HuR promotes myogenesis by stabilizing the MyoD, Myogenin and p21 mRNAs during the fusion of muscle cells to form myotubes. Here we show that HuR, via a novel mRNA destabilizing activity, promotes the early steps of myogenesis by reducing the expression of the cell cycle promoter nucleophosmin (NPM). Depletion of HuR stabilizes the NPM mRNA, increases NPM protein levels and inhibits myogenesis, while its overexpression elicits the opposite effects. NPM mRNA destabilization involves the association of HuR with the decay factor KSRP as well as the ribonuclease PARN and the exosome. The C-terminus of HuR mediates the formation of the HuR-KSRP complex and is sufficient for maintaining a low level of the NPM mRNA as well as promoting the commitment of muscle cells to myogenesis. We therefore propose a model whereby the downregulation of the NPM mRNA, mediated by HuR, KSRP and its associated ribonucleases, is required for proper myogenesis.

Long noncoding RNAs (lncRNAs) interact with protein factors to regulate different layers of gene expression transcriptionally or posttranscriptionally. Here we report on the functional consequences of the unanticipated interaction of the RNA binding protein K homology-type splicing regulatory protein (KSRP) with the H19 lncRNA (H19). KSRP directly binds to H19 in the cytoplasm of undifferentiated multipotent mesenchymal C2C12 cells, and this interaction favors KSRP-mediated destabilization of labile transcripts such as myogenin. AKT activation induces KSRP dismissal from H19 and, as a consequence, myogenin mRNA is stabilized while KSRP is repurposed to promote maturation of myogenic microRNAs, thus favoring myogenic differentiation. Our data indicate that H19 operates as a molecular scaffold that facilitates effective association of KSRP with myogenin and other labile transcripts, and we propose that H19 works with KSRP to optimize an AKT-regulated posttranscriptional switch that controls myogenic differentiation. Among small noncoding RNAs, microRNAs (miRNAs) repress gene expression by inhibiting translation and/or promoting decay of target mRNAs (1), with RBPs being able to control miRNA maturation from precursors or influence miRNA function (1, 2). A new class of transcripts referred to as long noncoding RNAs (lncRNAs, arbitrarily defined as longer than 200 nt) has recently moved to the forefront of regulatory RNA research (3). lncRNAs had been originally considered epigenetic regulators of gene expression, but the emphasis placed on the ways they regulate chromatin state likely obscures the full repertoire of their functions (4-6). Other roles of lncRNAs include posttranscriptional regulation, posttranslational control of protein activity, organization of protein complexes, as well as cell-cell signaling (4, 6). lncRNAs have been implicated in cellular events as different as cell cycle regulation, pluripotency, apoptosis, and DNA damage response, to name just a few (5, 6). Not surprisingly, lncRNA expression is altered in cancer, and it is becoming clear that some lncRNAs can control cell transformation by regulating vital cellular functions (7). Nevertheless, the composition and function of ribonucleoprotein complexes, including lncRNAs, is generally uncharacterized.Studies performed in primary and cultured cells as well as in mice proved that the K homology (KH)-type splicing regulatory protein (KSRP), a single-stranded RBP that interacts with nucleic acids through four hnRNP KH domains, is able to integrate different levels of gene expression and is required for proper immune response, lipid metabolism, cell fate decisions, and tissue regeneration (see ref. 8 for a recent review). We and others have found that KSRP negatively regulates gene expression via at least two distinct and integrated posttranscriptional mechanisms: (i) by promoting decay of unstable mRNAs [mainly targeting AU-rich elements (AREs) in their 3′UTRs] (8, 9) and (ii) by favoring maturation of select miRNAs from precursors (8, 10). Brie...

Skeletal myogenesis is orchestrated by distinct regulatory signaling pathways, including PI3K/AKT, that ultimately control muscle gene expression. Recently discovered myogenic micro-RNAs (miRNAs) are deeply implicated in muscle biology. Processing of miRNAs from their primary transcripts is emerging as a major step in the control of miRNA levels and might be well suited to be regulated by extracellular signals. Here we report that the RNA binding protein KSRP is required for the correct processing of primary myogenic miRNAs upon PI3K/AKT activation in myoblasts C2C12 and in the course of injury-induced muscle regeneration, as revealed by Ksrp knock-out mice analysis. PI3K/AKT activation regulates in opposite ways two distinct KSRP functions inhibiting its ability to promote decay of myogenin mRNA and activating its ability to favor maturation of myogenic miRNAs. This dynamic regulatory switch eventually contributes to the activation of the myogenic program.

KSRP is a single-strand nucleic acids binding protein that affects RNA fate at multiple levels. KSRP modular structure and its complex pattern of post-translational modifications underpin the interaction with a wide spectrum of RNA target sequences, as well as with other RNA-binding proteins and molecular adaptors. These interactions are important to the regulation of different steps of mRNA metabolism and, in turn, modulate several aspects of cellular proliferation and differentiation. In this review we will discuss in detail KSRP ability to i) promote decay of labile mRNAs interacting with some components of the mRNA decay machinery and ii) favor the maturation of a select group of microRNA precursors.

Tumor microenvironment is fundamental for cancer progression and chemoresistance. Among stromal cells tumor-associated macrophages (TAMs) represent the largest population of infiltrating inflammatory cells in malignant tumors, promoting their growth, invasion, and immune evasion. M2-polarized TAMs are endowed with the nitric oxide (NO)-generating enzyme inducible nitric oxide synthase (iNOS). NO has divergent effects on tumors, since it can either stimulate tumor cells growth or promote their death depending on the source of it; likewise the role of iNOS in cancer differs depending on the cell type. The role of NO generated by TAMs has not been investigated. Using different tumor models in vitro and in vivo we found that NO generated by iNOS of M2-polarized TAMs is able to protect tumor cells from apoptosis induced by the chemotherapeutic agent cisplatin (CDDP). Here, we demonstrate that the protective effect of NO depends on the inhibition of acid sphingomyelinase (A-SMase), which is activated by CDDP in a pathway involving the death receptor CD95. Mechanistic insights indicate that NO actions occur via generation of cyclic GMP and activation of protein kinase G (PKG), inducing phosphorylation of syntaxin 4 (synt4), a SNARE protein responsible for A-SMase trafficking and activation. Noteworthy, phosphorylation of synt4 at serine 78 by PKG is responsible for the proteasome-dependent degradation of synt4, which limits the CDDP-induced exposure of A-SMase to the plasma membrane of tumor cells. This inhibits the cytotoxic mechanism of CDDP reducing A-SMase-triggered apoptosis. This is the first demonstration that endogenous NO system is a key mechanism through which TAMs protect tumor cells from chemotherapeutic drug-induced apoptosis. The identification of the pathway responsible for A-SMase activity downregulation in tumors leading to chemoresistance warrants further investigations as a means to identify new anti-cancer molecules capable of specifically inhibiting synt4 degradation.

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SEPN1-related myopathy (SEPN1-RM) is a muscle disorder due to mutations of the SEPN1 gene, which is characterized by muscle weakness and fatigue leading to scoliosis and life-threatening respiratory failure. Core lesions, focal areas of mitochondria depletion in skeletal muscle fibers, are the most common histopathological lesion. SEPN1-RM underlying mechanisms and the precise role of SEPN1 in muscle remained incompletely understood, hindering the development of biomarkers and therapies for this untreatable disease. To investigate the pathophysiological pathways in SEPN1-RM, we performed metabolic studies, calcium and ATP measurements, super-resolution and electron microscopy on in vivo and in vitro models of SEPN1 deficiency as well as muscle biopsies from SEPN1-RM patients. Mouse models of SEPN1 deficiency showed marked alterations in mitochondrial physiology and energy metabolism, suggesting that SEPN1 controls mitochondrial bioenergetics. Moreover, we found that SEPN1 was enriched at the mitochondria-associated membranes (MAM), and was needed for calcium transients between ER and mitochondria, as well as for the integrity of ER-mitochondria contacts. Consistently, loss of SEPN1 in patients was associated with alterations in body composition which correlated with the severity of muscle weakness, and with impaired ER-mitochondria contacts and low ATP levels. Our results indicate a role of SEPN1 as a novel MAM protein involved in mitochondrial bioenergetics. They also identify a systemic bioenergetic component in SEPN1-RM and establish mitochondria as a novel therapeutic target. This role of SEPN1 contributes to explain the fatigue and core lesions in skeletal muscle as well as the body composition abnormalities identified as part of the SEPN1-RM phenotype. Finally, these results point out to an unrecognized interplay between mitochondrial bioenergetics and ER homeostasis in skeletal muscle. They could therefore pave the way to the identification of biomarkers and therapeutic drugs for SEPN1-RM and for other disorders in which muscle ER-mitochondria cross-talk are impaired.

Brown adipose tissue oxidizes chemical energy for heat generation and energy expenditure. Promoting brown-like transformation in white adipose tissue (WAT) is a promising strategy for combating obesity. Here, we find that targeted deletion of KH-type splicing regulatory protein (KSRP), an RNA-binding protein that regulates gene expression at multiple levels, causes a reduction in body adiposity. The expression of brown fat–selective genes is increased in subcutaneous/inguinal WAT (iWAT) of Ksrp−/− mice because of the elevated expression of PR domain containing 16 and peroxisome proliferator–activated receptor gamma coactivator 1α, which are key regulators promoting the brown fat gene program. The expression of microRNA (miR)-150 in iWAT is decreased due to impaired primary miR-150 processing in the absence of KSRP. We show that miR-150 directly targets and represses Prdm16 and Ppargc1a, and that forced expression of miR-150 attenuates the elevated expression of brown fat genes caused by KSRP deletion. This study reveals the in vivo function of KSRP in controlling brown-like transformation of iWAT through post-transcriptional regulation of miR-150 expression.