Biology subjects

Silke, J.

Publications and source records attributed to Silke, J..

8 recordsLinked to original sources

bioRxiv2026

Termination dynamics set RNAPII elongation rate and gate the response to CDK12 inactivation

The transcriptional fidelity of RNA polymerase II (RNAPII) is governed by a tight equilibrium between elongation and termination activities, a balance frequently disrupted in human diseases such as cancer. The transcriptional cyclin-dependent kinase 12 (CDK12) maintains RNAPII elongation rate and processivity throughout the gene body. Inactivation of CDK12 disrupts this homeostatic balance and causes elongation stress, slowing RNAPII and triggering premature termination at intronic polyadenylation sites (IPAs). Despite this, the precise executors of intronic premature termination under CDK12-inactivation-induced elongation stress remain poorly understood. Using genome-wide CRISPR screening combined with chemical-genetic approach, we identified a pro-termination mechanism at intronic checkpoints; upon CDK12 inactivation, SCAF4 recruits the cleavage and polyadenylation (CPA) complex through its catalytic endonuclease CPSF3 to execute premature cleavage at IPAs. Disruption of SCAF4-CPA axis prevents early termination, restores full length transcription and confers resistance to CDK12/cyclin K inhibition. Genetic loss of SCAF4 restores the RNAPII elongation rate under CDK12 inhibition, revealing that termination dynamics actively shape the rate of transcription. Supporting this model, we uncovered an anti-termination mechanism driven by KHDRBS1/SAM68, whose depletion promotes proximal termination and sensitises cells to CDK12 targeting. Our findings mechanistically couple elongation and termination activities at intronic checkpoints as joint contributors to both the processivity and elongation rate of RNAPII. This establishes termination dynamics as an active and tractable axis of the cellular response to elongation stress.

molecular biology↗

bioRxiv2026

Necroptotic Signalling Diverts Keratinocyte Fate to Promote Differentiation and Slow Wound Healing

Necroptosis is best known as a lytic, proinflammatory cell-death pathway mediated by RIPK3 and MLKL. Effective wound repair requires the rapid resolution of inflammation, and ongoing necroptotic activity would only exacerbate tissue damage, delaying healing. However, damaged skin presents a trigger-rich environment for necroptotic signalling, an apparent paradox that remains unresolved. Using genetic ablation and pharmacological inhibition across multiple wound models, we show that inhibiting necroptosis accelerates wound closure, revealing that necroptotic signalling normally restrains repair. Surprisingly, we found that MLKL activation in wild-type keratinocytes induces differentiation and membrane repair rather than cell lysis. This adaptive, non-lethal mode of necroptotic signalling preserves barrier integrity but slows re-epithelialisation. Our findings redefine epidermal necroptotic signalling as a stress-responsive program that modulates keratinocyte fate in a trigger-rich environment. Temporarily dampening this pathway may enhance regeneration after barrier loss without compromising immune defence, revealing necroptosis as a tunable mechanism balancing tissue repair and inflammation.

cell biology↗

bioRxiv2023

An immunohistochemical atlas of necroptotic pathway expression

Necroptosis is a lytic form of regulated cell death reported to contribute to inflammatory diseases of the gut, skin and lung, as well as ischemic-reperfusion injuries of the kidney, heart and brain. However, precise identification of the cells and tissues that undergo necroptotic cell death in vivo has proven challenging in the absence of robust protocols for immunohistochemical detection. Here, we provide automated immunohistochemistry protocols to detect core necroptosis regulators - Caspase-8, RIPK1, RIPK3 and MLKL - in formalin-fixed mouse and human tissues. We observed surprising heterogeneity in protein expression within tissues, whereby short-lived immune barrier cells were replete with necroptotic effectors, whereas long-lived cells lacked RIPK3 or MLKL expression. Local changes in the expression of necroptotic effectors occurred in response to insults such as inflammation, dysbiosis or immune challenge, consistent with necroptosis being dysregulated in disease contexts. These methods will facilitate the precise localisation and evaluation of necroptotic signaling in vivo. HighlightsO_LI13 automated immunohistochemistry protocols for detecting the necroptotic pathway C_LIO_LINecroptotic pathway expression is confined to fast-cycling immune barriers C_LIO_LINecroptotic pathway expression changes at sites of immunoinflammatory challenge C_LIO_LIImmunodetection of necrosomes in IBD patients is a putative new diagnostic tool C_LI

cell biology↗

bioRxiv2022

The unifying catalytic mechanism of the RING-between-RING E3 ligase family

The RING-between-RING (RBR) E3 ubiquitin ligase family in humans comprises 14 members and is defined by a two-step catalytic mechanism in which ubiquitin is first transferred from an E2 to the RBR active site and then to the substrate. To define the core features of this catalytic mechanism, we structurally and biochemically characterise the two RBRs HOIL-1 and RNF216. Crystal structures of both enzymes in their RBR/E2-Ub/Ub transthiolation complexes capturing the first catalytic step, in combination with complementary functional experiments, reveal the defining features of the RBR catalytic mechanism. RBRs catalyse ubiquitination via a conserved transthiolation complex structure that enables efficient E2-to-RBR ubiquitin transfer. Our data also highlight a conserved RBR allosteric mechanism by distinct ubiquitin linkages that suggests that RBRs employ a feed-forward mechanism accelerating their signalling pathways. We finally identify that the HOIL-1 RING2 domain contains an unusual Zn2/Cys6 bivalent zinc cluster that may represent a specific adaptation enabling HOIL-1s atypical substrate specificity.

biochemistry↗

bioRxiv2022

A common human MLKL polymorphism confers resistance to negative regulation by phosphorylation

Across the globe, 2-3% of humans carry the p.Ser132Pro single nucleotide polymorphism in MLKL, the terminal effector protein of the inflammatory form of programmed cell death, necroptosis. We show that this substitution confers a gain in necroptotic function in human cells, with more rapid accumulation of activated MLKLS132P in biological membranes and MLKLS132P overriding pharmacological and endogenous inhibition of MLKL. In mouse cells, the equivalent Mlkl S131P mutation confers a gene dosage dependent reduction in sensitivity to TNF-induced necroptosis in both hematopoietic and non-hematopoietic cells, but enhanced sensitivity to IFN-{beta} induced death in non-hematopoietic cells. In vivo, MlklS131P homozygosity reduces the capacity to clear Salmonella from major organs and retards recovery of hematopoietic stem cells. Thus, by dysregulating necroptosis, the S131P substitution impairs the return to homeostasis after systemic challenge. Present day carriers of the MLKL S132P polymorphism may be the key to understanding how MLKL and necroptosis modulate the progression of complex polygenic human disease.

immunology↗

bioRxiv2021

Oligomerization-driven MLKL ubiquitylation antagonises necroptosis

Mixed lineage kinase domain-like (MLKL) is the executioner in the caspase-independent form of programmed cell death called necroptosis. Receptor Interacting serine/threonine Protein Kinase 3 (RIPK3) phosphorylates MLKL, triggering MLKL oligomerization, membrane translocation and membrane disruption. MLKL also undergoes ubiquitylation during necroptosis, yet neither the mechanism nor significance of this event have been demonstrated. Here we show that necroptosis-specific, multi-mono-ubiquitylation of MLKL occurs following its activation and oligomerization. Ubiquitylated MLKL accumulates in a digitonin insoluble cell fraction comprising plasma/organellar membranes and protein aggregates. This ubiquitylated form is diminished by a plasma membrane located deubiquitylating enzyme. MLKL is ubiquitylated on at least 4 separate lysine residues once oligomerized, and this correlates with proteasome- and lysosome-dependent turnover. Using a MLKL-DUB fusion strategy, we show that constitutive removal of ubiquitin from MLKL licences MLKL auto-activity independent of necroptosis signalling in mouse and human cells. Therefore, besides its role in the kinetic regulation of MLKL-induced death following an exogenous necroptotic stimulus, ubiquitylation also contributes to the restraint of basal levels of activated MLKL to avoid errant cell death.

molecular biology↗

bioRxiv2021

Tankyrase-mediated ADP-ribosylation is a novel regulator of TNF-induced death

Tumor necrosis factor (TNF) is an inflammatory cytokine that, upon binding to its receptor TNFR1, can drive cytokine production, cell survival, or cell death and is a major component of an organisms anti-pathogen repetoire1,2. TNF stimulation leads to the formation of two distinct signalling complexes, a well-defined membrane bound complex (complex 1), and a less well characterised cytosolic death inducing complex (complex 2). Using mass spectrometry, we identified the ADP-ribosyltransferase, tankyrase-1 (TNKS1/TNKS/ARTD5/PARP5a) as a novel native complex 2 component. Following a TNF-induced death stimulus TNKS1 is recruited to complex 2, resulting in complex 2 poly(ADP-ribosyl)ation (PARylation). Tankyrase inhibitors sensitise cells to TNF-induced death, which is correlated with increased complex 2 assembly. Tankyrase-mediated PARylation promotes recruitment of the E3 ligase RNF146 and RNF146 deficiency or proteasome inhibition results in increased levels of complex 2, suggesting that RNF146 causes proteasomal degradation of complex 2. Several viruses express ADP-ribose binding macrodomain proteins, and expression of the SARS-CoV-2 or VEEV macrodomain markedly sensitises cells to TNF-induced death. This suggests that ADP-ribosylation serves as yet another mechanism to detect pathogenic interference of TNF signalling and retaliate with an inflammatory cell death.

cell biology↗

bioRxiv2020

A regulatory interface on RIPK2 is required for XIAP binding and NOD signaling activity

Signaling via the intracellular pathogen receptors Nucleotide-binding oligomerization domain-containing proteins NOD1 and NOD2 requires Receptor Interacting Kinase 2 (RIPK2), an adaptor kinase that can be targeted for the treatment of various inflammatory diseases. However, the molecular mechanisms of how RIPK2 contributes to NOD signaling are not completely understood. We generated FLAG-tagged RIPK2 knock-in mice using CRISPR/Cas9 technology to study NOD signaling mechanisms at the endogenous level. Using cells from these mice we were able to generate a detailed map of post-translational modifications on RIPK2 during NOD signaling and we identified a new regulatory interface on RIPK2, which dictates the crucial interaction with the E3 ligase XIAP.

cell biology↗