bims-mitran 2021-10-17 papers

Issue of 2021–10–17
nine papers selected by
Andreas Kohler, Stockholm University

Trends Cell Biol. 2021 Oct 08. pii: S0962-8924(21)00183-5. [Epub ahead of print]

Hierarchical folding of the catalytic core during mitochondrial ribosome biogenesis.

Elena Lavdovskaia, Hauke S Hillen, Ricarda Richter-Dennerlein.

Final maturation steps during ribosome biogenesis require the assistance of assembly and quality control factors to ensure the folding of rRNA and proteins into a functional translation machinery. Here we integrate several recent structural snapshots of native large ribosomal subunit intermediates into the complex pathway of mitochondrial ribosome assembly.

DOI: https://doi.org/10.1016/j.tcb.2021.09.004

Nucleic Acids Res. 2021 Oct 11. pii: gkab789. [Epub ahead of print]

Translational activators and mitoribosomal isoforms cooperate to mediate mRNA-specific translation in Schizosaccharomyces pombe mitochondria.

Christopher J Herbert, Sylvie Labarre-Mariotte, David Cornu, Cyrielle Sophie, Cristina Panozzo, Thomas Michel, Geneviève Dujardin, Nathalie Bonnefoy.

Mitochondrial mRNAs encode key subunits of the oxidative phosphorylation complexes that produce energy for the cell. In Saccharomyces cerevisiae, mitochondrial translation is under the control of translational activators, specific to each mRNA. In Schizosaccharomyces pombe, which more closely resembles the human system by its mitochondrial DNA structure and physiology, most translational activators appear to be either lacking, or recruited for post-translational functions. By combining bioinformatics, genetic and biochemical approaches we identified two interacting factors, Cbp7 and Cbp8, controlling Cytb production in S. pombe. We show that their absence affects cytb mRNA stability and impairs the detection of the Cytb protein. We further identified two classes of Cbp7/Cbp8 partners and showed that they modulated Cytb or Cox1 synthesis. First, two isoforms of bS1m, a protein of the small mitoribosomal subunit, that appear mutually exclusive and confer translational specificity. Second, a complex of four proteins dedicated to Cox1 synthesis, which includes an RNA helicase that interacts with the mitochondrial ribosome. Our results suggest that S. pombe contains, in addition to complexes of translational activators, a heterogeneous population of mitochondrial ribosomes that could specifically modulate translation depending on the mRNA translated, in order to optimally balance the production of different respiratory complex subunits.

DOI: https://doi.org/10.1093/nar/gkab789

STAR Protoc. 2021 Dec 17. 2(4): 100850

Generating stable isolated mitochondrial recipient clones in mammalian cells using MitoPunch mitochondrial transfer.

Alexander J Sercel, Alexander J Napior, Alexander N Patananan, Ting-Hsiang Wu, Pei-Yu Chiou, Michael A Teitell.

This protocol describes the assembly and use of MitoPunch to deliver mitochondria containing mitochondrial DNA (mtDNA) into cells lacking mtDNA (ρ0 cells). MitoPunch generates stable isolated mitochondrial recipient clones with restored mtDNA and recovered respiration, enabling investigation of mtDNA mutations and mtDNA-nuclear DNA interactions in a range of cell types. For complete details on the use and execution of this protocol, please refer to Sercel et al. (2021) and Patananan et al. (2020).

Keywords: Biotechnology and bioengineering; Cell Biology; Cell culture; Cell-based Assays; Metabolism

DOI: https://doi.org/10.1016/j.xpro.2021.100850

Sci Rep. 2021 Oct 15. 11(1): 20510

A benchmarking of human mitochondrial DNA haplogroup classifiers from whole-genome and whole-exome sequence data.

Víctor García-Olivares, Adrián Muñoz-Barrera, José M Lorenzo-Salazar, Carlos Zaragoza-Trello, Luis A Rubio-Rodríguez, Ana Díaz-de Usera, David Jáspez, Antonio Iñigo-Campos, Rafaela González-Montelongo, Carlos Flores.

The mitochondrial genome (mtDNA) is of interest for a range of fields including evolutionary, forensic, and medical genetics. Human mitogenomes can be classified into evolutionary related haplogroups that provide ancestral information and pedigree relationships. Because of this and the advent of high-throughput sequencing (HTS) technology, there is a diversity of bioinformatic tools for haplogroup classification. We present a benchmarking of the 11 most salient tools for human mtDNA classification using empirical whole-genome (WGS) and whole-exome (WES) short-read sequencing data from 36 unrelated donors. We also assessed the best performing tool in third-generation long noisy read WGS data obtained with nanopore technology for a subset of the donors. We found that, for short-read WGS, most of the tools exhibit high accuracy for haplogroup classification irrespective of the input file used for the analysis. However, for short-read WES, Haplocheck and MixEmt were the most accurate tools. Based on the performance shown for WGS and WES, and the accompanying qualitative assessment, Haplocheck stands out as the most complete tool. For third-generation HTS data, we also showed that Haplocheck was able to accurately retrieve mtDNA haplogroups for all samples assessed, although only after following assembly-based approaches (either based on a referenced-based assembly or a hybrid de novo assembly). Taken together, our results provide guidance for researchers to select the most suitable tool to conduct the mtDNA analyses from HTS data.

DOI: https://doi.org/10.1038/s41598-021-99895-5

Biochemistry. 2021 Oct 15.

Ribosome Elongation Kinetics of Consecutively Charged Residues Are Coupled to Electrostatic Force.

Sarah E Leininger, Judith Rodriguez, Quyen V Vu, Yang Jiang, Mai Suan Li, Carol Deutsch, Edward P O'Brien.

The speed of protein synthesis can dramatically change when consecutively charged residues are incorporated into an elongating nascent protein by the ribosome. The molecular origins of this class of allosteric coupling remain unknown. We demonstrate, using multiscale simulations, that positively charged residues generate large forces that move the P-site amino acid away from the A-site amino acid. Negatively charged residues generate forces of similar magnitude but move the A- and P-sites closer together. These conformational changes, respectively, increase and decrease the transition state barrier height to peptide bond formation, explaining how charged residues mechanochemically alter translation speed. This mechanochemical mechanism is consistent with in vivo ribosome profiling data exhibiting proportionality between translation speed and the number of charged residues, experimental data characterizing nascent chain conformations, and a previously published cryo-EM structure of a ribosome-nascent chain complex containing consecutive lysines. These results expand the role of mechanochemistry in translation and provide a framework for interpreting experimental results on translation speed.

DOI: https://doi.org/10.1021/acs.biochem.1c00507

Nat Commun. 2021 Oct 11. 12(1): 5933

Structural mechanism of GTPase-powered ribosome-tRNA movement.

Valentyn Petrychenko, Bee-Zen Peng, Ana C de A P Schwarzer, Frank Peske, Marina V Rodnina, Niels Fischer.

GTPases are regulators of cell signaling acting as molecular switches. The translational GTPase EF-G stands out, as it uses GTP hydrolysis to generate force and promote the movement of the ribosome along the mRNA. The key unresolved question is how GTP hydrolysis drives molecular movement. Here, we visualize the GTPase-powered step of ongoing translocation by time-resolved cryo-EM. EF-G in the active GDP-Pi form stabilizes the rotated conformation of ribosomal subunits and induces twisting of the sarcin-ricin loop of the 23 S rRNA. Refolding of the GTPase switch regions upon Pi release initiates a large-scale rigid-body rotation of EF-G pivoting around the sarcin-ricin loop that facilitates back rotation of the ribosomal subunits and forward swiveling of the head domain of the small subunit, ultimately driving tRNA forward movement. The findings demonstrate how a GTPase orchestrates spontaneous thermal fluctuations of a large RNA-protein complex into force-generating molecular movement.

DOI: https://doi.org/10.1038/s41467-021-26133-x

Nat Chem. 2021 Oct 14.

Interactions between nascent proteins and the ribosome surface inhibit co-translational folding.

Anaïs M E Cassaignau, Tomasz Włodarski, Sammy H S Chan, Lauren F Woodburn, Ivana V Bukvin, Julian O Streit, Lisa D Cabrita, Christopher A Waudby, John Christodoulou.

Most proteins begin to fold during biosynthesis on the ribosome. It has been suggested that interactions between the emerging polypeptide and the ribosome surface might allow the ribosome itself to modulate co-translational folding. Here we combine protein engineering and NMR spectroscopy to characterize a series of interactions between the ribosome surface and unfolded nascent chains of the immunoglobulin-like FLN5 filamin domain. The strongest interactions are found for a C-terminal segment that is essential for folding, and we demonstrate quantitative agreement between the strength of this interaction and the energetics of the co-translational folding process itself. Mutations in this region that reduce the extent of binding result in a shift in the co-translational folding equilibrium towards the native state. Our results therefore demonstrate that a competition between folding and binding provides a simple, dynamic mechanism for the modulation of co-translational folding by the ribosome.

DOI: https://doi.org/10.1038/s41557-021-00796-x

Front Aging Neurosci. 2021 ;13 713084

Nanopore Single-Molecule Sequencing for Mitochondrial DNA Methylation Analysis: Investigating Parkin-Associated Parkinsonism as a Proof of Concept.

Theresa Lüth, Kobi Wasner, Christine Klein, Susen Schaake, Ronnie Tse, Sandro L Pereira, Joshua Laß, Lasse Sinkkonen, Anne Grünewald, Joanne Trinh.

Objective: To establish a workflow for mitochondrial DNA (mtDNA) CpG methylation using Nanopore whole-genome sequencing and perform first pilot experiments on affected Parkin biallelic mutation carriers (Parkin-PD) and healthy controls. Background: Mitochondria, including mtDNA, are established key players in Parkinson's disease (PD) pathogenesis. Mutations in Parkin, essential for degradation of damaged mitochondria, cause early-onset PD. However, mtDNA methylation and its implication in PD is understudied. Herein, we establish a workflow using Nanopore sequencing to directly detect mtDNA CpG methylation and compare mtDNA methylation between Parkin-related PD and healthy individuals. Methods: To obtain mtDNA, whole-genome Nanopore sequencing was performed on blood-derived from five Parkin-PD and three control subjects. In addition, induced pluripotent stem cell (iPSC)-derived midbrain neurons from four of these patients with PD and the three control subjects were investigated. The workflow was validated, using methylated and unmethylated 897 bp synthetic DNA samples at different dilution ratios (0, 50, 100% methylation) and mtDNA without methylation. MtDNA CpG methylation frequency (MF) was detected using Nanopolish and Megalodon. Results: Across all blood-derived samples, we obtained a mean coverage of 250.3X (SD ± 80.5X) and across all neuron-derived samples 830X (SD ± 465X) of the mitochondrial genome. We detected overall low-level CpG methylation from the blood-derived DNA (mean MF ± SD = 0.029 ± 0.041) and neuron-derived DNA (mean MF ± SD = 0.019 ± 0.035). Validation of the workflow, using synthetic DNA samples showed that highly methylated DNA molecules were prone to lower Guppy Phred quality scores and thereby more likely to fail Guppy base-calling. CpG methylation in blood- and neuron-derived DNA was significantly lower in Parkin-PD compared to controls (Mann-Whitney U-test p < 0.05). Conclusion: Nanopore sequencing is a useful method to investigate mtDNA methylation architecture, including Guppy-failed reads is of importance when investigating highly methylated sites. We present a mtDNA methylation workflow and suggest methylation variability across different tissues and between Parkin-PD patients and controls as an initial model to investigate.

Keywords: Nanopore; Parkin; Parkinson's disease; methylation; mitochondrial DNA; third-generation sequencing

DOI: https://doi.org/10.3389/fnagi.2021.713084