bims-mitran 2021-09-05 papers

Issue of 2021–09–05
five papers selected by
Andreas Kohler, Stockholm University

Bio Protoc. 2021 Aug 05. 11(15): e4110

Purification of Mitochondrial Ribosomes with the Translocase Oxa1L from HEK Cells.

Mitochondrial ribosomes (mitoribosomes) perform protein synthesis inside mitochondria, the organelles responsible for energy conversion and adenosine triphosphate (ATP) production in eukaryotic cells. To investigate their functions and structures, large-scale purification of intact mitoribosomes from mitochondria-rich animal tissues or HEK cells have been developed. However, the fast purification of mitoribosomes anchored to the mitochondrial inner membrane in complex with the Oxa1L translocase remains particularly challenging. Herein, we present a protocol recently developed and modified in our lab that provides details for the efficient isolation of intact mitoribosomes with its translocase Oxa1L. We combined the cell culture of PDE12-/- or wild-type HEK293 cell lines with the isolation of mitochondria and the purification steps used for the biochemical and structural studies of mitoribosomes and Oxa1L. Graphic abstract: Schematic procedure for the purification of mitoribosomes from HEK cells. The protocol described herein includes two main sections: 1) isolation of mitochondria from HEK cells; and 2) purification of mitoribosome-Oxa1L from mitochondria. RB: Resuspension Buffer (see Recipes) (Created with BioRender.com).

Keywords: Biochemistry; Cryo-EM; Mitochondria; Mitoribosome; Oxa1L; Ribosome purification

DOI: https://doi.org/10.21769/BioProtoc.4110

Nat Commun. 2021 Sep 02. 12(1): 5241

Cell reprogramming shapes the mitochondrial DNA landscape.

Wei Wei, Daniel J Gaffney, Patrick F Chinnery.

Individual induced pluripotent stem cells (iPSCs) show considerable phenotypic heterogeneity, but the reasons for this are not fully understood. Comprehensively analysing the mitochondrial genome (mtDNA) in 146 iPSC and fibroblast lines from 151 donors, we show that most age-related fibroblast mtDNA mutations are lost during reprogramming. However, iPSC-specific mutations are seen in 76.6% (108/141) of iPSC lines at a mutation rate of 8.62 × 10-5/base pair. The mutations observed in iPSC lines affect a higher proportion of mtDNA molecules, favouring non-synonymous protein-coding and tRNA variants, including known disease-causing mutations. Analysing 11,538 single cells shows stable heteroplasmy in sub-clones derived from the original donor during differentiation, with mtDNA variants influencing the expression of key genes involved in mitochondrial metabolism and epidermal cell differentiation. Thus, the dynamic mtDNA landscape contributes to the heterogeneity of human iPSCs and should be considered when using reprogrammed cells experimentally or as a therapy.

DOI: https://doi.org/10.1038/s41467-021-25482-x

Life Sci Alliance. 2021 Nov;pii: e202101034. [Epub ahead of print]4(11):

High levels of TFAM repress mammalian mitochondrial DNA transcription in vivo.

Nina A Bonekamp, Min Jiang, Elisa Motori, Rodolfo Garcia Villegas, Camilla Koolmeister, Ilian Atanassov, Andrea Mesaros, Chan Bae Park, Nils-Göran Larsson.

Mitochondrial transcription factor A (TFAM) is compacting mitochondrial DNA (dmtDNA) into nucleoids and directly controls mtDNA copy number. Here, we show that the TFAM-to-mtDNA ratio is critical for maintaining normal mtDNA expression in different mouse tissues. Moderately increased TFAM protein levels increase mtDNA copy number but a normal TFAM-to-mtDNA ratio is maintained resulting in unaltered mtDNA expression and normal whole animal metabolism. Mice ubiquitously expressing very high TFAM levels develop pathology leading to deficient oxidative phosphorylation (OXPHOS) and early postnatal lethality. The TFAM-to-mtDNA ratio varies widely between tissues in these mice and is very high in skeletal muscle leading to strong repression of mtDNA expression and OXPHOS deficiency. In the heart, increased mtDNA copy number results in a near normal TFAM-to-mtDNA ratio and maintained OXPHOS capacity. In liver, induction of LONP1 protease and mitochondrial RNA polymerase expression counteracts the silencing effect of high TFAM levels. TFAM thus acts as a general repressor of mtDNA expression and this effect can be counterbalanced by tissue-specific expression of regulatory factors.

DOI: https://doi.org/10.26508/lsa.202101034

Cell Rep. 2021 Aug 31. pii: S2211-1247(21)01085-8. [Epub ahead of print]36(9): 109642

iRQC, a surveillance pathway for 40S ribosomal quality control during mRNA translation initiation.

Danielle M Garshott, Heeseon An, Elayanambi Sundaramoorthy, Marilyn Leonard, Alison Vicary, J Wade Harper, Eric J Bennett.

Post-translational modification of ribosomal proteins enables rapid and dynamic regulation of protein biogenesis. Site-specific ubiquitylation of 40S ribosomal proteins uS10 and eS10 plays a key role during ribosome-associated quality control (RQC). Distinct, and previously functionally ambiguous, ubiquitylation events on the 40S proteins uS3 and uS5 are induced by diverse proteostasis stressors that impact translation activity. Here, we identify the ubiquitin ligase RNF10 and the deubiquitylating enzyme USP10 as the key enzymes that regulate uS3 and uS5 ubiquitylation. Prolonged uS3 and uS5 ubiquitylation results in 40S, but not 60S, ribosomal protein degradation in a manner independent of canonical autophagy. We show that blocking progression of either scanning or elongating ribosomes past the start codon triggers site-specific ubiquitylation events on ribosomal proteins uS5 and uS3. This study identifies and characterizes a distinct arm in the RQC pathway, initiation RQC (iRQC), that acts on 40S ribosomes during translation initiation to modulate translation activity and capacity.

Keywords: RNF10; protein homeostasis; ribosome-associated quality control; translation initiation; ubiquitin

DOI: https://doi.org/10.1016/j.celrep.2021.109642

Nat Methods. 2021 Sep 03.

Nascent Ribo-Seq measures ribosomal loading time and reveals kinetic impact on ribosome density.

Johanna Schott, Sonja Reitter, Doris Lindner, Jan Grosser, Marius Bruer, Anjana Shenoy, Tamar Geiger, Arthur Mathes, Gergana Dobreva, Georg Stoecklin.

In general, mRNAs are assumed to be loaded with ribosomes instantly upon entry into the cytoplasm. To measure ribosome density (RD) on nascent mRNA, we developed nascent Ribo-Seq by combining Ribo-Seq with progressive 4-thiouridine labeling. In mouse macrophages, we determined experimentally the lag between the appearance of nascent mRNA and its association with ribosomes, which was calculated to be 20-22 min for bulk mRNA. In mouse embryonic stem cells, nRibo-Seq revealed an even stronger lag of 35-38 min in ribosome loading. After stimulation of macrophages with lipopolysaccharide, the lag between cytoplasmic and translated mRNA leads to uncoupling between input and ribosome-protected fragments, which gives rise to distorted RD measurements under conditions where mRNA amounts are far from steady-state expression. As a result, we demonstrate that transcriptional changes affect RD in a passive way.

DOI: https://doi.org/10.1038/s41592-021-01250-z