Biology subjects

Tibble, R. W.

Publications and source records attributed to Tibble, R. W..

5 recordsLinked to original sources

bioRxiv2026

Kaposi sarcoma herpesvirus vFLIP induces lipid droplet formation in Tert-immortalized microvascular endothelial cells via NF-κB activation

Kaposi sarcoma herpesvirus (KSHV) is the etiologic agent of Kaposi sarcoma (KS) and preferentially establishes a latent infection state in both the characteristic spindle cells of the KS tumor and cultured endothelial cells. KSHV has been described to broadly alter cellular metabolism during latent infection of endothelial cells. In Tert-immortalized microvascular endothelial (TIME) cells, our lab has shown that latent infection increases the abundance of long chain fatty acids in the cell, as well as formation of lipid droplets, which is dependent on viral gene expression. Here, we show that acyl-CoA synthetase activity, crucial for lipid droplet formation, is essential for cell survival during latent infection of TIME cells. Expression of the major genetic locus expressed during KSHV latency is sufficient to induce lipid droplet formation. Virus mutants lacking expression of the latent viral gene vFLIP, or the expression of the latent viral microRNAs, do not induce lipid droplet formation. Expression of vFLIP in isolation is sufficient to induce lipid droplet formation and sensitivity to ACSL inhibition, but a mutant version of vFLIP with decreased NF-{kappa}B signaling capacity is unable to increase lipid droplet formation to the same degree as its wild-type counterpart. Lipid droplet formation is also limited in the context of co-expression with a mutant version of I{kappa}B resistant to degradation. We conclude that vFLIP drives the increase in lipid droplet formation observed during latent KSHV infection, in an NF-{kappa}B signaling dependent fashion and that ACSL function is important for the survival of latently infected endothelial cells.

microbiology↗

bioRxiv2021

Structure of the poxvirus decapping enzyme D9 reveals its mechanism of cap recognition and catalysis

Poxviruses encode decapping enzymes that remove the protective 5 cap from both host and viral mRNAs to commit transcripts for decay by the cellular exonuclease Xrn1. Decapping by these enzymes is critical for poxvirus pathogenicity by means of simultaneously suppressing host protein synthesis and limiting the accumulation of viral dsRNA, a trigger for antiviral responses. Here we present the first high resolution structural view of the vaccinia virus decapping enzyme D9. This Nudix enzyme contains a novel domain organization in which a three-helix bundle is inserted into the catalytic Nudix domain. The 5 mRNA cap is positioned in a bipartite active site at the interface of the two domains. Specificity for the methylated guanosine cap is achieved by stacking between conserved aromatic residues in a manner similar to that observed in canonical cap binding proteins VP39, eIF4E, and CBP20 and distinct from eukaryotic decapping enzyme Dcp2.

biophysics↗

bioRxiv2019

Pat1 activates late steps in mRNA decay by multiple mechanisms

Pat1 is a hub for mRNA metabolism, acting in pre-mRNA splicing, translation repression and mRNA decay. A critical step in all 5-3 mRNA decay pathways is removal of the 5 cap structure, which precedes and permits digestion of the RNA body by conserved exonucleases. During bulk 5-3 decay, the Pat1/Lsm1-7 complex engages mRNA at the 3 end and promotes hydrolysis of the cap structure by Dcp1/Dcp2 at the 5 end through an unknown mechanism. We reconstitute Pat1 with 5 and 3 decay factors and show how it activates multiple steps in late mRNA decay. First, we find that Pat1 stabilizes binding of the Lsm1-7 complex to RNA using two conserved short-linear interaction motifs. Secondly, Pat1 directly activates decapping by binding elements in the disordered C-terminal extension of Dcp2, alleviating autoinhibition and promoting substrate binding. Our results uncover the molecular mechanism of how separate domains of Pat1 coordinate the assembly and activation of a decapping mRNP that promotes 5-3 mRNA degradation.

biochemistry↗

bioRxiv2018

Disorganization of the histone core promotes organization of heterochromatin into phase-separated droplets

The heterochromatin protein HP1 is proposed to enable chromatin compaction via liquid droplet formation. Yet, a connection between phase separation and chromatin compaction has not been experimentally demonstrated. More fundamentally, how HP1 action at the level of a single nucleosome drives chromatin compaction remains poorly understood. Here we directly demonstrate that the S. pombe HP1 protein, Swi6, compacts arrays of multiple nucleosomes into phase-separated droplets. Using hydrogen-deuterium exchange, NMR, and mass-spectrometry, we further find that Swi6 substantially increases the accessibility and dynamics of buried histone residues within a mononucleosome. Restraining these dynamics via site-specific disulfide bonds impairs the compaction of nucleosome arrays into phase-separated droplets. Our results indicate that chromatin compaction and phase separation can be highly coupled processes. Further, we find that such coupling is promoted by a counter-intuitive function of Swi6, namely disorganization of the octamer core. Phase separation is canonically mediated by weak and dynamic multivalent interactions. We propose that dynamic exposure of buried histone residues increases opportunities for multivalent interactions between nucleosomes, thereby coupling chromatin compaction to phase separation. We anticipate that this new model for chromatin organization may more generally explain the formation of highly compacted chromatin assemblies beyond heterochromatin.

biophysics↗

bioRxiv2018

Control of mRNA decapping by autoinhibition

5 mediated cytoplasmic RNA decay is a conserved cellular process in eukaryotes. While the functions of the structured core domains in this pathway are understood, the role of abundant intrinsically disordered regions (IDRs) is lacking. Here we reconstitute the Dcp1:Dcp2 complex containing a portion of the disordered C-terminus and show its activity is autoinhibited by linear interaction motifs. Enhancers of decapping (Edc) 1 and 3 cooperate to activate decapping by different mechanisms: Edc3 alleviates auto-inhibition by binding IDRs and destabilizing an inactive form of the enzyme, whereas Edc1 stabilizes the transition state for catalysis. Both activators are required to fully stimulate an autoinhibited Dcp1:Dcp2 as Edc1 alone cannot overcome the decrease in activity attributed to the C-terminal extension. Our data provide a mechanistic framework for combinatorial control of decapping by protein cofactors, a principle that is likely conserved in multiple 5 mRNA decay pathways.

biochemistry↗