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Low-input RNase footprinting for simultaneous quantification of cytosolic and mitochondrial translation.

ABSTRACT: We describe a low-input RNase footprinting approach for the rapid quantification of ribosome-protected fragments with as few as 1000 cultured cells. The assay uses a simplified procedure to selectively capture ribosome footprints based on optimized RNase digestion. It simultaneously maps cytosolic and mitochondrial translation with single-nucleotide resolution. We applied it to reveal selective functions of the elongation factor TUFM in mitochondrial translation, as well as synchronized repression of cytosolic translation after TUFM perturbation. We show the assay is applicable to small amounts of primary tissue samples with low protein synthesis rates, including snap-frozen tissues and immune cells from an individual's blood draw. We showed its feasibility to characterize the personalized immuno-translatome. Our analyses revealed that thousands of genes show lower translation efficiency in monocytes compared with lymphocytes, and identified thousands of translated noncanonical open reading frames (ORFs). Altogether, our RNase footprinting approach opens an avenue to assay transcriptome-wide translation using low-input samples from a wide range of physiological conditions.

SUBMITTER: Li Q

PROVIDER: S-EPMC8896460 | biostudies-literature | 2022 Mar

REPOSITORIES: biostudies-literature

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Low-input RNase footprinting for simultaneous quantification of cytosolic and mitochondrial translation.

Li Qianru Q Yang Haiwang H Stroup Emily K EK Wang Hongbin H Ji Zhe Z

Genome research 20220222 3

We describe a low-input RNase footprinting approach for the rapid quantification of ribosome-protected fragments with as few as 1000 cultured cells. The assay uses a simplified procedure to selectively capture ribosome footprints based on optimized RNase digestion. It simultaneously maps cytosolic and mitochondrial translation with single-nucleotide resolution. We applied it to reveal selective functions of the elongation factor TUFM in mitochondrial translation, as well as synchronized repressi ...[more]

PMID: 35193938

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Project description:Mitochondrial respiratory chain (RC) disease therapies directed at intra-mitochondrial pathology are largely ineffective. Recognizing that RC dysfunction invokes pronounced extra-mitochondrial transcriptional adaptations, particularly involving dysregulated translation, we hypothesized that translational dysregulation is itself contributing to the pathophysiology of RC disease. Here, we investigated the activities, and effects from direct inhibition, of a central translational regulator (mTORC1) and its downstream biological processes in diverse genetic and pharmacological models of RC disease. Our data identify novel mechanisms underlying the cellular pathogenesis of RC dysfunction, including the combined induction of proteotoxic stress, the ER stress response and autophagy. mTORC1 inhibition with rapamycin partially ameliorated renal disease in B6.Pdss2(kd/kd) mice with complexes I-III/II-III deficiencies, improved viability and mitochondrial physiology in gas-1(fc21) nematodes with complex I deficiency, and rescued viability across a variety of RC-inhibited human cells. Even more effective was probucol, a PPAR-activating anti-lipid drug that we show also inhibits mTORC1. However, directly inhibiting mTORC1-regulated downstream activities yielded the most pronounced and sustained benefit. Partial inhibition of translation by cycloheximide, or of autophagy by lithium chloride, rescued viability, preserved cellular respiratory capacity and induced mitochondrial translation and biogenesis. Cycloheximide also ameliorated proteotoxic stress via a uniquely selective reduction of cytosolic protein translation. RNAseq-based transcriptome profiling of treatment effects in gas-1(fc21) mutants provide further evidence that these therapies effectively restored altered translation and autophagy pathways toward that of wild-type animals. Overall, partially inhibiting cytosolic translation and autophagy offer novel treatment strategies to improve health across the diverse array of human diseases whose pathogenesis involves RC dysfunction.

Dataset Information

Low-input RNase footprinting for simultaneous quantification of cytosolic and mitochondrial translation.

Publications

Low-input RNase footprinting for simultaneous quantification of cytosolic and mitochondrial translation.

Similar Datasets

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