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SummaryRecently, a new regulatory circuitry has been identified in which RNAs can crosstalk with each other by competing for shared microRNAs. Such competing endogenous RNAs (ceRNAs) regulate the distribution of miRNA molecules on their targets and thereby impose an additional level of post-transcriptional regulation. Here we identify a muscle-specific long noncoding RNA, linc-MD1, which governs the time of muscle differentiation by acting as a ceRNA in mouse and human myoblasts. Downregulation or overexpression of linc-MD1 correlate with retardation or anticipation of the muscle differentiation program, respectively. We show that linc-MD1 “sponges” miR-133 and miR-135 to regulate the expression of MAML1 and MEF2C, transcription factors that activate muscle-specific gene expression. Finally, we demonstrate that linc-MD1 exerts the same control over differentiation timing in human myoblasts, and that its levels are strongly reduced in Duchenne muscle cells. We conclude that the ceRNA network plays an important role in muscle differentiation.

N6-methyladenosine (m6A) is a common modification of mRNA, with potential roles in fine-tuning the RNA life-cycle. Here, we identify a dense network of proteins interacting with METTL3, a component of the methyltransferase complex, and show that three of them, WTAP, METTL14 and KIAA1429, are required for methylation. Monitoring m6A levels upon WTAP depletion allowed the definition of accurate and near single-nucleotide resolution methylation maps, and their classification into WTAP-dependent and independent sites. WTAP-dependent sites are located at internal positions in transcripts, are topologically static across a variety of systems we surveyed, and are inversely correlated with mRNA stability, consistent with a role in establishing ‘basal’ degradation rates. WTAP-independent sites form at the first transcribed base as part of the cap structure, and are present at thousands of sites, forming a previously unappreciated layer of transcriptome complexity. Our data sheds new light on proteomic and transcriptional underpinnings of this epitranscriptomic modification.

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Dystrophin absence in Duchenne muscular dystrophy (DMD) causes severe muscle degeneration. We describe that, as consequence of fibre damage, specific muscle-miRNAs are released in to the bloodstream of DMD patients and their levels correlate with the severity of the disease. The same miRNAs are abundant also in the blood of mdx mice and recover to wild-type levels in animals ‘cured’ through exon skipping. Even though creatine kinase (CK) blood levels have been utilized as diagnostic markers of several neuromuscular diseases, including DMD, we demonstrate that they correlate less well with the disease severity. Although the analysis of a larger number of patients should allow to obtain more refined correlations with the different stages of disease progression, we propose that miR-1, miR-133, and miR-206 are new and valuable biomarkers for the diagnosis of DMD and possibly also for monitoring the outcomes of therapeutic interventions in humans. Despite many different DMD therapeutic approaches are now entering clinical trials, a unifying method for assessing the benefit of different treatments is still lacking.

Summary Induced pluripotency is a promising avenue for disease modeling and therapy, but the molecular principles underlying this process, particularly in human cells, remain poorly understood due to donor-to-donor variability and intercellular heterogeneity. Here we constructed and characterized a clonal, inducible human reprogramming system that provides a reliable source of cells at any stage of the process. This system enabled integrative transcriptional and epigenomic analysis across the human reprogramming timeline at high resolution. We observed distinct waves of gene network activation, including the ordered reactivation of broad developmental regulators followed by early embryonic patterning genes and culminating in the emergence of a signature reminiscent of pre-implantation stages. Moreover, complementary functional analyses allowed us to identify and validate novel regulators of the reprogramming process. Altogether, this study sheds light on the molecular underpinnings of induced pluripotency in human cells and provides a robust cell platform for further studies.

In Duchenne muscular dystrophy (DMD) the absence of dystrophin at the sarcolemma delocalizes and downregulates nitric oxide synthase (nNOS); this alters S-nitrosylation of HDAC2 and its chromatin association. We show that the differential HDAC2 nitrosylation state in Duchenne versus wild-type conditions deregulates the expression of a specific subset of microRNA genes. Several circuitries controlled by the identified microRNAs, such as the one linking miR-1 to the G6PD enzyme and the redox state of cell, or miR-29 to extracellular proteins and the fibrotic process, explain some of the DMD pathogenetic traits. We also show that, at variance with other myomiRs, miR-206 escapes from the dystrophin-nNOS control being produced in activated satellite cells before dystrophin expression; in these cells, it contributes to muscle regeneration through repression of the satellite specific factor, Pax7. We conclude that the pathway activated by dystrophin/nNOS controls several important circuitries increasing the robustness of the muscle differentiation program.

The RNA-binding proteins LIN28A and LIN28B play critical roles in embryonic development, tumorigenesis, and pluripotency, but their exact functions are poorly understood. Here, we show that, like LIN28A, LIN28B can function effectively with NANOG, OCT4, and SOX2 in reprogramming to pluripotency and that reactivation of both endogenous LIN28A and LIN28B loci are required for maximal reprogramming efficiency. In human fibroblasts, LIN28B is activated early during reprogramming, while LIN28A is activated later during the transition to bona fide induced pluripotent stem cells (iPSCs). In murine cells, LIN28A and LIN28B facilitate conversion from naive to primed pluripotency. Proteomic and metabolomic analysis highlighted roles for LIN28 in maintaining the low mitochondrial function associated with primed pluripotency and in regulating one-carbon metabolism, nucleotide metabolism, and histone methylation. LIN28 binds to mRNAs of proteins important for oxidative phosphorylation and modulates protein abundance. Thus, LIN28A and LIN28B play cooperative roles in regulating reprogramming, naive/primed pluripotency, and stem cell metabolism.