Dataset Information

Identification of proteins associated with the human mitochondrial degradosome

ABSTRACT: The human mitochondrial degradosome is a complex of the ribonuclease PNPase (Q8TCS8, encoded by PNPT1 gene) and RNA helicase SUV3 (Q8IYB8, encoded by SUPV3L1 gene). The aim of the project was to identify proteins which co-purify with both subunits of the degradosome. To this end we used human 293 cells stably expressing hSuv3 or PNPase with a C-terminal TAP tag or TAP tag fused to a mitochondria targeting sequence (control bait) (cell lines are described in details in (PMID: 19864255). Mitochondria were isolated from the cells, lysed and the protein extracts were subjected to affinity chromatography. Co-purified proteins were identified by mass spectrometry and their amounts were quantified by a label-free approach (MaxQuant). These experiments are part of the project “Dedicated surveillance mechanism controls G-quadruplex forming non-coding RNAs in human mitochondria”.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Dominik Cysewski

LAB HEAD: Andrzej Dziembowski

PROVIDER: PXD009826 | Pride | 2018-07-06

REPOSITORIES: Pride

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Dedicated surveillance mechanism controls G-quadruplex forming non-coding RNAs in human mitochondria.

Pietras Zbigniew Z Wojcik Magdalena A MA Borowski Lukasz S LS Szewczyk Maciej M Kulinski Tomasz M TM Cysewski Dominik D Stepien Piotr P PP Dziembowski Andrzej A Szczesny Roman J RJ

Nature communications 20180702 1

The GC skew in vertebrate mitochondrial genomes results in synthesis of RNAs that are prone to form G-quadruplexes (G4s). Such RNAs, although mostly non-coding, are transcribed at high rates and are degraded by an unknown mechanism. Here we describe a dedicated mechanism of degradation of G4-containing RNAs, which is based on cooperation between mitochondrial degradosome and quasi-RNA recognition motif (qRRM) protein GRSF1. This cooperation prevents accumulation of G4-containing transcripts in h ...[more]

PMID: 29967381

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Project description:ABSTRACT Rationale Premature senescence is conducive to aging and cardiovascular diseases. Nrf2 transcription factor, the master orchestrator of adoptive response to cellular stress, has been implicated in regulation of premature senescence in fibroblasts, neural and mesenchymal stem cells by transactivation of antioxidant gene expression. However, as we show here, human primary endothelial cells (ECs) devoid of Nrf2 and murine Nrf2 transcriptional knockout (tKO) aortas are senescent but do not encounter oxidative stress and damage, what contradicts this mechanism. Moreover, a molecular switch between normal, senescent and apoptotic fate remains unknown. Objective To elucidate the mechanism of Nrf2-related premature senescence of vascular system, to understand why Nrf2 deregulation does not cause oxidative stress exclusionary in ECs and to indicate a molecular switch determining ECs fate. Methods and Results Herein we evidence that ECs deficient in Nrf2 protein, or with limited Nrf2 activity in shear stress conditions, exhibit excessive S-nitrosylation of proteins. It is also a characteristic of Nrf2 tKO murine aortas, as determined by biotin switch assay in situ. Mass spectrometry analysis reveals that NOX4 is S-nitrosylated exclusively in ECs devoid of Nrf2. A functional role of S-nitrosylation is protection of ECs from death by inhibition of NOX4-mediated oxidative damage. As a result Nrf2-deficient ECs preserve oxidative balance but are redirected to premature senescence. The same phenotype is seen in Nrf2 tKO aortas. These effects are mediated by Keap1, a direct binding partner of Nrf2 and repressor of its transcriptional activity, remaining in cytoplasm unrestrained by Nrf2. S-nitrosylation, followed by senescence, can also be triggered in smooth muscle cells (SMCs) by EC-derived paracrine induction of iNOS. Conclusions Collectively, Keap1-dependent S-nitrosylation of NOX4 hampers oxidative detriment in ECs with disturbed Nrf2 signaling and may provide defence in the adjacent aortic cells. Overabundance of unrestrained Keap1 in the cytoplasm determines fate of ECs.

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