is recruited to stalled ribosomes to promote NGD. Ribosome profiling and biochemistry provide strong evidence that Cue2 cleaves mRNA within the A site of the colliding ribosome. Finally, we show that NGD primarily proceeds via Xrn1-mediated exonucleolytic decay. Cue2-mediated endonucleolytic decay normally constitutes a secondary decay pathway, but becomes a major contributor in cells depleted of factors required for the resolution of stalled ribosome complexes (the RQT factors including Slh1). Together these results provide insights into how multiple decay processes converge to process problematic mRNAs in eukaryotic cells.\n\nOne Sentence SummaryCue2 is the endonuclease that cleaves mRNA at ribosome stall sites.

molecular biology↗