Mitochondrial stress is involved in many pathological conditions and triggers the integrated stress response (ISR). The ISR is initiated by phosphorylation of the eukaryotic translation initiation factor (eIF) 2α and results in global inhibition of protein synthesis, while the production of specific proteins important for the stress response and recovery is favored. The stalled translation preinitiation complexes phase-separate together with local RNA binding proteins into cytoplasmic stress granules (SG), which are important for regulation of cell signaling and survival under stress conditions. Here we found that mitochondrial inhibition by sodium azide (NaN₃) in mammalian cells leads to translational inhibition and formation of SGs, as previously shown in yeast. Although mammalian NaN₃-induced SGs are very small, they still contain the canonical SG proteins Caprin 1, eIF4A, eIF4E, eIF4G and eIF3B. Similar to FCCP and oligomycine, other mitochodrial stressors that cause SG formation, NaN₃-induced SGs are formed by an eIF2α phosphorylation-independent mechanisms. Finally, we discovered that as shown for arsenite (ASN), but unlike FCCP or heatshock stress, Thioredoxin 1 (Trx1) is required for formation of NaN₃-induced SGs.

Myotonic dystrophy type 1 (DM1) is a progressive multisystemic disease caused by a CTG repeat expansion in the DMPK gene. The toxic mutant mRNA sequesters MBNL proteins, disrupting global RNA metabolism. Although alternative splicing in DM1 skeletal muscle pathology has been extensively studied, early-stage transcriptomic changes remained uncharacterized. To gain deeper and contextual insight into DM1 transcriptome, we performed the first Weighted Gene Co-expression Network Analysis (WGCNA) on skeletal muscle RNA sequencing data from the widely used DM1 mouse model HSA^LR (~250 CTG repeats). We identified 532 core genes using data from 16-week-old mice, an age before the onset of muscle weakness. Additional differential expression analysis across multiple HSA^LR datasets revealed 42 common up-regulated coding and non-coding genes. Within identified core genes, the pathway gene-pair signature analysis enabled contextual selection of functionally related genes involved in maintaining proteostasis, including endoplasmic reticulum (ER) protein processing, the ubiquitin-proteasome system (UPS), macroautophagy and mitophagy, and muscle contraction. The enrichment of ER protein processing with prevailing core genes related to ER-associated degradation suggests adaptive chaperone and UPS activation, while core genes such as Ambra1, Mfn2, and Usp30 indicate adaptations in mitochondrial quality control. Coordinated early alterations in processes maintaining protein homeostasis, critical for muscle mass and function, possibly reflect a response to cellular stress due to repeat expansion and appears before muscle weakness development. Although the study relies exclusively on transcriptomic analyses, it offers a comprehensive, hypothesis-generating perspective that pinpoints candidate pathways, preceding muscle weakness, for future mechanistic validation.

Ribosomes, the catalytic machinery required for protein synthesis, are comprised of 4 ribosomal RNAs and about 80 ribosomal proteins in mammals. Ribosomes further interact with numerous associated factors that regulate their biogenesis and function. As mutations of ribosomal proteins and ribosome-associated proteins cause many diseases, it is important to develop tools by which ribosomes can be purified efficiently and with high specificity. Here, we designed a method to purify ribosomes from human cell lines by C-terminally tagging human RPS9, a protein of the small ribosomal subunit. The tag consists of a flag peptide and a streptavidin-binding peptide (SBP) separated by the tobacco etch virus (TEV) protease cleavage site. We demonstrate that RPS9-Flag-TEV-SBP (FTS) is efficiently incorporated into the ribosome without interfering with regular protein synthesis. Using HeLa-GFP-G3BP1 cells stably expressing RPS9-FTS or, as a negative control, mCherry-FTS, we show that complete ribosomes as well as numerous ribosome-associated proteins are efficiently and specifically purified following pull-down of RPS9-FTS using streptavidin beads. This tool will be helpful for the characterization of human ribosome heterogeneity, post-translational modifications of ribosomal proteins, and changes in ribosome-associated factors after exposing human cells to different stimuli and conditions.