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Tetracyclines Modify Translation By Targeting Key Human rRNA Substructures

ABSTRACT: Mortison JD, Schenone M, Myers JA, Zhang Z, Chen L, Ciarlo C, Comer E, Natchiar SK, Carr SA, Klaholz BP, Myers AG. Cell Chem Bio 2018. Apart from their antimicrobial properties, tetracyclines demonstrate clinically validated effects in the amelioration of pathological inflammation and human cancer. Delineation of the target(s) and mechanism(s) responsible for these effects, however, has remained elusive. Here, employing quantitative mass spectrometry-based proteomics, we identified human 80S ribosomes as targets of the tetracyclines Col-3 and doxycycline. We then developed in-cell click selective crosslinking with RNA sequence profiling (icCL-Seq) to map binding sites for these tetracyclines on key human rRNA substructures at nucleotide resolution. Importantly, we found that structurally and phenotypically variant tetracycline analogs could chemically discriminate these rRNA binding sites. We also found that tetracyclines both subtly modify human ribosomal translation and selectively activate the cellular integrated stress response (ISR). Together, the data reveal that targeting of specific rRNA substructures, activation of the ISR, and inhibition of translation are correlated with the anti-proliferative properties of tetracyclines in human cancer cell lines.

INSTRUMENT(S): LTQ Orbitrap

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Steven A. Carr

PROVIDER: MSV000082819 | MassIVE | Wed Aug 15 09:02:00 BST 2018

REPOSITORIES: MassIVE

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Project description:Background: The orphan nuclear receptor TR4 (human testicular receptor 4 or NR2C2) plays a pivotal role in a variety of biological and metabolic processes. With no known ligand and few known target genes, the mode of TR4 function was unclear. Results: We report the first genome-wide identification and characterization of TR4 in vivo binding. Using chromatin immunoprecipitation followed by high throughput sequencing (ChIP-seq), we identified TR4 binding sites in 4 different human cell types and found that the majority of target genes were shared among different cells. TR4 target genes are involved in fundamental biological processes such as RNA metabolism and protein translation. In addition, we found that a subset of TR4 target genes exerts cell-type specific functions. Analysis of the TR4 binding sites revealed that less than 30% of the peaks from any of the cell types contained the DR1 motif previously derived from in vitro studies, suggesting that TR4 may be recruited to the genome via interaction with other proteins. A bioinformatics analysis of the TR4 binding sites predicted a cis regulatory module involving TR4 and ETS transcription factors. To test this prediction, we performed ChIP-seq for the ETS factor ELK4 and found that 30% of TR4 binding sites were also bound by ELK4. Motif analysis of the sites bound by both factors revealed a lack of the DR1 element, suggesting that TR4 binding at a subset of sites is facilitated through the ETS transcription factor ELK4. Further studies will be required to investigate the functional interdependence of these two factors. Conclusions: Our data suggest that TR4 plays a pivotal role in fundamental biological processes across different cell types. In addition, the identification of cell type specific TR4 binding sites enables future studies of the pathways underlying TR4 action and its possible role in metabolic diseases. This is one part of the study that involved the examination of HepG2 gene expression. Biological triplicate Illumina Sentrex bead arrays.

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Tetracyclines Modify Translation By Targeting Key Human rRNA Substructures

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