Biology subjects

Meyers, R.

Publications and source records attributed to Meyers, R..

4 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↗

bioRxiv2023

Interaction between host G3BP and viral nucleocapsid protein regulates SARS-CoV-2 replication

G3BP1/2 are paralogous proteins that promote stress granule formation in response to cellular stresses, including viral infection. G3BP1/2 are prominent interactors of the nucleocapsid (N) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, the functional consequences of the G3BP1-N interaction in the context of viral infection remain unclear. Here we used structural and biochemical analyses to define the residues required for G3BP1-N interaction, followed by structure-guided mutagenesis of G3BP1 and N to selectively and reciprocally disrupt their interaction. We found that mutation of F17 within the N protein led to selective loss of interaction with G3BP1 and consequent failure of the N protein to disrupt stress granule assembly. Introduction of SARS-CoV-2 bearing an F17A mutation resulted in a significant decrease in viral replication and pathogenesis in vivo, indicating that the G3BP1-N interaction promotes infection by suppressing the ability of G3BP1 to form stress granules.

cell biology↗

bioRxiv2023

Identification of small molecule inhibitors of G3BP-driven stress granule formation

Stress granule formation is triggered by the release of mRNAs from polysomes and is promoted by the action of the paralogs G3BP1 and G3BP2. G3BP1/2 proteins bind mRNAs and thereby promote the condensation of mRNPs into stress granules. Stress granules have been implicated in several disease states, including cancer and neurodegeneration. Consequently, compounds that limit stress granule formation or promote their dissolution have potential as both experimental tools and novel therapeutics. Herein, we describe two small molecules, referred to as G3BP inhibitor a and b (G3Ia and G3Ib), designed to bind to a specific pocket in G3BP1/2 that is known to be targeted by viral inhibitors of G3BP1/2 function. In addition to disrupting co-condensation of RNA, G3BP1, and caprin 1 in vitro, these compounds inhibit stress granule formation in cells treated prior to or concurrent with stress, and dissolve pre-existing stress granules when added to cells after stress granule formation. These effects are consistent across multiple cell types and a variety of initiating stressors. Thus, these compounds represent ideal tools to probe the biology of stress granules and hold promise for therapeutic interventions designed to modulate stress granule formation.

cell biology↗

bioRxiv2022

Combination of Volasertib and Rapamycin Inhibits the Regrowth of TSC2-Deficient Tumors

Mutations in TSC1 and TSC2 lead to hyperactivation of mTORC1 and cause Tuberous Sclerosis Complex (TSC) and pulmonary Lymphangioleiomyomatosis (LAM). Rapamycin and rapalogs are potent inhibitors of mTORC1 activity and are approved for the treatment of TSC and LAM. Nevertheless, rapalogs do not cause tumor cell death, and cessation of therapy leads to tumor regrowth. Polo-like kinase 1 (PLK1) interacts with and phosphorylates TSC1, and PLK1 inhibition induces apoptosis and attenuates autophagy in TSC1/TSC2-deficient cells. Here we report that the PLK1 inhibitor volasertib decreases the viability and survival and induces apoptosis in 621-101 cells, a TSC-deficient renal angiomyolipoma cell line from a LAM patient. Combined with rapamycin, volasertib further decreased the survival of 621-101 cells. In vivo, volasertib reduced short-term mouse lung colonization by TSC2-deficient cells and decreased the growth of TSC2-deficient subcutaneous tumors. Mice treated with a combination of volasertib and rapamycin had slower tumor relapse after discontinuation of treatment, compared to rapamycin only. Approximately 35 days after discontinuation of treatment, we observed persistent apoptotic markers and gene expression changes for type I interferon signaling in the regrowth tumors from combination-treated mice, compared to rapamycin only. Notably, combination treatment potently inhibited the growth of subcutaneous tumors derived from the rapamycin-refractory cell line ELT3-245. Taken together, our current work demonstrates that for the management of TSC and LAM disease combination of mTORC1 and PLK1 inhibitors in some cases may be advantageous over currently used rapalog monotherapies.

cancer biology↗