Using ribosome profiling in Haloferax volcanii to determine sRNA targets for translational regulation in response to oxidative stress

ATP-binding cassette subfamily E member 1 (ABCE1) belongs to the ABC protein family of transporters; however, it does not behave as a drug transporter. Instead, ABCE1 actively participates in different stages of translation and is also associated with oncogenic functions. Ribosome profiling analysis in colorectal cancer cells has revealed a high ribosome occupancy in the human ABCE1 mRNA 5′-leader sequence, indicating the presence of translatable upstream open reading frames (uORFs). These cis-acting translational regulatory elements usually act as repressors of translation of the main coding sequence. In the present study, we dissect the regulatory function of the five AUG and five non-AUG uORFs identified in the human ABCE1 mRNA 5′-leader sequence. We show that the expression of the main coding sequence is tightly regulated by the ABCE1 AUG uORFs in colorectal cells. Our results are consistent with a model wherein uORF1 is efficiently translated, behaving as a barrier to downstream uORF translation. The few ribosomes that can bypass uORF1 (and/or uORF2) must probably initiate at the inhibitory uORF3 or uORF5 that efficiently repress translation of the main ORF. This inhibitory property is slightly overcome in conditions of endoplasmic reticulum stress. In addition, we observed that these potent translation-inhibitory AUG uORFs function equally in cancer and in non-tumorigenic colorectal cells, which is consistent with a lack of oncogenic function. In conclusion, we establish human ABCE1 as an additional example of uORF-mediated translational regulation and that this tight regulation contributes to control ABCE1 protein levels in different cell environments.

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Monitoring mitochondrial translation by pulse SILAC

10.1101/2021.01.31.428997 ◽

2021 ◽

Author(s):

Koshi Imami ◽

Matthias Selbach ◽

Yasushi Ishihama

Keyword(s):

Oxidative Stress ◽

Amino Acids ◽

Membrane Proteins ◽

Translational Regulation ◽

Isotope Labeling ◽

Protein Complexes ◽

Complex Iii ◽

Mitochondrial Translation ◽

Mitochondrial Ribosomes ◽

Hydrophobic Nature

SummaryMitochondrial ribosomes are specialized to translate the 13 membrane proteins encoded in the mitochondrial genome, but it is challenging to quantify mitochondrial translation products due to their hydrophobic nature. Here, we introduce a proteomic method that combines biochemical isolation of mitochondria with pulse stable isotope labeling by amino acids in cell culture (pSILAC). Our method provides the highest protein coverage (quantifying 12 out of the 13 inner-membrane proteins; average 2-fold improvement over previous studies) with the shortest measurement time. We applied this method to uncover the global picture of (post)translational regulation of both mitochondrial- and nuclear-encoded proteins involved in the assembly of protein complexes that mediate oxidative phosphorylation (OXPHOS). The results allow us to infer the assembly order of complex components and/or partners, as exemplified by complex III. This method should be applicable to study mitochondrial translation programs in many contexts, including oxidative stress and mitochondrial disease.

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Ribosome Profiling Reveals Genome-wide Cellular Translational Regulation upon Heat Stress in Escherichia coli

Genomics Proteomics & Bioinformatics ◽

10.1016/j.gpb.2017.04.005 ◽

2017 ◽

Vol 15 (5) ◽

pp. 324-330 ◽

Cited By ~ 8

Author(s):

Yanqing Zhang ◽

Zhengtao Xiao ◽

Qin Zou ◽

Jianhuo Fang ◽

Qifan Wang ◽

...

Keyword(s):

Escherichia Coli ◽

Heat Stress ◽

Translational Regulation ◽

Ribosome Profiling ◽

Genome Wide

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TrmB, a tRNA m7G46 methyltransferase, plays a role in hydrogen peroxide resistance and positively modulates the translation of katA and katB mRNAs in Pseudomonas aeruginosa

Nucleic Acids Research ◽

10.1093/nar/gkz702 ◽

2019 ◽

Vol 47 (17) ◽

pp. 9271-9281 ◽

Cited By ~ 5

Author(s):

Narumon Thongdee ◽

Juthamas Jaroensuk ◽

Sopapan Atichartpongkul ◽

Jurairat Chittrakanwong ◽

Kamonchanok Chooyoung ◽

...

Keyword(s):

Oxidative Stress ◽

Pseudomonas Aeruginosa ◽

Stress Response ◽

Translational Regulation ◽

Cellular Response ◽

Oxidative Stress Response ◽

Translation Efficiency ◽

Trna Modification ◽

Negative Effect

Abstract Cellular response to oxidative stress is a crucial mechanism that promotes the survival of Pseudomonas aeruginosa during infection. However, the translational regulation of oxidative stress response remains largely unknown. Here, we reveal a tRNA modification-mediated translational response to H2O2 in P. aeruginosa. We demonstrated that the P. aeruginosa trmB gene encodes a tRNA guanine (46)-N7-methyltransferase that catalyzes the formation of m7G46 in the tRNA variable loop. Twenty-three tRNA substrates of TrmB with a guanosine residue at position 46 were identified, including 11 novel tRNA substrates. We showed that loss of trmB had a strong negative effect on the translation of Phe- and Asp-enriched mRNAs. The trmB-mediated m7G modification modulated the expression of the catalase genes katA and katB, which are enriched with Phe/Asp codons at the translational level. In response to H2O2 exposure, the level of m7G modification increased, consistent with the increased translation efficiency of Phe- and Asp-enriched mRNAs. Inactivation of trmB led to decreased KatA and KatB protein abundance and decreased catalase activity, resulting in H2O2-sensitive phenotype. Taken together, our observations reveal a novel role of m7G46 tRNA modification in oxidative stress response through translational regulation of Phe- and Asp-enriched genes, such as katA and katB.

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Recent advances in ribosome profiling for deciphering translational regulation

Methods ◽

10.1016/j.ymeth.2019.05.011 ◽

2020 ◽

Vol 176 ◽

pp. 46-54 ◽

Cited By ~ 6

Author(s):

Yirong Wang ◽

Hong Zhang ◽

Jian Lu

Keyword(s):

Translational Regulation ◽

Ribosome Profiling ◽

Recent Advances

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Translational Regulation of Pmt1 and Pmt2 by Bfr1 Affects Unfolded Protein O-Mannosylation

International Journal of Molecular Sciences ◽

10.3390/ijms20246220 ◽

2019 ◽

Vol 20 (24) ◽

pp. 6220 ◽

Cited By ~ 2

Author(s):

Joan Castells-Ballester ◽

Natalie Rinis ◽

Ilgin Kotan ◽

Lihi Gal ◽

Daniela Bausewein ◽

...

Keyword(s):

Quality Control ◽

Translational Regulation ◽

Secretory Pathway ◽

Rna Binding ◽

Protein Quality ◽

Fluorescent Protein ◽

Brefeldin A ◽

Protein Quality Control ◽

Ribosome Profiling ◽

Unfolded Protein

O-mannosylation is implicated in protein quality control in Saccharomyces cerevisiae due to the attachment of mannose to serine and threonine residues of un- or misfolded proteins in the endoplasmic reticulum (ER). This process also designated as unfolded protein O-mannosylation (UPOM) that ends futile folding cycles and saves cellular resources is mainly mediated by protein O-mannosyltransferases Pmt1 and Pmt2. Here we describe a genetic screen for factors that influence O-mannosylation in yeast, using slow-folding green fluorescent protein (GFP) as a reporter. Our screening identifies the RNA binding protein brefeldin A resistance factor 1 (Bfr1) that has not been linked to O-mannosylation and ER protein quality control before. We find that Bfr1 affects O-mannosylation through changes in Pmt1 and Pmt2 protein abundance but has no effect on PMT1 and PMT2 transcript levels, mRNA localization to the ER membrane or protein stability. Ribosome profiling reveals that Bfr1 is a crucial factor for Pmt1 and Pmt2 translation thereby affecting unfolded protein O-mannosylation. Our results uncover a new level of regulation of protein quality control in the secretory pathway.

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