Reprogramming Efficiency Research Articles

Soft Matrix Promotes Cardiac Reprogramming via Inhibition of YAP/TAZ and Suppression of Fibroblast Signatures.

SummaryDirect cardiac reprogramming holds great potential for regenerative medicine. However, it remains inefficient, and induced cardiomyocytes (iCMs) generated in vitro are less mature than those in vivo, suggesting that undefined extrinsic factors may regulate cardiac reprogramming. Previous in vitro studies mainly used hard polystyrene dishes, yet the effect of substrate rigidity on cardiac reprogramming remains unclear. Thus, we developed a Matrigel-based hydrogel culture system to determine the roles of matrix stiffness and mechanotransduction in cardiac reprogramming. We found that soft matrix comparable with native myocardium promoted the efficiency and quality of cardiac reprogramming. Mechanistically, soft matrix enhanced cardiac reprogramming via inhibition of integrin, Rho/ROCK, actomyosin, and YAP/TAZ signaling and suppression of fibroblast programs, which were activated on rigid substrates. Soft substrate further enhanced cardiac reprogramming with Sendai virus vectors via YAP/TAZ suppression, increasing the reprogramming efficiency up to ∼15%. Thus, mechanotransduction could provide new targets for improving cardiac reprogramming.

Developmental changes in the accessible chromatin, transcriptome and Ascl1-binding correlate with the loss in M\xfcller Glial regenerative potential

Diseases and damage to the retina lead to losses in retinal neurons and eventual visual impairment. Although the mammalian retina has no inherent regenerative capabilities, fish have robust regeneration from Müller glia (MG). Recently, we have shown that driving expression of Ascl1 in adult mouse MG stimulates neural regeneration. The regeneration observed in the mouse is limited in the variety of neurons that can be derived from MG; Ascl1-expressing MG primarily generate bipolar cells. To better understand the limits of MG-based regeneration in mouse retinas, we used ATAC- and RNA-seq to compare newborn progenitors, immature MG (P8-P12), and mature MG. Our analysis demonstrated developmental differences in gene expression and accessible chromatin between progenitors and MG, primarily in neurogenic genes. Overexpression of Ascl1 is more effective in reprogramming immature MG, than mature MG, consistent with a more progenitor-like epigenetic landscape in the former. We also used ASCL1 ChIPseq to compare the differences in ASCL1 binding in progenitors and reprogrammed MG. We find that bipolar-specific accessible regions are more frequently linked to bHLH motifs and ASCL1 binding. Overall, our analysis indicates a loss of neurogenic gene expression and motif accessibility during glial maturation that may prevent efficient reprogramming.

Abstract 221: Tannic Acid Coated Nanoparticles for Cardiac Regeneration

The advance of nanomedicines has recently offered novel approaches to tackle cardiovascular diseases and, in particular, myocardial infarction (MI). However, the constant pumping of the heart and the still poor knowledge of targetable moieties, prevented the application of nanomedicines in the cardiovascular field to rise. Tannic acid, a polyphenol derived from plants, has showed affinity for components of the extracellular matrix, in particular elastin, allowing the retention of protein aggregates in the cardiac tissue. Here, we explored the heart targeting abilities of tannic acid by using it to coat spermine modified-acetalated dextran (AcDXSp) nanoparticles (NPs). Briefly, particles were prepared by single emulsion technique and then coated with tannic acid by complexation of the polyphenol with Fe 3+ ions, resulting in the formation of a capsule around the AcDXSp NPs. The nanoparticles were loaded with two small hydrophobic compounds, CHIR99021 and SB431542, which were both proven to increase the efficiency of direct reprogramming of fibroblasts into cardiomyocytes. The biocompatibility of the nanosystem and cellular uptake were performed on both primary rat cardiomyocytes and fibroblasts. The nanoparticles were taken-up by both the cell types and were safe towards primary cardiomyocytes, while the tannic acid coating showed anti-fibrotic effects on primary fibroblasts. Anti-fibrotic effect was further confirmed by RT-qPCR and the effect of the loaded compounds was assessed by β-catenin and Smad3 immunostainings, which demonstrated the ability of the system to induce direct reprogramming of fibroblasts into cardiomyocytes. In particular, the system stabilized β-catenin and prevented the translocation of Smad3 to the nucleus of myo(fibroblasts). In conclusion this nanosystems exhibited potential to tackle the negative fibrosis process occurring after myocardial infarction by both contrasting it, due to the anti-fibrotic effects showed by the tannic acid coating and by potentially regenerating the cardiac tissue, due to the efficient direct reprogramming of fibroblasts into cardiomyocytes exerted by the loaded drugs.

Mthfd2 Modulates Mitochondrial Function and DNA Repair to Maintain the Pluripotency of Mouse Stem Cells.

SummaryThe pluripotency of stem cells determines their developmental potential. While the pluripotency states of pluripotent stem cells are variable and interconvertible, the mechanisms underlying the acquisition and maintenance of pluripotency remain largely elusive. Here, we identified that methylenetetrahydrofolate dehydrogenase (NAD+-dependent), methenyltetrahydrofolate cyclohydrolase (Mthfd2) plays an essential role in maintaining embryonic stem cell pluripotency and promoting complete reprogramming of induced pluripotent stem cells. Mechanistically, in mitochondria, Mthfd2 maintains the integrity of the mitochondrial respiratory chain and prevents mitochondrial dysfunction. In the nucleus, Mthfd2 stabilizes the phosphorylation of EXO1 to support DNA end resection and promote homologous recombination repair. Our results revealed that Mthfd2 is a dual-function factor in determining the pluripotency of pluripotent stem cells through both mitochondrial and nuclear pathways, ultimately ensuring safe application of pluripotent stem cells.

Analysis of Menstrual Blood Stromal Cells Reveals SOX15 Triggers Oocyte-Based Human Cell Reprogramming.

SummaryCell reprogramming has revolutionized cell and regenerative biology field. However, human iPS derivation remains inefficient and variable. A better knowledge of molecular processes and the rationale underlying the importance of somatic cell origin is crucial to uncover reprogramming mechanisms. Here, we analyze the molecular profile of different human somatic cell types. We show menstrual blood-derived stromal cells (MnSCs) have a distinct, reprogramming prone, profile, and we identify SOX15 from their oocyte-related signature as a prominent responsible candidate. SOX15 orchestrates an efficient oocyte-based reprogramming combination when overexpressed with the also oocyte-enriched histone chaperone ASF1A and OCT4 and, through specific mechanism, generates iPSCs with distinguishable pluripotent state that further present higher differentiation capacity than canonical iPSCs. Our work supports the presence of different pluripotency states in reprogramming and the importance of using metaphase-II oocyte and MnSCs information to provide alternative reprogramming combinations and, importantly, to improve and understand pluripotency acquisition.

Chaetocin Improves Pig Cloning Efficiency by Enhancing Epigenetic Reprogramming and Autophagic Activity.

Efficient epigenetic reprogramming is crucial for the in vitro development of mammalian somatic cell nuclear transfer (SCNT) embryos. The aberrant levels of histone H3 lysine 9 trimethylation (H3K9me3) is an epigenetic barrier. In this study, we evaluated the effects of chaetocin, an H3K9me3-specific methyltransferase inhibitor, on the epigenetic reprogramming and developmental competence of porcine SCNT embryos. The SCNT embryos showed abnormal levels of H3K9me3 at the pronuclear, two-cell, and four-cell stages compared to in vitro fertilized embryos. Moreover, the expression levels of H3K9me3-specific methyltransferases (suv39h1 and suv39h2) and DNA methyltransferases (DNMT1, DNMT3a, and DNMT3b) were higher in SCNT embryos. Treatment with 0.5 nM chaetocin for 24 h after activation significantly increased the developmental competence of SCNT embryos in terms of the cleavage rate, blastocyst formation rate, hatching rate, cell number, expression of pluripotency-related genes, and cell survival rate. In particular, chaetocin enhanced epigenetic reprogramming by reducing the H3K9me3 and 5-methylcytosine levels and restoring the abnormal expression of H3K9me3-specific methyltransferases and DNA methyltransferases. Chaetocin induced autophagic activity, leading to a significant reduction in maternal mRNA levels in embryos at the pronuclear and two-cell stages. These findings revealed that chaetocin enhanced the developmental competence of porcine SCNT embryos by regulating epigenetic reprogramming and autophagic activity and so could be used to enhance the production of transgenic pigs for biomedical research.

Epitranscriptomic N6-Methyladenosine Modification Is Required for Direct Lineage Reprogramming into Neurons.

N6-methyladenosine (m6A), a conserved epitranscriptomic modification of eukaryotic mRNA (mRNA), plays a critical role in a variety of biological processes. Here, we report that m6A modification plays a key role in governing direct lineage reprogramming into induced neuronal cells (iNs). We found that m6A modification is required for the remodeling of specific mRNAs required for the neuronal direct conversion. Inhibition of m6A methylation by Mettl3 knockdown decreased the efficiency of direct lineage reprogramming, whereas increased m6A methylation by Mettl3 overexpression increased the efficiency of iN generation. Moreover, we found that transcription factor Btg2 is a functional target of m6A modification for efficient iN generation. Taken together, our results suggest the importance of establishing epitranscriptomic remodeling for the cell fate conversion into iNs.

Mapping regulators of cell fate determination: Approaches and challenges.

Given the limited regenerative capacities of most organs, strategies are needed to efficiently generate large numbers of parenchymal cells capable of integration into the diseased organ. Although it was initially thought that terminally differentiated cells lacked the ability to transdifferentiate, it has since been shown that cellular reprogramming of stromal cells to parenchymal cells through direct lineage conversion holds great potential for the replacement of post-mitotic parenchymal cells lost to disease. To this end, an assortment of genetic, chemical, and mechanical cues have been identified to reprogram cells to different lineages both in vitro and in vivo. However, some key challenges persist that limit broader applications of reprogramming technologies. These include: (1) low reprogramming efficiencies; (2) incomplete functional maturation of derived cells; and (3) difficulty in determining the typically multi-factor combinatorial recipes required for successful transdifferentiation. To improve efficiency by comprehensively identifying factors that regulate cell fate, large scale genetic and chemical screening methods have thus been utilized. Here, we provide an overview of the underlying concept of cell reprogramming as well as the rationale, considerations, and limitations of high throughput screening methods. We next follow with a summary of unique hits that have been identified by high throughput screens to induce reprogramming to various parenchymal lineages. Finally, we discuss future directions of applying this technology toward human disease biology via disease modeling, drug screening, and regenerative medicine.

289 Highly efficient RNA-based reprogramming of renal epithelial cells derived from recessive dystrophic epidermolysis bullosa patients into induced pluripotent stem cells

Recessive Dystrophic Epidermolysis Bullosa (RDEB) is a severe skin blistering disease caused by mutations in type VII collagen, resulting in impairment of the anchoring fibrils at the dermal-epidermal junction. There is no cure for RDEB, and current therapy is limited to wound care. Induced pluripotent stem cells (iPSCs) have immense value in furthering the study of this disease and developing new treatment options. The use of iPSCs, however, is limited by the availability of primary cells for reprogramming, the length of transfection regimens, and low reprogramming efficiency. iPSCs can be generated from a variety of cell sources including skin punch biopsies, blood and urine samples. However, due to the severe fragility of RDEB patients’ skin, it is incredibly difficult to draw blood or obtain skin biopsies from these patients. Exfoliated renal epithelial cells (RECs) found in urine samples can provide an alternative, non-invasive and easily accessible source of RDEB somatic cells for reprogramming. Previously published methods for REC reprogramming rely on electroporation of non-integrating plasmids or the use of the Sendai viral vector. These methods require time-consuming regimens and result in poor efficiency of reprogramming. Here, we report a highly efficient, non-integrating RNA-based method for reprogramming of disease-associated RECs into high quality iPSCs with an efficiency and kinetics that surpass all previously published studies. The approach depends on highly – tuned transfections of RECs with reprogramming modified mRNAs and mature miRNA mimics in combination with optimized culturing conditions. The resulting renal-derived iPSCs exhibit normal karyotypes and display a pluripotent phenotype. Thus, our approach is ideal for generating high quality patient-derived iPSCs from a non-invasive source of somatic cells to be used for modeling RDEB and other diseases and for potential clinical applications.

High-Efficiency Cellular Reprogramming by Nanoscale Puncturing.

Induced pluripotent stem cells (iPSCs) bear great potential for disease modeling, drug discovery, and regenerative medicine; however, the wide adoption of iPSC for clinically relevant applications has been hindered by the extremely low reprogramming efficiency. Here, we describe a high-efficiency cellular reprogramming strategy by puncturing cells with an array of diamond nanoneedles, which is applied to temporally disrupt the cell membrane in a reversible and minimally invasive format. This method enables high-efficiency cytoplasmic delivery of mini-intronic plasmid vectors to initiate the conversion of human fibroblast cells to either primed iPSCs or naı̈ve iPSCs. The nanopuncturing operation is directly performed on cells in adherent culture without any cell lift-off and is completed within just 5 min. The treated cells are then cultured in feeder-free medium to achieve a reprogramming efficiency of 1.17 ± 0.28%, which is more than 2 orders of magnitude higher than the typical results from common methods involving plasmid delivery.

The function of a heterozygous p53 mutation in a Li-Fraumeni syndrome patient.

p53 is one of the most extensively studied proteins in cancer research. Mutations in p53 generally abolish normal p53 function, and some mutants can gain new oncogenic functions. However, the mechanisms underlying p53 mutation-driven cancer remains to be elucidated. Our study investigated the function of a heterozygous p53 mutation (p.Asn268Glufs*4) in a Li-Fraumeni syndrome (LFS) patient. We used episomal technology to perform somatic reprogramming, and used molecular and cell biology methods to determine the p53 mutation levels in patient-originated induced pluripotent stem (iPS) cells at the RNA and protein levels. We found that p53 protein expression was not increased in this patient's somatic cells compared with those of a healthy control. p53 mutation facilitates the proliferation of tumor cells by inhibiting apoptosis and promoting cell division. It can inhibit the efficiency of somatic reprogramming by inhibiting OCT4 expression during reprogramming stage. Moreover, not all p53 mutant iPS cell lines have mutant p53 RNA sequences. A small percentage of mutant p53 mRNA is present in the somatic cells from the patient and his mother. In summary, this p53 mutation can promote tumor cell proliferation, inhibit somatic reprogramming, and exhibit random p53 allelic expression of heterozygous mutations in the patient and iPS cells which may be one of the reasons why the people with p53 mutations develop cancer at random. This finding suggested that mutant p53 allelic expression should be added to the risk forecasting of cancer.

Lactate preconditioning promotes a HIF-1\u03b1-mediated metabolic shift from OXPHOS to glycolysis in normal human diploid fibroblasts

Recent evidence has emerged that cancer cells can use various metabolites as fuel sources. Restricting cultured cancer cells to sole metabolite fuel sources can promote metabolic changes leading to enhanced glycolysis or mitochondrial OXPHOS. However, the effect of metabolite-restriction on non-transformed cells remains largely unexplored. Here we examined the effect of restricting media fuel sources, including glucose, pyruvate or lactate, on the metabolic state of cultured human dermal fibroblasts. Fibroblasts cultured in lactate-only medium exhibited reduced PDH phosphorylation, indicative of OXPHOS, and a concurrent elevation of ROS. Lactate exposure primed fibroblasts to switch to glycolysis by increasing transcript abundance of genes encoding glycolytic enzymes and, upon exposure to glucose, increasing glycolytic enzyme levels. Furthermore, lactate treatment stabilized HIF-1α, a master regulator of glycolysis, in a manner attenuated by antioxidant exposure. Our findings indicate that lactate preconditioning primes fibroblasts to switch from OXPHOS to glycolysis metabolism, in part, through ROS-mediated HIF-1α stabilization. Interestingly, we found that lactate preconditioning results in increased transcript abundance of MYC and SNAI1, key facilitators of early somatic cell reprogramming. Defined metabolite treatment may represent a novel approach to increasing somatic cell reprogramming efficiency by amplifying a critical metabolic switch that occurs during iPSC generation.

Generation of hepatocyte-like cells from human urinary epithelial cells and the role of autophagy during direct reprogramming

Somatic cells can be directly reprogrammed into other cell lineages, which holds great promise for regenerative medicine. However, low efficiency and obscure mechanism hinder the application of direct reprogramming. Here, we show that overexpressing the hepatic-specific transcription factors (TFs) HNF1α, FOXA3, and GATA4 was sufficient to convert human urinary epithelial cells (hUCs) into induced hepatocyte-like cells (iHeps). The obtained iHeps were confirmed to express various hepatocyte-specific genes with multiple mature hepatocyte functions. Moreover, autophagy-related genes P62, ULK1, BECN1, VPS34, and LC3B were upregulated in the early stage of reprogramming and knockout of P62 and BECN1 in hUCs with CRISPR/Cas9 technology increased the efficiency of direct reprogramming. Collectively, we established a non-invasive approach to convert hUCs into iHeps and provided a glimpse into the role of autophagy in this process.

ADAR1-Dependent RNA Editing Promotes MET and iPSC Reprogramming by Alleviating ER Stress.

RNA editing of adenosine to inosine (A to I) is catalyzed by ADAR1 and dramatically alters the cellular transcriptome, although its functional roles in somatic cell reprogramming are largely unexplored. Here, we show that loss of ADAR1-mediated A-to-I editing disrupts mesenchymal-to-epithelial transition (MET) during induced pluripotent stem cell (iPSC) reprogramming and impedes acquisition of induced pluripotency. Using chemical and genetic approaches, we show that absence of ADAR1-dependent RNA editinginducesaberrant innate immune responses through the double-stranded RNA (dsRNA) sensor MDA5, unleashing endoplasmic reticulum (ER) stress and hindering epithelial fate acquisition. We found that A-to-I editing impedes MDA5 sensing and sequestration of dsRNAs encoding membrane proteins, which promote ER homeostasis by activating the PERK-dependent unfolded protein response pathway to consequently facilitate MET. This study therefore establishes a critical role for ADAR1 and its A-to-I editing activity duringcell fate transitions and delineates a key regulatory layer underlying MET to control efficient reprogramming.

Histone demethylase KDM6A promotes somatic cell reprogramming by epigenetically regulating the PTEN and IL-6 signal pathways.

Aberrant epigenetic reprogramming is one of the major barriers for somatic cell reprogramming. Although our previous study has indicated that H3K27me3 demethylase KDM6A can improve the nuclear reprogramming efficiency, the mechanism remains unclear. In this study, we demonstrate that the overexpression of Kdm6a may improve induced pluripotent stem cell (iPSC) reprogramming efficiency in a demethylase enzymatic activity-dependent manner. KDM6A erased H3K27me3 on pluripotency- and metabolism-related genes, and consequently facilitated changing the gene expression profile and metabolic pattern to an intermediate state. Furthermore, KDM6A may promote IL-6 expression, and the secreted IL-6 may further improve iPSC reprogramming efficiency. In addition, KDM6A may promote PTEN expression to decrease p-AKT and p-mTOR levels, which in turn facilitates reprogramming. Overall, our results reveal that KDM6A may promote iPSC reprogramming efficiency by accelerating changes in the gene expression profile and the metabolic pattern in a demethylation-activity-dependent manner. These results may provide an insight into the relationship between epigenomics, transcriptomics, metabolomics, and reprogramming.

RS-1 enhanced the efficiency of somatic cell reprogramming via activation of RAD51 in somatic cell nuclear transfer

Background & Aim Reprogramming efficiency of somatic cell nuclei is very low in both somatic cell nuclear transfer (SCNT) and iPSCs technologies. Recently, it was reported that the genetic instability and DNA damage arise prior to transcriptional activity during reprogramming. Subsequently, in the absence of mitotic nuclear remodeling, DNA replication is delayed and errors are exacerbated (Egli D et al., 2017). Methods, Results & Conclusion Here, in order to find a regulator of DNA damage during reprogramming events, we observed the effect of RAD51 stimulatory compound (RS-1) supplements in DNA repair process of mouse SCNT embryos in the early embryogenesis and compared with in vitro fertilization (IVF)-derived embryos. First, Rad51 mRNA expression in both embryos of SCNT and IVF groups was observed at 1-cell, 2-cell, and 4-cell stages. In IVF group, relative Rad51 mRNA expression was markedly decreased from 1-cell to 2-cell stages and then restored in 4 cell stage. However, in SCNT group, its expression decreased from 1-cell to 2-cell stages at a similar rate with IVF group, but not recovered at 4-cell stage. Based on these results, we have observed DNA damage detected by rH2AX and DNA repair of double strand breaks by Rad51 expressions. The rH2AX expression was highly localized in the nucleus of IVF embryo when compared with SCNT groups until 10 h after reconstruction of embryos. However Rad51 was not expressed in the nucleus until 10 h after SCNT moreover, at 12h after SCNT, rH2AX showed highly dotted in nuclear in both IVF and SCNT+RS-1 group. A large number of embryos were observed Rad51 expression only in IVF and SCNT+RS-1 groups. In this study, we found different expression of some cell cycle-related genes among IVF, SCNT and SCNT+RS-1 groups by RNA-sequencing. The SCNT+RS-1 group significantly enhanced blastocyst formation rate of the cloned embryos when compared to those of the RS-1-untreated group, and reduced the rate of DNA fragmentation. Furthermore SCNT+RS-1 group improved full term clone mice rates and significantly increased the derivation efficiency of pluripotent stem cells from cloned embryos (SCNT-PSCs). Based on these results, we suggest that DNA damage and Rad51-mediated DNA repair during DNA replication could complement each other and its regulation has plays an important role in somatic cell nuclei reprogramming process.

Bmi1 inhibitor PTC-209 promotes Chemically-induced Direct Cardiac Reprogramming of cardiac fibroblasts into cardiomyocytes

The development of therapeutic approaches based on direct cardiac reprogramming of fibroblasts into induced-cardiomyocytes (iCM) has emerged as an attractive strategy to repair the injured myocardium. The identification of the mechanisms driving lineage conversion represents a crucial step toward the development of new and more efficient regenerative strategies. To this aim, here we show that pre-treatment with the Bmi1 inhibitor PTC-209 is sufficient to increase the efficiency of Chemical-induced Direct Cardiac Reprogramming both in mouse embryonic fibroblasts and adult cardiac fibroblasts. PTC-209 induces an overall increase of spontaneously beating iCM at end-stage of reprogramming, expressing high levels of late cardiac markers Troponin T and myosin muscle light chain-2v. The inhibition of Bmi1 expression occurring upon PTC-209 pre-treatment was maintained throughout the reprogramming protocol, contributing to a significant gene expression de-regulation. RNA profiling revealed that, upon Bmi1 inhibition a significant down-regulation of genes associated with immune and inflammatory signalling pathways occurred, with repression of different genes involved in interleukin, cytokine and chemokine pathways. Accordingly, we observed the down-regulation of both JAK/STAT3 and MAPK/ERK1-2 pathway activation, highlighting the crucial role of these pathways as a barrier for cardiac reprogramming. These findings have significant implications for the development of new cardiac regenerative therapies.

Sirt1 is regulated by miR-135a and involved in DNA damage repair during mouse cellular reprogramming.

Sirt1 facilitates the reprogramming of mouse somatic cells into induced pluripotent stem cells (iPSCs). It is regulated by micro-RNA and reported to be a target of miR-135a. However, their relationship and roles on cellular reprogramming remain unknown. In this study, we found negative correlations between miR-135a and Sirt1 during mouse embryonic stem cells differentiation and mouse embryonic fibroblasts reprogramming. We further found that the reprogramming efficiency was reduced by the overexpression of miR-135a precursor but induced by the miR-135a inhibitor. Co-immunoprecipitation followed by mass spectrometry identified 21 SIRT1 interacting proteins including KU70 and WRN, which were highly enriched for DNA damage repair. In accordance, Sirt1 activator resveratrol reduced DNA damage during the reprogramming process. Wrn was regulated by miR-135a and resveratrol partly rescued the impaired reprogramming efficiency induced by Wrn knockdown. This study showed Sirt1, being partly regulated by miR-135a, bound proteins involved in DNA damage repair and enhanced the iPSCs production.

Planar Mechanical Metamaterials with Embedded Permanent Magnets.

The design space of mechanical metamaterials can be drastically enriched by the employment of non-mechanical interactions between unit cells. Here, the mechanical behavior of planar metamaterials consisting of rotating squares is controlled through the periodic embedment of modified elementary cells with attractive and repulsive configurations of the magnets. The proposed design of mechanical metamaterials produced by three-dimensional printing enables the efficient and quick reprogramming of their mechanical properties through the insertion of the magnets into various locations within the metamaterial. Experimental and numerical studies reveal that under equibiaxial compression various mechanical characteristics, such as buckling strain and post-buckling stiffness, can be finely tuned through the rational placement of the magnets. Moreover, this strategy is shown to be efficient in introducing bistability into the metamaterial with an initially single equilibrium state.

CLUH granules coordinate translation of mitochondrial proteins with mTORC1 signaling and mitophagy

Mitochondria house anabolic and catabolic processes that must be balanced and adjusted to meet cellular demands. The RNA‐binding protein CLUH (clustered mitochondria homolog) binds mRNAs of nuclear‐encoded mitochondrial proteins and is highly expressed in the liver, where it regulates metabolic plasticity. Here, we show that in primary hepatocytes, CLUH coalesces in specific ribonucleoprotein particles that define the translational fate of target mRNAs, such as Pcx, Hadha, and Hmgcs2, to match nutrient availability. Moreover, CLUH granules play signaling roles, by recruiting mTOR kinase and the RNA‐binding proteins G3BP1 and G3BP2. Upon starvation, CLUH regulates translation of Hmgcs2, involved in ketogenesis, inhibits mTORC1 activation and mitochondrial anabolic pathways, and promotes mitochondrial turnover, thus allowing efficient reprograming of metabolic function. In the absence of CLUH, a mitophagy block causes mitochondrial clustering that is rescued by rapamycin treatment or depletion of G3BP1 and G3BP2. Our data demonstrate that metabolic adaptation of liver mitochondria to nutrient availability depends on a compartmentalized CLUH‐dependent post‐transcriptional mechanism that controls both mTORC1 and G3BP signaling and ensures survival.