Mitochondria are dynamic organelles that must precisely control their protein composition according to cellular energy demand. Although nuclear-encoded mRNAs can be localized to the mitochondrial surface, the importance of this localization is unclear. As yeast switch to respiratory metabolism, there is an increase in the fraction of the cytoplasm that is mitochondrial. Our data point to this change in mitochondrial volume fraction increasing the localization of certain nuclear-encoded mRNAs to the surface of the mitochondria. We show that mitochondrial mRNA localization is necessary and sufficient to increase protein production to levels required during respiratory growth. Furthermore, we find that ribosome stalling impacts mRNA sensitivity to mitochondrial volume fraction and counterintuitively leads to enhanced protein synthesis by increasing mRNA localization to mitochondria. This points to a mechanism by which cells are able to use translation elongation and the geometric constraints of the cell to fine-tune organelle-specific gene expression through mRNA localization.

During times of unpredictable stress, organisms must adapt their gene expression to maximize survival. Along with changes in transcription, one conserved means of gene regulation during conditions that quickly represses translation is the formation of cytoplasmic phase-separated mRNP granules such as P-bodies and stress granules. Previously, we identified that distinct steps in gene expression can be coupled during glucose starvation as promoter sequences in the nucleus are able to direct the subcellular localization and translatability of mRNAs in the cytosol. Here, we report that Rvb1 and Rvb2, conserved ATPase proteins implicated as protein assembly chaperones and chromatin remodelers, were enriched at the promoters and mRNAs of genes involved in alternative glucose metabolism pathways that we previously found to be transcriptionally upregulated but translationally downregulated during glucose starvation in yeast. Engineered Rvb1/Rvb2-binding on mRNAs was sufficient to sequester mRNAs into mRNP granules and repress their translation. Additionally, this Rvb-tethering to the mRNA drove further transcriptional upregulation of the target genes. Further we found that depletion of Rvb2 caused decreased alternative glucose metabolism gene mRNA induction, but upregulation of protein synthesis during glucose starvation. Overall, our results point to Rvb1/Rvb2 coupling transcription, mRNA granular localization, and translatability of mRNAs during glucose starvation. This Rvb-mediated rapid gene regulation could potentially serve as an efficient recovery plan for cells after stress removal.

Mitochondria are dynamic in their size and morphology yet must also precisely control their protein composition according to cellular energy demand. Although nuclear-encoded mRNAs can be localized to the mitochondrial outer membrane, the importance of this localization in altering mitochondrial protein composition is unclear. We have found that, as yeast switch from fermentative to respiratory metabolism, there is an increase in the fraction of the cytoplasm that is mitochondrial. This drives the localization of certain nuclear-encoded mitochondrial mRNAs to the surface of the mitochondria. Through tethering experiments, we show that mitochondrial mRNA localization is necessary and sufficient to increase protein production to levels required during respiratory growth. Furthermore, we find that ribosome stalling impacts mRNA sensitivity to mitochondrial volume fraction and counterintuitively leads to enhanced protein synthesis by increasing mRNA localization to the mitochondria. This points to a mechanism by which cells are able to use translation elongation and the geometric constraints of the cell to fine-tune organelle-specific gene expression through mRNA localization while potentially circumventing the need to directly coordinate with the nuclear genome. mitochondria | geometric constraints | mRNA localization | translation control

Diseases caused by emerging swine viruses had a great economic impact, constituting a new challenge for researchers and practicing veterinarians. Innate immune control of viral pathogen invasion is mediated by interferons (IFNs), resulting in transcriptional elevation of hundreds of IFN-stimulated genes (ISGs). However, the ISG family is vast and species-specific, and despite remarkable advancements in uncovering the breadth of IFN-induced gene expression in mouse and human, it is less characterized with respect to the repertoire of porcine ISGs and their functional annotation. Herein, with the application of RNA-sequencing (RNA-Seq) gene profiling, the breadth of IFN-induced gene expression in the context of type I IFN stimulation was explored by using IBRS-2 cell, a commonly used high-efficient cultivation system for porcine picornaviruses. By establishing inclusion criteria, a total of 359 ISGs were selected. Aiming to identify key effectors mediating type I IFN inhibition of swine viruses, a CRISPR/Cas9 knockout library of 1908 sgRNAs targeting 5’ constitutive exons of 359 ISGs with an average of 5 to 6 sgRNAs per gene was constructed. Using VSV-eGFP (vesicular stomatitis virus, fused with GFP) as a model virus, a subset of highest-ranking candidates were identified, including previously validated anti-VSV genes IRF9, IFITM3, LOC100519082 and REC8, as well as several novel hits. This approach attains a high level of feasibility and reliability, and a high rate of hit identification, providing a forward-looking platform to systematically profile the effectors of type I IFN antiviral response against porcine viruses.

Background Cistanche tubulosa is an editable and medicinal traditional Chinese herb and phenylethanoid glycosides are its major components, which have shown various beneficial effects such as anti-tumor, anti-oxidant and neuroprotective activities. However, the anti-obesity effect of C. tubulosa phenylethanoid glycosides (CTPG) and their regulatory effect on gut microbiota are still unclear. In the present study, we investigated its anti-obesity effect and regulatory effect on gut microbiota by 3T3-L1 cell model and obesity mouse model. Methods 3T3-L1 adipocytes were used to evaluate CTPG effects on adipogenesis and lipids accumulation. Insulin resistant 3T3-L1 cells were induced and used to measure CTPG effects on glucose consumption and insulin sensitivity. High-fat diet (HFD)-induced C57BL/6 obese mice were used to investigate CTPG effects on fat deposition, glucose and lipid metabolism, insulin resistance and intestinal microorganism. Results In vitro data showed that CTPG significantly decreased the triglyceride (TG) and non-esterified fatty acid (NEFA) contents of the differentiated 3T3-L1 adipocytes in a concentration-dependent manner without cytotoxicity, and high concentration (100 µg/ml) of CTPG treatment dramatically suppressed the level of monocyte chemoattractant protein-1 (MCP-1) in 3T3-L1 mature adipocytes. Meanwhile, CTPG increased glucose consumption and decreased NEFA level in insulin resistant 3T3-L1 cells. We further found that CTPG protected mice from the development of obesity by inhibiting the expansion of adipose tissue and adipocyte hypertrophy, and improved hepatic steatosis by activating AMPKα to reduce hepatic fat accumulation. CTPG ameliorated HFD-induced hyperinsulinemia, hyperglycemia, inflammation and insulin resistance by activating IRS1/Akt/GLUT4 insulin signaling pathway in white adipose tissue. Moreover, gut microbiota structure and metabolic functions in HFD-induced obese mice was changed by CTPG, especially short chain fatty acids-producing bacteria including Blautia, Roseburia, Butyrivibrio and Bacteriodes were significantly increased by CTPG treatment. Conclusions CTPG effectively suppressed adipogenesis and lipid accumulation in 3T3-L1 adipocytes and ameliorated HFD-induced obesity and insulin resistance through activating AMPKα and IRS1/AKT/GLUT4 signaling pathway and regulating the composition and metabolic functions of gut microbiota.