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Douglas, C. J.

Publications and source records attributed to Douglas, C. J..

4 recordsLinked to original sources

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↗

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↗

DeSciDe: A tool for unbiased literature searching and gene list cu-ration unveils a new role for the acidic patch mutation H2A E92K

Omics analysis has become an indispensable tool for researchers in the life sciences, enabling the study of DNA, RNA, and proteins and how they respond to cellular stimulus. Many methods of data analysis exist for the generation and characterization of gene lists, however, selection of genes for further investigation is still heavily influenced by prior knowledge, with practitioners often studying well characterized genes, reinforcing bias in the literature. Here, we have developed an open-source, R package for impartial ranking of gene lists derived from omics analysis that we term Deciphering Scientific Discoveries (DeSciDe). We applied a pipeline that sorts a gene list first by precedence, which we define as co-occurrence of the gene with pre-defined search terms in publications. We then rank gene lists by connectivity, an underutilized metric for how related a gene is to other enriched genes. The combination of these rankings by scatterplot provides a method for gene selection by simple visual analysis. We apply this analysis methods to published Omics datasets, identifying novel avenues for investigation. Further, using this method we have been able to assign a novel loss of function role for the histone mutation H2A E92K.

bioinformatics↗

Mesoscale proximity labeling to study macro changes to chromatin occupancy

Proximity labeling traditionally identifies interactomes of a single protein or RNA, though this approach limits mechanistic understanding of biomolecules functioning within complex systems. Here, we demonstrate a strategy for deciphering ligand-induced changes to global biomolecular interactions by enabling proximity labelling at the mesoscale, across an entire cellular system. By inserting nanoscale proximity labelling catalysts throughout chromatin, this system, MesoMap, provided new insights into how HDAC inhibitors regulate gene expression. Furthermore, it revealed that the orphaned drug candidate, SR-1815, regulates disease-linked Syngap1 gene expression through direct inhibition of kinases implicated in both neurological disorders and cancer. Through precise mapping of global chromatin mobility, MesoMap promotes insights into how drug-like chemical probes induce transcriptional dynamics within healthy and disease-associated cellular states.

molecular biology↗