bims-tricox 2023-01-08 papers

Issue of 2023‒01‒08
four papers selected by
Yash Verma
University of Delhi South Campus

J Biol Chem. 2023 Jan 02. pii: S0021-9258(22)01308-4. [Epub ahead of print] 102865

Monitoring mitochondrial translation by pulse SILAC.

Koshi Imami, Matthias Selbach, Yasushi Ishihama.

Mitochondrial ribosomes are specialized to translate the 13 membrane proteins encoded in the mitochondrial genome, which shapes the oxidative phosphorylation (OXPHOS) complexes essential for cellular energy metabolism. Despite the importance of mitochondrial translation control, it is challenging to identify and quantify the mitochondrial-encoded proteins due to their hydrophobic nature and low abundance. Here, we introduce a mass spectrometry-based 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 identification rate with the shortest measurement time among currently available methods, enabling us to quantify 12 out of the 13 mitochondrial-encoded proteins. We applied this method to uncover the global picture of (post-)translational regulation of both mitochondrial- and nuclear-encoded subunits of OXPHOS complexes. We found that inhibition of mitochondrial translation led to degradation of orphan nuclear-encoded subunits that are considered to form subcomplexes with the mitochondrial-encoded subunits. This method should be readily applicable to study mitochondrial translation programs in many contexts, including oxidative stress and mitochondrial disease.

Keywords: Mitochondria; OXPHOS; Protein complex; Proteomics; Translation; pulse SILAC

DOI: https://doi.org/10.1016/j.jbc.2022.102865

Nat Commun. 2023 Jan 03. 14(1): 30

Human mitochondria require mtRF1 for translation termination at non-canonical stop codons.

Annika Krüger, Cristina Remes, Dmitrii Igorevich Shiriaev, Yong Liu, Henrik Spåhr, Rolf Wibom, Ilian Atanassov, Minh Duc Nguyen, Barry S Cooperman, Joanna Rorbach.

The mitochondrial translation machinery highly diverged from its bacterial counterpart. This includes deviation from the universal genetic code, with AGA and AGG codons lacking cognate tRNAs in human mitochondria. The locations of these codons at the end of COX1 and ND6 open reading frames, respectively, suggest they might function as stop codons. However, while the canonical stop codons UAA and UAG are known to be recognized by mtRF1a, the release mechanism at AGA and AGG codons remains a debated issue. Here, we show that upon the loss of another member of the mitochondrial release factor family, mtRF1, mitoribosomes accumulate specifically at AGA and AGG codons. Stalling of mitoribosomes alters COX1 transcript and protein levels, but not ND6 synthesis. In addition, using an in vitro reconstituted mitochondrial translation system, we demonstrate the specific peptide release activity of mtRF1 at the AGA and AGG codons. Together, our results reveal the role of mtRF1 in translation termination at non-canonical stop codons in mitochondria.

DOI: https://doi.org/10.1038/s41467-022-35684-6

STAR Protoc. 2022 Dec 16. pii: S2666-1667(22)00742-0. [Epub ahead of print]3(4): 101862

Optimized protocol for quantifying 5' UTR-mediated translation initiation in S. cerevisiae using direct analysis of ribosome targeting.

Cole J T Lewis, Rachel O Niederer, Ritam Neupane, Wendy V Gilbert.

Direct analysis of ribosome targeting (DART) allows investigators to measure the translation initiation potential of thousands of RNAs in parallel. Here, we describe an optimized protocol for generating active translation extract from S. cerevisiae, followed by in vitro translation, purification of ribosome-bound RNAs, and subsequent library preparation and sequencing. This protocol can be applied to a variety of cell types and will enable high-throughput interrogation of translational determinants. For complete details on the use and execution of this protocol, please refer to Niederer et al. (2022).1.

Keywords: Gene Expression; Molecular Biology; Sequencing; Systems biology

DOI: https://doi.org/10.1016/j.xpro.2022.101862

Genetics. 2023 Jan 04. pii: iyac191. [Epub ahead of print]

Saccharomyces Genome Database Update: Server Architecture, Pan-Genome Nomenclature, and External Resources.

Edith D Wong, Stuart R Miyasato, Suzi Aleksander, Kalpana Karra, Robert S Nash, Marek S Skrzypek, Shuai Weng, Stacia R Engel, J Michael Cherry.

As one of the first model organism knowledgebases, Saccharomyces Genome Database (SGD) has been supporting the scientific research community since 1993. As technologies and research evolve, so does SGD: from updates in software architecture, to curation of novel data types, to incorporation of data from, and collaboration with, other knowledgebases. We are continuing to make steps toward providing the community with an S. cerevisiae pan-genome. Here we describe software upgrades, a new nomenclature system for genes not found in the reference strain, and additions to gene pages. With these improvements, we aim to remain a leading resource for students, researchers, and the broader scientific community.

Keywords: Saccharomyces cerevisiae ; MOD; SGD; budding yeast; knowledgebase; model organism database

DOI: https://doi.org/10.1093/genetics/iyac191