The ribosome-associated protein quality control (RQC) pathway acts as a translational surveillance mechanism to maintain proteostasis. In mammalian cells, the cytoplasmic RQC pathway involves nuclear export mediator factor (NEMF)-dependent recruitment of the E3 ligase Listerin to ubiquitinate ribosome-stalled nascent polypeptides on the lysine residue for degradation. However, the quality control of ribosome-stalled nuclear-encoded mitochondrial nascent polypeptides remains elusive, as these peptides can be partially imported into mitochondria through translocons, restricting accessibility to the lysine by Listerin. Here, we identify a Listerin-independent organelle-specific mitochondrial RQC pathway that acts on NEMF-mediated carboxy-terminal poly-alanine modification. In the pathway, mitochondrial proteins carrying C-end poly-Ala tails are recognized by the cytosolic E3 ligase Pirh2 and the ClpXP protease in the mitochondria, which coordinately clear ribosome-stalled mitochondrial nascent polypeptides. Defects in this elimination pathway result in NEMF-mediated aggregates and mitochondrial integrity failure, thus providing a potential molecular mechanism of the RQC pathway in mitochondrial-associated human diseases.

F‑box only protein 31 (FBXO31), initially identified in 2005, is a novel subunit of the S‑phase kinase associated protein 1‑Cullin 1‑F‑box ubiquitin ligase. As with other F‑box proteins, FBXO31 may interact with several proteins to promote their ubquitination and subsequent degradation in an F‑box‑dependent manner. It has been revealed that FBXO31 serves a crucial role in DNA damage response and tumorigenesis. However, the expression and function of FBXO31 varies in different types of human cancer. To the best of our knowledge, the present review is the first to summarize the role of FBXO31 in different types of human cancer and determine its underlying mechanisms, thereby paving the road for the design of FBXO31‑targeted anticancer therapies.

Type 1 diabetes mellitus (T1DM) is a chronic autoimmune disease that is characterized by autoimmunity and its mediated β-cell damage. Chronic exposure of β-cells to proinflammatory cytokines is known to regulate the expression of many genes, subsequently resulting in the impairment of some signaling pathways involved with insulin production and secretion and/or β-cell apoptosis. In our study, RNA sequencing technology was applied to identify differentially expressed mRNAs in MIN6 cells treated with a mix of cytokines, including IL-1β, TNF-α, and IFN-γ. The results showed 809 upregulated and 946 downregulated protein-coding mRNAs in MIN6 cells upon the stimulation of cytokines. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) biological pathway analyses were performed to predict the functions of dysregulated genes. The networks of circRNA-mRNA were constructed between differentially mRNAs and dysregulated expressed circRNAs in our previous study. In addition, we selected 8 dysregulated mRNAs for further validation by quantitative real-time PCR. The RNA sequencing data showed 809 upregulated and 946 downregulated protein-coding mRNAs. GO analysis showed that the top 10 significant biological processes, cellular components, and molecular functions for upregulated mRNAs include immune system process, inflammatory response, and innate immune response and the top 10 for downregulated mRNAs include cell cycle, mitotic cytokinesis, and cytoplasm. KEGG analysis showed that these differentially expressed genes were involved with antigen processing and presentation, TNF signaling pathway and type 1 diabetes, cell cycle, necroptosis, and Rap1 signaling pathway. We also constructed the networks of differentially expressed circRNAs and mRNAs. We observed that upregulated circRNA 006029 and downregulated circRNA 000286 and 017277 were associated with the vast majority of selected dysregulated mRNAs, while circRNA 013053 was only related to the protein-coding gene, Slc7a2. To the summary, these data indicated that differentially expressed mRNAs may play key or partial roles in cytokine-mediated β-cell dysfunction and gave us the hint that circRNAs might regulate mRNAs, thereby contributing to the development of T1DM. The current study provided a systematic perspective on the potential functions and possible regulatory mechanisms of mRNAs in proinflammatory cytokine-induced β-cell destruction.

Background: Non-small cell lung cancer (NSCLC) patients with double-driver gene mutations are reported with poorer survival outcomes and reduced therapy responses compared to single-mutant (SM) patients. While substantial progress has been made in treating cancers with single-driver gene alterations, the therapeutic implications and tumor microenvironment of double-mutant (DM) NSCLC remain largely unexplored because of its rarity and poor prognosis. This study aims to delineate the landscape of the immune microenvironment within DM NSCLC. Methods: We employed single-cell RNA sequencing (scRNA-seq) to generate a comprehensive transcriptomic atlas from 25 EGFR mutant NSCLC samples. To assess immune microenvironment changes over time, time-series scRNA-seq and multiplex immunofluorescence analyses were performed in two DM patients. In vitro, EGFR-mutant cell lines were constructed to assess potential therapeutic responses for future clinical applications. Results: The scRNA-seq platform scFocuSCOPE accurately identified and characterized rare, mutation-bearing cancer cells at the single-cell level. DM cancer cells exhibited a strong tendency toward angiogenesis, suggesting an invasive phenotype. DM patients had a more suppressed immune microenvironment, with fewer dysfunctional T lymphocytes. The observation of fewer immune cells and high programmed death ligand-1 (PD-L1) expression in DM cases, probably related to immune evasion and poorer prognosis. In one DM patient, PD-L1 expression remained unchanged after targeted therapy but decreased after immunotherapy. In vitro, EGFR/ERBB2 DM cells showed greater sensitivity to dual-targeted therapies than to single-agent treatments. Conclusions: scFocuSCOPE precisely delineated tumor heterogeneity and immune suppression in EGFR-DM NSCLC. The complex immune landscape of EGFR-DM tumors offers valuable insights for future mechanistic studies and personalized therapies.

The ribosome-associated protein quality control (RQC) core factor nuclear export mediator factor (NEMF) appends C-terminal extended sequences (CESs) to ribosome-stalled nascent chains (NCs). Specific CESs compositions could be directly recognized by enzymes and facilitate NC degradation. Yet, NEMF-mediated CESs remains largely unidentified. Here, we present a protocol for identifying and characterizing NEMF-mediated C-terminal modifications on mitochondrial NCs (mitoNCs) via tandem mass spectrometry (MS/MS) analysis. We describe strategies aimed at constructing a customized MS/MS spectra database for unknown CESs and detail the steps for CES-modified sample preparation. For complete details on the use and execution of this protocol, please refer to Lv et al.¹.