Biology subjects

Burke, J.

Publications and source records attributed to Burke, J..

At least 19 recordsLinked to original sources

bioRxiv2026

Differential assembly of RNP granules via activation of distinct dsRNA sensors by adenovirus mutants

Recognition of dsRNA triggers antiviral defense mediated by PKR and OAS3/RNase L pathways through translational arrest and RNA decay. This is accompanied by assembly of distinct cytoplasmic ribonucleoprotein (RNP) condensates termed stress granules (SGs) and RNase L-dependent bodies (RLBs). Here we show that adenovirus infection differentially modulates dsRNA sensors and RNP granule assembly. Infection with splicing-defective {Delta}E4 mutant leads to dsRNA accumulation and activation of both PKR and OAS3/RNase L, promoting formation of RLB-like granules. In contrast, mutants lacking virus-associated (VA) RNAs trigger PKR activation and assembly of SGs despite absence of detectable dsRNA. Proteomic analysis revealed distinct protein compositions of canonical SGs and RLBs, which were reflected in virus-induced granules. While {Delta}VA-induced granules were PKR-dependent, {Delta}E4 mutants induced RLB-like granules independently of PKR and RNase L. In these cells, granule assembly coincided with translational arrest independent of eIF2 phosphorylation, indicating additional pathways linking nuclear dsRNA sensing to translational control and RNP granule assembly during viral infection. These findings provide novel insights into how distinct dsRNA sensors modulate translation and RNP condensates in response to stress.

cell biology↗

bioRxiv2025

SARS-CoV-2 and MERS-CoV disrupt host protein synthesis via nsp1 with differential effects on the integrated stress response

Coronaviruses pose a serious threat to public health, driving the need for antiviral therapeutics and vaccines. Therefore, it is paramount to understand how this family of viruses evades cellular antiviral responses and establishes productive infection. The conserved coronavirus non-structural protein (nsp)1 has been shown to inhibit host protein synthesis and promote host mRNA degradation while viral mRNAs are protected. We showed previously that SARS-CoV-2 induces activation of host integrated stress response (ISR) kinases PKR and PERK, which promote phosphorylation of eIF2 and consequent inhibition of host protein synthesis. In contrast, eIF2 remains unphosphorylated during MERS-CoV infection. To investigate the interactions of nsp1 and the ISR kinases, we utilized recombinant SARS-CoV-2 and MERS-CoV expressing nsp1 with mutations in each of two conserved domains. Upon infection with SARS-CoV-2 nsp1 mutants, translation was shut down in wildtype (WT) and PKR knockout (KO) cells but rescued in PERK KO cells, likely due to reduced p-eIF2. In contrast, translation was rescued during infection with the analogous MERS-CoV nsp1 mutants even in WT cells. Moreover, SARS-CoV-2 WT suppressed expression of GADD34, a negative regulator of eIF2 phosphorylation, while SARS-CoV-2 nsp1 mutants induced GADD34. In contrast MERS-CoV WT induced GADD34. Utilizing single-molecule fluorescence in situ hybridization, we found that SARS-CoV-2 and MERS-CoV nsp1 promote host mRNA degradation during WT, but not nsp1 mutant, infection. Finally, while SARS-CoV-2 WT suppressed stress granule formation, nsp1 mutants induced stress granules containing host RNA. Thus, SARS-CoV-2 and MERS-CoV differ in interactions with the ISR and nsp1 control of host protein synthesis. SignificanceCoronaviruses cause disease across a wide range of animal species, and the human coronaviruses SARS-CoV-2 and MERS-CoV have caused epidemics of severe respiratory illness. Thus, it is imperative to understand how these viruses antagonize host responses and cause lethal disease. We show here that the betacoronavirus non-structural protein (nsp)1 promotes shutdown of host protein synthesis while preserving viral protein synthesis and, in addition, promotes degradation of host mRNAs. However, SARS-CoV-2 and MERS-CoV differ in their ability to manipulate the host integrated stress response, indicating that it is important to understand detailed coronavirus-host interactions and how they differ even between lethal coronaviruses. Such insights will inform the development of antiviral therapeutics to treat and prevent current and future coronavirus outbreaks.

microbiology↗

bioRxiv2025

Mutant p53 binds RNA to drive mitochondrial dysfunction

Tumor suppressor protein 53 (p53) is a transcription factor that is deregulated in 50% of cancers. Often termed the guardian of the genome, p53 is responsible for maintenance of genomic stability, cell cycle arrest, DNA repair, senescence, and apoptosis. In cancer cells, deregulation of p53 often occurs through mutations in the DNA binding domain which lead to a loss of the transcriptional activity. While 100s of somatic mutations in the DNA binding domain are known, a small number of mutants are enriched in cancer, suggesting a gain-of-function role. Here we deploy an intein-based approach to localize {micro}Map photoproximity labeling to p53 to define novel interactions contributing to the loss and gain of function roles of 5 separate hotspot mutants. These data revealed that G245S and R273H binds to RNA through its C-terminal domain. We show through CLIP experiments that mutant p53 has an RNA binding motif that conserved across mutants and is enriched in 3UTRs, promoting ribosomal localization and labeling of proteins at the mitochondrial surface. We further demonstrate that the RNA binding ability of mutant p53 promotes altered miRNA processing and mitochondrial dysfunction providing mechanistic rationale for historically reported but poorly understood phenotypes.

molecular biology↗

bioRxiv2025

A simple circuit to sustain intact tumor microenvironments for complex drug interrogations

Deep learning and large language models can integrate complex datasets to uncover biological insights that are often undetectable through conventional analyses. With application to translational cancer research, these computational tools have positioned 3D patient-derived tumor avatars front and center as crucial data input sources. However, a major challenge remains: the lack of standardization in media composition in 3D patient-derived tumor models unpredictably affects cell behavior and limit the utility beyond predicting treatment responses. To address this unmet need, we developed a simple, reproducible perfusion circuit system to approximate in vivo physiology using autologous patient plasma. With peritoneal metastases and core needle biopsies across multiple tumor histologies, we demonstrate preservation of the tumor microenvironment for up to 48 hours using multi-modal interrogation techniques. With proof-of-concept experiments, we display the systems ability to unveil complex drug-dependent biology within this time window. Standardizable, physiologically relevant platforms for 3D patient-derived tumor avatars will yield unprecedented insights through the integration of data from broad groups of patients and the use of an expanding armamentarium of artificial intelligence capabilities.

cancer biology↗

bioRxiv2025

Macrocyclic Peptides Containing an Imidazopyridinium (IP+) Unit Display Enhanced Passive Cell Permeability

Macrocyclic peptides (MPs) have emerged as interesting therapeutic candidates due to their ability to engage difficult protein targets with high affinity and selectivity. However, their application to intracellular targets is limited by the poor passive membrane permeability of most MPs. We previously showed that incorporation of an imidazopyridinium (IP+) moiety into an MP significantly boosted passive membrane permeability, as measured by the parallel artificial membrane permeability assay (PAMPA)(Li, et al. (2024) J. Amer. Chem. Soc. 146, 14633-14644). In this study we report a detailed analysis of the entry of IP+-containing MPs into living cells. Chloroalkane penetration assay (CAPA) data show that IP+ MPs access the cytoplasm rapidly, often at rates approaching those of drug-like small molecules. Mechanistic studies, including live-cell imaging, ATP-depletion experiments, and organelle colocalization analyses, indicate that IP+ MPs traverse the plasma membrane primarily via passive diffusion, avoiding endosomal entrapment. IP+ MPs do not localize to mitochondria, as is the case for many positively charged molecules. We show that incorporation of an IP+ unit transforms a previously described membrane impermeable macrocyclic antagonist of the p53-MDM2 interaction into a bioactive inhibitor of MCF-7 proliferation. Collectively, these results establish that IP+ incorporation is an effective strategy for the development of bioactive MPs targeting intracellular proteins.

biochemistry↗

bioRxiv2025

RNase L regulates the antiviral proteome by accelerating mRNA1decay, inhibiting nuclear mRNA export, and repressing RNAPII-2mediated transcription

Ribonuclease L (RNase L) is an antiviral endoribonuclease that triggers widespread degradation of cellular mRNAs. Here, we show that the degradation of cellular mRNA by RNase L is a conserved response to flaviviruses, including Zika virus (ZIKV), dengue virus serotype 2 (DENV-2), and West Nile virus (WNV). Quantitative mass spectrometry in response to dsRNA or ZIKV infection shows that RNase L broadly downregulates the cellular proteome, reducing proteins with short half-lives involved in cell cycle progression, cellular metabolism, and protein synthesis. The mRNAs encoded by interferon-stimulated genes (ISGs) evade mRNA decay by RNase L, allowing for protein synthesis of ISG-encoding mRNAs. However, RNase L dampens ISG protein synthesis by triggering a block in nuclear mRNA export and repressing RNAPII-mediated transcription at later times during the antiviral response. These findings implicate reprograming of the cellular proteome as primary means by which RNase L combats viral infection, tumorigenesis, and immune dysregulation. HIGHLIGHTSO_LIRNase L initiates widespread decay of cellular mRNA in response to flaviviruses C_LIO_LIRNase L-mediated mRNA decay broadly downregulates the homoeostatic proteome C_LIO_LIAntiviral mRNAs are translated due to their ability to evade RNase L-mediated decay C_LIO_LIRNase L reduces antiviral proteins by inhibiting mRNA export and RNAPII-mediated transcription C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=194 SRC="FIGDIR/small/665572v1_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@13c4940org.highwire.dtl.DTLVardef@ed1bd9org.highwire.dtl.DTLVardef@bf5057org.highwire.dtl.DTLVardef@33bca9_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2025

Assessing the Suitability of Deubiquitylases As Substrates For Targeted Protein Degradation

The development of selective inhibitors of Deubiquitylase enzymes (DUBs) is difficult due to a high level of homology in the active sites of the {approx} 100 such enzymes in the human proteome. A potential way to achieve this in a more facile manner would be to develop PROTACs or molecular glues that engage the target DUB in a less conserved region outside of the catalytic domain. However, this raises the concern that auto-deubiquitylation would make DUBs poor substrates for this modality. Here we describe a chemical genetics system to evaluate this issue. We find that some DUBs are readily degradable via the Ubiquitin-proteasome pathway and some are not. Of the latter category, some resist turnover through auto-deubiquitylation and some are simply poor proteasome substrates. SignificanceDeubiquitylases (DUBs) are a family of specialized proteases that hydrolyze the isopeptide bond between a lysine and the C-terminal carboxylate of Ubiquitin. DUBs are involved in a myriad of cellular processes and many are attractive drug targets. However, it is difficult to develop selective orthosteric inhibitors due to the high degree of homology between DUB active sites. Targeted protein degradation using a proteolysis-targeting chimera (PROTAC) that recognizes the DUB in a less conserved region outside of the catalytic domain constitutes an attractive alternative strategy for selectively inhibiting a given DUB. Such ligands may not block the catalytic activity of the enzyme, raising the concern that auto-deubiquitylation will make DUBs inherently poor substrates for PROTACs of this type. Since drug-like ligands that engage DUBs outside of the active site are extremely rare, this issue is difficult to address in a straightforward fashion. In this study we establish a generally applicable chemical genetics workflow to evaluate the degradability of DUBs by a PROTAC. The data indicate that some DUBs are readily degradable and some are not. In particular, USP11, an attractive drug target in various cancers and Alzheimers disease, is shown to be rapidly degradable, while its paralogs, USP4 and USP15 resist degradation through auto-deubiquitylation.

biochemistry↗

bioRxiv2024

The Zymoseptoria tritici effector Zt-11 contributes to aggressiveness in wheat

Zymoseptoria tritici is an ascomycete fungus and the causal agent of Septoria tritici leaf blotch (STB) in wheat. Z. tritici secretes an array of effector proteins that are likely to facilitate host infection, colonisation and pycnidia production. In this study we demonstrate a role for Zt-11 as a Z. tritici effector during disease progression. Zt-11 is upregulated during the transition of the pathogen from the biotrophic to necrotrophic phase of wheat infection. Deletion of Zt-11 delayed disease development in wheat, reducing the number and size of pycnidia, as well as the number of macropycnidiospores produced by Z. tritici. This delayed disease development by the {Delta}Zt-11 mutants was accompanied by a lower induction of PR genes in wheat, when compared to infection with wildtype Z. tritici. Overall, these data suggest that Zt-11 plays a role in Z. tritici aggressiveness and STB disease progression possibly via a salicylic acid associated pathway.

plant biology↗

bioRxiv2024

RNase L-induced bodies sequester subgenomic flavivirus RNAs and re-establish host RNA decay

Subgenomic flavivirus RNAs (sfRNAs) are structured RNA elements encoded in the 3-UTR of flaviviruses that promote viral infection by inhibiting cellular RNA decay machinery. Herein, we analyze the production of sfRNAs using single-molecule RNA fluorescence in situ hybridization (smRNA-FISH) and super-resolution microscopy during West Nile virus, Zika virus, or Dengue virus serotype 2 infection. We show that sfRNAs are initially localized diffusely in the cytosol or in processing bodies (P-bodies). However, upon activation of the host antiviral endoribonuclease, Ribonuclease L (RNase L), nearly all sfRNAs re-localize to antiviral biological condensates known as RNase L-induced bodies (RLBs). RLB-mediated sequestration of sfRNAs reduces sfRNA association with RNA decay machinery in P-bodies, which coincides with increased viral RNA decay. These findings establish a role of RLBs in promoting viral RNA decay, demonstrating the complex host-pathogen interactions at the level of RNA decay and biological condensation. HighlightsO_LISingle-molecule imaging of sfRNA production and localization C_LIO_LIsfRNAs localize to RNase L-induced bodies C_LIO_LIRNase L-induced bodies sequester sfRNAs away from P-bodies C_LIO_LISequestration of sfRNAs by RNase L-induced bodies enhances decay of viral genomes C_LI

cell biology↗

bioRxiv2023

Hardwired to attack: Transcriptionally defined amygdala subpopulations play distinct roles in innate social behaviors.

Social behaviors are innate and supported by dedicated neural circuits, but it remains unclear whether these circuits are developmentally hardwired or established through social experience. Here, we revealed distinct response patterns and functions in social behavior of medial amygdala (MeA) cells originating from two embryonically parcellated developmental lineages. MeA cells in male mice that express the transcription factor Foxp2 (MeAFoxp2) are specialized for processing male conspecific cues even before puberty and are essential for adult inter-male aggression. In contrast, MeA cells derived from the Dbx1-lineage (MeADbx1) respond broadly to social cues and are non-essential for male aggression. Furthermore, MeAFoxp2 and MeADbx1 cells show differential anatomical and functional connectivity. Altogether, our results support a developmentally hardwired aggression circuit at the level of the MeA and we propose a lineage-based circuit organization by which a cells embryonic transcription factor profile determines its social information representation and behavior relevance during adulthood. HighlightsO_LIMeAFoxp2 cells in male mice show highly specific responses to male conspecific cues and during attack while MeADbx1 cells are broadly tuned to social cues. C_LIO_LIThe male-specific response of MeAFoxp2 cells is present in naive adult males and adult social experience refines the response by increasing its trial-to-trial reliability and temporal precision. C_LIO_LIMeAFoxp2 cells show biased response to males even before puberty. C_LIO_LIActivation of MeAFoxp2, but not MeADbx1, cells promote inter-male aggression in naive male mice. C_LIO_LIInactivation of MeAFoxp2, but not MeADbx1, cells suppresses inter-male aggression. C_LIO_LIMeAFoxp2 and MeADbx1 cells show differential connectivity at both the input and output levels. C_LI

neuroscience↗

bioRxiv2022

Diverse RNA viruses associated with diatom, eustigmatophyte, dinoflagellate and rhodophyte microalgae cultures

Unicellular microalgae are of immense ecological importance with growing commercial potential in industries such as renewable energy, food and pharmacology. Viral infections can have a profound impact on the growth and evolution of their hosts. However, very little is known of the diversity within, and effect of, unicellular microalgal RNA viruses. In addition, identifying RNA viruses in these organisms that could have originated more than a billion years ago constitutes a robust data set to dissect molecular events and address fundamental questions on virus evolution. We assessed the diversity of RNA viruses in eight microalgal cultures including representatives from the diatom, eustigmatophyte, dinoflagellate, red algae and euglenid groups. Using metatranscriptomic sequencing combined with bioinformatic approaches optimised to detect highly divergent RNA viruses, we identified ten RNA virus sequences, with nine constituting new viral species. Most of the newly identified RNA viruses belonged to the double-stranded Totiviridae, Endornaviridae and Partitiviridae, greatly expanding the reported host range for these families. Two new species belonging to the single-stranded RNA viral clade Marnaviridae, commonly associated with microalgal hosts, were also identified. This study highlights that a great diversity of RNA viruses likely exists undetected within the unicellular microalgae. It also highlights the necessity for RNA viral characterisation and to investigate the effects of viral infections on microalgal physiology, biology and growth, considering their environmental and industrial roles. ImportanceIn comparison to animals or plants, our knowledge of the diversity of RNA viruses infecting microbial algae - the microalgae - is minimal. Yet describing the RNA viruses infecting these organisms is of primary importance at both the ecological and economical levels because of the fundamental roles these organisms play in aquatic environments and their growing value across a range of industrial fields. Using metatranscriptomic sequencing we aimed to reveal the RNA viruses present in cultures of eight microalgae species belonging to the diatom, dinoflagellate, eustigmatophyte, rhodophyte and euglena major clades of algae. This work identified ten new divergent RNA virus species, belonging to RNA virus families as diverse as the double-stranded Totiviridae, Endornaviridae, Partitiviridae and the single-stranded Marnaviridae. By expanding the known diversity of RNA viruses infecting unicellular eukaryotes, this study contributes to a better understanding of the early evolution of the virosphere and will inform the use of microalgae in industrial applications.

microbiology↗

bioRxiv2020

Phylogeny of docks and sorrels (Rumex, Polygonaceae) reveals plasticity of reproductive systems

The genus Rumex is a unique member of the Polygonaceae (Buckwheat) family of plants. A source of intrigue for Rumex lies in the diversity of the reproductive systems associated with the subgenera, species, and subspecies within this genus. Four previously circumscribed subgenera, some 200 species, and a number of subspecies comprise the collective Rumex genus. These species exhibit monoecious, dioecious, synoecious (hermaphroditic), and polygamous reproductive systems. Moreover, some of the dioecious species contain sex chromosomes, a phenomenon that is very rare in angiosperms. Apart from the confirmed morphological and phytogeographical distinctions, two of the four described subgenera, Acetosa and Acetosella, are distinctive in their exhibited sex chromosome systems. For this study, we used three chloroplast markers, rbcL, trnH-psbA, trnL-F, and dense taxon sampling, to reconstruct a molecular phylogeny for Rumex.The reconstructed phylogeny for this work resolves six major clades and one large grade in Rumex. In addition, the species with known dioecious reproductive systems derived from unique sex chromosome systems are resolved in two different clades nested within "the dioecious clade". These results suggest that the species with divergent sexual systems are more closely related to each other than to other species comprising the rest of the Rumex genus. Furthermore, some species with known synoecious reproductive systems are resolved in a single clade which is also nested within "the dioecious clade". These results imply a possible reversal occurring over time which suggests the highly plastic nature of reproductive systems among Rumex species.

plant biology↗

bioRxiv2020

Subcutaneous nanotherapy repurposes the immunosuppressive mechanism of rapamycin to enhance allogeneic islet graft viability

Oral rapamycin administration rapamycin is plagued by poor bioavailability and wide biodistribution. Thus, this pleotropic mTOR inhibitor has a narrow therapeutic window, numerous side effects and provides inadequate transplantation protection. Parental formulation was not possible due to rapamycins hydrophobicity (log P 4.3). Here, we demonstrate that subcutaneous rapamycin delivery via poly(ethylene glycol)-b-poly(propylene sulfide)(PEG-b-PPS) polymersome (PS) nanocarriers modulates cellular biodistribution of rapamycin to change its immunosuppressive mechanism for enhanced efficacy while minimizing side effects. While oral rapamycin inhibits naive T cell proliferation directly, subcutaneously administered rapamycin-loaded polymersomes (rPS) instead modulated Ly-6Clow monocytes and tolerogenic semi-mature dendritic cells, with immunosuppression mediated by CD8+ Tregs and rare CD4+ CD8+ double-positive T cells. As PEG-b-PPS PS are uniquely non-inflammatory, background immunostimulation from the vehicle was avoided, allowing immunomodulation to be primarily attributed to rapamycins cellular biodistribution. Repurposing mTOR inhibition significantly improved maintenance of normoglycemia in a clinically relevant, MHC-mismatched, allogeneic, intraportal (liver) islet transplantation model. These results demonstrate the ability of engineered nanocarriers to repurpose drugs for alternate routes of administration by rationally controlling cellular biodistribution.

bioengineering↗

bioRxiv2019

Fertility of Pedicellate Spikelets in Sorghum is Controlled by a Jasmonic Acid Regulatory Module

As in other cereal crops, the panicles of sorghum (Sorghum bicolor (L.) Moench) comprise two types of floral spikelets (grass flowers). Only sessile spikelets (SSs) are capable of producing viable grains, whereas pedicellate spikelets (PSs) cease development after initiation and eventually abort. Consequently, grain number per panicle (GNP) is lower than the total number of flowers produced per panicle. The mechanism underlying this differential fertility is not well understood. To investigate this issue, we isolated a series of EMS-induced multiseeded (msd) mutants that result in full spikelet fertility, effectively doubling GNP. Previously, we showed that MSD1 is a TCP (Teosinte branched/Cycloidea/PCF) transcription factor that regulates jasmonic acid (JA) biosynthesis, and ultimately floral sex organ development. Here, we show that MSD2 encodes a lipoxygenase (LOX) that catalyzes the first committed step of JA biosynthesis. Further, we demonstrate that MSD1 binds to the promoters of MSD2 and other JA pathway genes. Together, these results show that a JA-induced module regulates sorghum panicle development and spikelet fertility. The findings advance our understanding of inflorescence development and could lead to new strategies for increasing GNP and grain yield in sorghum and other cereal crops.\n\nSignificanceThrough a single base pair mutation, grain number can be increased by ~200% in the globally important crop Sorghum bicolor. This mutation affects the expression of an enzyme, MSD2, that catalyzes the jasmonic acid pathway in developing floral meristems. The global gene expression profile in this enzymatic mutant is similar to that of a transcription factor mutant, msd1, indicating that disturbing any component of this regulatory module disrupts a positive feedback loop that occurs normally due to regular developmental perception of jasmonic acid. Additionally, the MSD1 transcription factor is able to regulate MSD2 in addition to other jasmonic acid pathway genes, suggesting that it is a primary transcriptional regulator of this hormone signaling pathway in floral meristems.

plant biology↗

bioRxiv2018

Norovirus infection results in assembly of virus-specific G3BP1 granules and evasion of eIF2α signaling.

During viral infection, the accumulation of RNA replication intermediates or viral proteins imposes major stress on the host cell. In response, cellular stress pathways can rapidly impose defence mechanisms by shutting off the protein synthesis machinery, which viruses depend on, and triggering the accumulation of mRNAs into stress granules to limit the use of energy and nutrients. Because this threatens viral gene expression, viruses need to evade these pathways to propagate. Human norovirus is responsible for gastroenteritis outbreaks worldwide. Previously we showed that murine norovirus (MNV) regulates the activity of eukaryotic initiation factors (eIFs). Here we examined how MNV interacts with the eIF2a signaling axis controlling translation and stress granules accumulation. We show that while MNV infection represses host cell translation, it results in the assembly of virus-specific granules rather than stress granules. Further mechanistic analyses revealed that eIF2a signaling is uncoupled from translational stalling. Moreover the interaction of the RNA-binding protein G3BP1 with viral factors together with a redistribution of its cellular interacting partners could explain norovirus evasion of stress granules assembly. These results identify novel strategies by which norovirus ensure efficient replication propagation by manipulating the host stress response.

microbiology↗

bioRxiv2018

Chromatin rewiring mediates programmed evolvability via aneuploidy

Eukaryotes have evolved elaborate mechanisms to ensure that chromosomes segregate with high fidelity during mitosis and meiosis1, and yet specific aneuploidies can be adaptive during environmental stress2,3. Here, we identify a chromatin-based system for inducible aneuploidy in a human pathogen. Candida albicans utilizes chromosome missegregation to acquire resistance to antifungal drugs4,5 and for ploidy reduction after mating6. We discovered that the ancestor of C. albicans and two related pathogens evolved a variant of histone H2A that lacks the conserved phosphorylation site for Bub1 kinase7, a key regulator of chromosome segregation1. Expression of this variant controls the rates of aneuploidy and antibiotic resistance in this species. Moreover, CENP-A/Cse4, the histone H3 that specifies centromeres, is depleted from tetraploid mating products and virtually eliminated from cells exposed to aneuploidy-promoting cues. Thus, changes in chromatin regulation can confer the capacity for rapid evolution in eukaryotes.

genetics↗

bioRxiv2017

Spliceosome profiling visualizes the operations of a dynamic RNP in vivo at nucleotide resolution

Tools to understand how the spliceosome functions in vivo have lagged behind advances in its structural biology. We describe methods to globally profile spliceosome-bound precursor, intermediates and products at nucleotide resolution. We apply these tools to three divergent yeast species that span 600 million years of evolution. The sensitivity of the approach enables detection of novel cases of non-canonical catalysis including interrupted, recursive and nested splicing. Employing statistical modeling to understand the quantitative relationships between RNA features and the data, we uncover independent roles for intron size, position and number in substrate progression through the two catalytic stages. These include species-specific inputs suggestive of spliceosome-transcriptome coevolution. Further investigations reveal ATP-dependent discard of numerous endogenous substrates at both the precursor and lariat-intermediate stages and connect discard to intron retention, a form of splicing regulation. Spliceosome profiling is a quantitative, generalizable global technology to investigate an RNP central to eukaryotic gene expression.\n\nHighlightsO_LIMeasurement of spliceosome-bound precursor and intermediate in three species\nC_LIO_LINon-canonical splicing events revealed\nC_LIO_LIStatistical modeling uncovers substrate features that predict catalytic efficiency\nC_LIO_LIDiscard of suboptimal substrates occurs in vivo and predicts intron-retained mRNAs\nC_LI

molecular biology↗

bioRxiv2017

Polymerase pausing induced by sequence-specific RNA binding protein drives heterochromatin assembly

Packaging of pericentromeric DNA into heterochromatin is crucial for genome stability, development and health, yet its endogenous triggers remain poorly understood1. A defining feature of pericentromeric heterochromatin is histone H3 lysine 9 methylation (H3K9me)2-4. In S. pombe, transcripts derived from the pericentromeric dg and dh repeat during S phase5-7 promote heterochromatin formation through two pathways: an RNAi-dependent mechanism involving recruitment of the Clr4 H3K9 methyltransferase complex (CLR-C) via the RITS complex8-13, and RNAi-independent mechanism involving an RNAPII-associated RNA-binding protein Seb1, the repressor complex SHREC, and RNA processing activities14-19. We show here that Seb1 promotes long-lived RNAPII pausing. Pause sites associated with sequence-specific Seb1 RNA binding events are significantly enriched in pericentromeric repeat regions and their presence correlates with the heterochromatin-triggering activities of the corresponding dg and dh DNA fragments. Remarkably, globally increasing RNAPII stalling by other means induces the formation of novel large ectopic heterochromatin domains. Such ectopic heterochromatin occurs even in cells lacking functional RITS, demonstrating that RNAPII pausing can be sufficient to trigger de novo heterochromatin independently of RNAi. These results uncover Seb1-mediated polymerase stalling as a new signal for nucleating heterochromatin assembly in repetitive DNA.

molecular biology↗