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Dodd, B.

Publications and source records attributed to Dodd, B..

4 recordsLinked to original sources

Temporal gating dictates stress-induced transcript export from the nucleus

Prior studies have largely focused on transcriptional and translational control during stress, but how regulated nuclear mRNA export contributes to the stress response remains unresolved. We show that nuclear mRNA export is progressively inhibited during arsenite and heat stress in human cells. In contrast to previous work largely in yeast that suggests nuclear export of stress-induced transcripts is prioritized through sequence-specific mechanisms, we demonstrate that temporal gating determines the nucleocytoplasmic distribution of mRNAs during stress. Using single molecule mRNA imaging and transcriptome-wide analyses, we find the majority of stress-induced mRNAs, including heat shock protein transcripts, accumulate in the nucleus during stress. However, a subset of stress-induced mRNAs, notably HMOX1, JUN, and FOS, escape nuclear retention. mRNAs transcribed early during stress, including those encoding immediate early genes, redox mediators, and protein chaperones, are exported from the nucleus prior to the global inhibition of mRNA export. In contrast, mRNAs transcribed later are retained in the nucleus until stress is resolved. Reporter RNA assays confirm that transcriptional timing determines mRNA export competence. This work reveals that the timing of transcription, rather than transcript-specific sequence features, is the major determinant of nuclear export efficiency of stress-induced transcripts in human cells.

molecular biology↗

tRNA synthetase activity is required for stress granule and P-body assembly

In response to stress, translation initiation is suppressed and ribosome runoff via translation elongation drives mRNA assembly into ribonucleoprotein (RNP) granules including stress granules and P-bodies. Defects in translation elongation activate the integrated stress response. If and how stalled ribosomes are removed from mRNAs during translation elongation stress to drive RNP granule assembly is not clear. We demonstrate the integrated stress response is induced upon tRNA synthetase inhibition in part via ribosome collision sensing. However, saturating levels of tRNA synthetase inhibitors do not induce stress granules or P-bodies and prevent RNP granule assembly upon exogenous stress. The loss of tRNA synthetase activity causes persistent ribosome stalls that can be released with puromycin but are not rescued by ribosome-associated quality control pathways. Therefore, tRNA synthetase activity is required for ribosomes to run off mRNAs during stress to scaffold cytoplasmic RNP granules. Our findings suggest ribosome stalls can persist in human cells and uniquely uncouple ribonucleoprotein condensate assembly from the integrated stress response.

molecular biology↗

The APE2 nuclease is essential for DNA double strand break repairby microhomology-mediated end-joining.

Microhomology-mediated end-joining (MMEJ) is an intrinsically mutagenic pathway of DNA double strand break repair essential for proliferation of homologous recombination (HR) deficient tumors. While targeting MMEJ has emerged as a powerful strategy to eliminate HR-deficient (HRD) cancers, this is limited by an incomplete understanding of the mechanism and factors required for MMEJ repair. Here, we identify the APE2 nuclease as a novel MMEJ effector. We show that loss of APE2 blocks the fusion of deprotected telomeres by MMEJ and inhibits MMEJ in DNA repair reporter assays to levels comparable to Pol Theta suppression. Mechanistically, we demonstrate that APE2 possesses intrinsic flap-cleaving activity, that its MMEJ function in cells depends on its nuclease domain and further identify uncharacterized domains required for recruitment to damaged DNA. We conclude that HR-deficient cells are addicted to APE2 due to a previously unappreciated role in MMEJ, which could be exploited in the treatment of cancer.

molecular biology↗

Aminoglycosides induce a bacterial senescent state that increases antibiotic tolerance in treatment-naïve cells

1.Bacterial evolution of antibiotic resistance is facilitated by non-genetic resistance that increases drug tolerance, buying time for evolutionary innovation. Escherichia coli treated with aminoglycosides permanently lose the ability to divide within four hours, yet we discovered a majority of cells maintain membrane integrity and metabolic activity greater than two days post treatment - a bacterial senescent-like state. These cells, which we term zombies, exhibit dynamic gene expression and metabolomic profiles, even after irreversible exit from the cell cycle. Our data reveal zombies upregulate the phage shock protein pathway to maintain membrane integrity. Remarkably, though unable to form new colonies, zombies increase the antibiotic tolerance of treatment-naive cells, implying chemical communication. Chemical supplementation and genetic knockouts show that zombies communicate with treatment-naive cells by secreting indole. In summary, our study revealed a bacterial senescent-like state, induced by aminoglycosides, that decreases the antibiotic susceptibility of multiple bacterial species. Thus, E. coli zombies utilize paracrine signaling to promote non-genetic antibiotic tolerance.

microbiology↗