bims-mitran 2021-08-29 papers

Issue of 2021–08–29
six papers selected by
Andreas Kohler, Stockholm University

Mol Biol Cell. 2021 Aug 25. mbcE20070457

MRX8, the conserved mitochondrial YihA GTPase family member is required for de novo Cox1 synthesis at suboptimal temperatures in Saccharomyces cerevisiae.

Yash Verma, Upasana Mehra, Dharmendra Kumar Pandey, Joy Kar, Xochitl Pérez-Martinez, Siddhartha S Jana, Kaustuv Datta.

The synthesis of Cox1, the conserved catalytic-core subunit of Complex IV, a multi-subunit machinery of the mitochondrial oxidative phosphorylation (OXPHOS) system under environmental stress is not sufficiently addressed. In this study, we show that the putative YihA superfamily GTPase, Mrx8 is a bonafide mitochondrial protein required for Cox1 translation initiation and elongation during suboptimal growth condition at 16°C. Mrx8 was found in a complex with mitochondrial ribosomes, consistent with a role in protein synthesis. Cells expressing mutant Mrx8 predicted to be defective in guanine nucleotide binding and hydrolysis were compromised for robust cellular respiration. We show that requirement of Pet309 and Mss51 for cellular respiration is not bypassed by overexpression of Mrx8 and vice versa. Consistently the ribosomal association of Mss51 is independent of Mrx8. Significantly, we find that GTPBP8, the human orthologue, complements the loss of cellular respiration in Δmrx8 cells and GTPBP8 localizes to the mitochondria in mammalian cells. This strongly suggest a universal role of MRX8 family of proteins in regulating mitochondrial function.

DOI: https://doi.org/10.1091/mbc.E20-07-0457

EMBO J. 2021 Aug 23. e107988

Mechanism of transcription initiation and primer generation at the mitochondrial replication origin OriL.

Azadeh Sarfallah, Angelica Zamudio-Ochoa, Michael Anikin, Dmitry Temiakov.

The intricate process of human mtDNA replication requires the coordinated action of both transcription and replication machineries. Transcription and replication events at the lagging strand of mtDNA prompt the formation of a stem-loop structure (OriL) and the synthesis of a ∼25 nt RNA primer by mitochondrial RNA polymerase (mtRNAP). The mechanisms by which mtRNAP recognizes OriL, initiates transcription, and transfers the primer to the replisome are poorly understood. We found that transcription initiation at OriL involves slippage of the nascent transcript. The transcript slippage is essential for initiation complex stability and its ability to translocate the mitochondrial DNA polymerase gamma, PolG, which pre-binds to OriL, downstream of the replication origin thus allowing for the primer synthesis. Our data suggest the primosome assembly at OriL-a complex of mtRNAP and PolG-can efficiently generate the primer, transfer it to the replisome, and protect it from degradation by mitochondrial endonucleases.

Keywords: POLRMT; PolG; mitochondrial replication; mitochondrial transcription; primosome

DOI: https://doi.org/10.15252/embj.2021107988

RNA. 2021 Aug 27. pii: rna.078852.121. [Epub ahead of print]

Development, validation, and application of the ribosome separation and reconstitution system for protein translation in vitro.

Brandon M Trainor, Dimitri G Pestov, Natalia Shcherbik.

Stress-induced molecular damage to ribosomes can impact protein synthesis in cells, but cell-based assays do not provide a clear way to distinguish the effects of ribosome damage from stress responses and damage to other parts of the translation machinery. Here we describe a detailed protocol for the separation of yeast ribosomes from other translational machinery constituents, followed by reconstitution of the translation mixture in vitro. This technique, which we refer to as Ribosome Separation and Reconstitution (RSR), allows chemical modifications of yeast ribosomes without compromising other key translational components. In addition to the characterization of stress-induced ribosome damage, RSR can be applied to a broad range of experimental problems in studies of yeast translation.

Keywords: Saccharomyces cerevisiae; centrifugation; in vitro translation; ribosome; ribosome modification

DOI: https://doi.org/10.1261/rna.078852.121

Nat Commun. 2021 08 24. 12(1): 5094

Humans and other commonly used model organisms are resistant to cycloheximide-mediated biases in ribosome profiling experiments.

Puneet Sharma, Jie Wu, Benedikt S Nilges, Sebastian A Leidel.

Ribosome profiling measures genome-wide translation dynamics at sub-codon resolution. Cycloheximide (CHX), a widely used translation inhibitor to arrest ribosomes in these experiments, has been shown to induce biases in yeast, questioning its use. However, whether such biases are present in datasets of other organisms including humans is unknown. Here we compare different CHX-treatment conditions in human cells and yeast in parallel experiments using an optimized protocol. We find that human ribosomes are not susceptible to conformational restrictions by CHX, nor does it distort gene-level measurements of ribosome occupancy, measured decoding speed or the translational ramp. Furthermore, CHX-induced codon-specific biases on ribosome occupancy are not detectable in human cells or other model organisms. This shows that reported biases of CHX are species-specific and that CHX does not affect the outcome of ribosome profiling experiments in most settings. Our findings provide a solid framework to conduct and analyze ribosome profiling experiments.

DOI: https://doi.org/10.1038/s41467-021-25411-y

Nat Med. 2021 Aug 23.

Mitochondrial DNA variants modulate N-formylmethionine, proteostasis and risk of late-onset human diseases.

ICICLE-PD Study Group

Mitochondrial DNA (mtDNA) variants influence the risk of late-onset human diseases, but the reasons for this are poorly understood. Undertaking a hypothesis-free analysis of 5,689 blood-derived biomarkers with mtDNA variants in 16,220 healthy donors, here we show that variants defining mtDNA haplogroups Uk and H4 modulate the level of circulating N-formylmethionine (fMet), which initiates mitochondrial protein translation. In human cytoplasmic hybrid (cybrid) lines, fMet modulated both mitochondrial and cytosolic proteins on multiple levels, through transcription, post-translational modification and proteolysis by an N-degron pathway, abolishing known differences between mtDNA haplogroups. In a further 11,966 individuals, fMet levels contributed to all-cause mortality and the disease risk of several common cardiovascular disorders. Together, these findings indicate that fMet plays a key role in common age-related disease through pleiotropic effects on cell proteostasis.

DOI: https://doi.org/10.1038/s41591-021-01441-3

Int J Biochem Cell Biol. 2021 Aug 21. pii: S1357-2725(21)00150-3. [Epub ahead of print] 106070

Deletion of ribosomal paralogs Rpl39 and Rpl39l compromises cell proliferation via protein synthesis and mitochondrial activity.

Qianxing Zou, Huayu Qi.

Accumulating evidences suggest that the composition and functional roles of ribosomes are heterogeneous in cells, partly due to the temporal-spatial expression of paralogous ribosomal proteins (RPs), of which functional relationships remain largely unexplored. In mouse, the X chromosome-linked RPL39 and its male germline specific paralog RPL39 L are thought to express mutually exclusively due to the meiotic sex chromosome inactivation, hinders the understanding of their functional relationships. In the present study, we investigated the expression and functional relations of Rpl39 and Rpl39l in a proliferative mouse cell line, in which both genes are expressed simultaneously, with the expression level of Rpl39 higher than that of Rpl39l. Disruption of Rpl39 via CRISPR/Cas9 method caused decreased cell proliferation, nascent protein synthesis and altered mitochondrial functions, whereas double mutations of Rpl39 and Rpl39l augmented these phenotypes, suggesting that both RPs contribute to the cellular physiology. Consistently, overexpression of Rpl39, Rpl39l or an alanine mutant of RPL39, rescued cell proliferation similarly in Rpl39-/-::Rpl39l-/- dual gene null cells. Deletion of Rpl39l induced compensatory expression of Rpl39, rendering the deleterious effects of Rpl39l mutation. Supporting this, Rpl39l mutation was more detrimental to cells under a low serum condition, under which the compensatory expression of Rpl39 was inhibited. Moreover, the low serum condition induced expression of both genes, suggesting that they possess stress responsive roles. Taken together, these data indicate that both RPL39 and RPL39 L influence cell proliferation via protein synthesis and mitochondrial functions, suggesting a link between protein translation and cellular metabolism through these ribosomal protein paralogs.

Keywords: RPL39; RPL39L; Ribosomal protein; cell proliferation; mitochondria; paralog

DOI: https://doi.org/10.1016/j.biocel.2021.106070