Sam68 splicing regulation contributes to motor unit establishment in the postnatal skeletal muscle.

RNA-binding proteins orchestrate the composite life of RNA molecules and impact most physiological processes, thus underlying complex phenotypes. The RNA-binding protein Sam68 regulates differentiation processes by modulating splicing, polyadenylation, and stability of select transcripts. Herein, we found that Sam68 -/- mice display altered regulation of alternative splicing in the spinal cord of key target genes involved in synaptic functions. Analysis of the motor units revealed that Sam68 ablation impairs the establishment of neuromuscular junctions and causes progressive loss of motor neurons in the spinal cord. Importantly, alterations of neuromuscular junction morphology and properties in Sam68 -/- mice correlate with defects in muscle and motor unit integrity. Sam68 -/- muscles display defects in postnatal development, with manifest signs of atrophy. Furthermore, fast-twitch muscles in Sam68 -/- mice show structural features typical of slow-twitch muscles, suggesting alterations in the metabolic and functional properties of myofibers. Collectively, our data identify a key role for Sam68 in muscle development and suggest that proper establishment of motor units requires timely expression of synaptic splice variants.

Aberrant Phase Transitions: Side Effects and Novel Therapeutic Strategies in Human Disease.

Phase separation is a physiological process occurring spontaneously when single-phase molecular complexes separate in two phases, a concentrated phase and a more diluted one. Eukaryotic cells employ phase transition strategies to promote the formation of intracellular territories not delimited by membranes with increased local RNA concentration, such as nucleolus, paraspeckles, P granules, Cajal bodies, P-bodies, and stress granules. These organelles contain both proteins and coding and non-coding RNAs and play important roles in different steps of the regulation of gene expression and in cellular signaling. Recently, it has been shown that most human RNA-binding proteins (RBPs) contain at least one low-complexity domain, called prion-like domain (PrLD), because proteins harboring them display aggregation properties like prion proteins. PrLDs support RBP function and contribute to liquid-liquid phase transitions that drive ribonucleoprotein granule assembly, but also render RBPs prone to misfolding by promoting the formation of pathological aggregates that lead to toxicity in specific cell types. Protein-protein and protein-RNA interactions within the separated phase can enhance the transition of RBPs into solid aberrant aggregates, thus causing diseases. In this review, we highlight the role of phase transition in human disease such as amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and in cancer. Moreover, we discuss novel therapeutic strategies focused to control phase transitions by preventing the conversion into aberrant aggregates. In this regard, the stimulation of chaperone machinery to disassemble membrane-less organelles, the induction of pathways that could inhibit aberrant phase separation, and the development of antisense oligonucleotides (ASOs) to knockdown RNAs could be evaluated as novel therapeutic strategies for the treatment of those human diseases characterized by aberrant phase transition aggregates.

Dysregulation of microRNA metabolism in motor neuron diseases: Novel biomarkers and potential therapeutics.

In the last 15 years, several classes of small regulatory RNAs have been identified, uncovering the widespread impact of non-coding elements in the human genome on cell homeostasis and human diseases. MicroRNAs (miRNAs) are a family of small, non-coding RNAs, which exert silencing of mRNA targets in a sequence-dependent fashion. Many miRNAs are specifically expressed in the central nervous system, where they display roles in differentiation, neuronal survival, neuronal plasticity and learning. On the other hand, deregulated miRNA/mRNA expression networks are deeply involved in neurodegeneration. Recent findings suggest a role for miRNAs in the pathogenesis of motor neuron diseases. In particular, cell-specific changes in miRNA profile are involved in the motor neuron disease phenotype and might be implicated in their selective vulnerability. Exploitation of noncoding RNAs, in particular miRNAs, for therapeutic strategies is being assessed for implementing current therapies. In this regard, the neuroprotective potential of certain miRNAs could represent a promising potential tool to improve therapies for motor-neuron diseases. This review focuses on emerging roles of miRNAs in motor neuron diseases and on their impact on neuron life-span and integrity.

hnRNPM guides an alternative splicing program in response to inhibition of the PI3K/AKT/mTOR pathway in Ewing sarcoma cells.

Ewing sarcomas (ES) are biologically aggressive tumors of bone and soft tissues for which no cure is currently available. Most ES patients do not respond to chemotherapeutic treatments or acquire resistance. Since the PI3K/AKT/mTOR axis is often deregulated in ES, its inhibition offers therapeutic perspective for these aggressive tumors. Herein, by using splicing sensitive arrays, we have uncovered an extensive splicing program activated upon inhibition of the PI3K/AKT/mTOR signaling pathway by BEZ235. Bioinformatics analyses identified hnRNPM as a key factor in this response. HnRNPM motifs were significantly enriched in introns flanking the regulated exons and proximity of binding represented a key determinant for hnRNPM-dependent splicing regulation. Knockdown of hnRNPM expression abolished a subset of BEZ235-induced splicing changes that contained hnRNPM binding sites, enhanced BEZ235 cytotoxicity and limited the clonogenicity of ES cells. Importantly, hnRNPM up-regulation correlates with poor outcome in sarcoma patients. These findings uncover an hnRNPM-dependent alternative splicing program set in motion by inhibition of the mTOR/AKT/PI3K pathway in ES cells that limits therapeutic efficacy of pharmacologic inhibitors, suggesting that combined inhibition of the PI3K/AKT/mTOR pathway and hnRNPM activity may represent a novel approach for ES treatment.

MiR-23-TrxR1 as a novel molecular axis in skeletal muscle differentiation.

Thioredoxin reductase 1 (TrxR1) is a selenocysteine-containing protein involved in cellular redox homeostasis which is downregulated in skeletal muscle differentiation. Here we show that TrxR1 decrease occurring during myogenesis is functionally involved in the coordination of this cellular process. Indeed, TrxR1 depletion reduces myoblasts growth by inducing an early myogenesis -related gene expression pattern which includes myogenin and Myf5 up-regulation and Cyclin D1 decrease. On the contrary, the overexpression of TrxR1 during differentiation delays myogenic process, by negatively affecting the expression of Myogenin and MyHC. Moreover, we found that miR-23a and miR-23b - whose expression was increased in the early stage of C2C12 differentiation - are involved in the regulation of TrxR1 expression through their direct binding to the 3' UTR of TrxR1 mRNA. Interestingly, the forced inhibition of miR-23a and miR-23b during C2C12 differentiation partially rescues TrxR1 levels and delays the expression of myogenic markers, suggesting the involvement of miR-23 in myogenesis via TrxR1 repression. Taken together, our results depict for the first time a novel molecular axis, which functionally acts in skeletal muscle differentiation through the modulation of TrxR1 by miR-23.

Post-transcriptional regulation of FUS and EWS protein expression by miR-141 during neural differentiation.

Brain development involves proliferation, migration and specification of neural progenitor cells, culminating in neuronal circuit formation. Mounting evidence indicates that improper regulation of RNA binding proteins (RBPs), including members of the FET (FUS, EWS, TAF15) family, results in defective cortical development and/or neurodegenerative disorders. However, in spite of their physiological relevance, the precise pattern of FET protein expression in developing neurons is largely unknown. Herein, we found that FUS, EWS and TAF15 expression is differentially regulated during brain development, both in time and in space. In particular, our study identifies a fine-tuned regulation of FUS and EWS during neuronal differentiation, whereas TAF15 appears to be more constitutively expressed. Mechanistically FUS and EWS protein expression is regulated at the post-transcriptional level during neuron differentiation and brain development. Moreover, we identified miR-141 as a key regulator of these FET proteins that modulate their expression levels in differentiating neuronal cells. Thus, our studies uncover a novel link between post-transcriptional regulation of FET proteins expression and neurogenesis.

The RNA helicase A in malignant transformation.

The RNA helicase A (RHA) is involved in several steps of RNA metabolism, such as RNA processing, cellular transit of viral molecules, ribosome assembly, regulation of transcription and translation of specific mRNAs. RHA is a multifunctional protein whose roles depend on the specific interaction with different molecular partners, which have been extensively characterized in physiological situations. More recently, the functional implication of RHA in human cancer has emerged. Interestingly, RHA was shown to cooperate with both tumor suppressors and oncoproteins in different tumours, indicating that its specific role in cancer is strongly influenced by the cellular context. For instance, silencing of RHA and/or disruption of its interaction with the oncoprotein EWS-FLI1 rendered Ewing sarcoma cells more sensitive to genotoxic stresses and affected tumor growth and maintenance, suggesting possible therapeutic implications. Herein, we review the recent advances in the cellular functions of RHA and discuss its implication in oncogenesis, providing a perspective for future studies and potential translational opportunities in human cancer.