microRNAs (miRNAs) are generated from long primary (pri-) RNA polymerase II (Pol II)-derived transcripts by two RNase III processing reactions: Drosha cleavage of nuclear pri-miRNAs and Dicer cleavage of cytoplasmic pre-miRNAs. Here we show that Drosha cleavage occurs during transcription acting on both independently transcribed and intron-encoded miRNAs. We also show that both 5'-3' and 3'-5' exonucleases associate with the sites where co-transcriptional Drosha cleavage occurs, promoting intron degradation before splicing. We finally demonstrate that miRNAs can also derive from 3' flanking transcripts of Pol II genes. Our results demonstrate that multiple miRNA-containing transcripts are co-transcriptionally cleaved during their synthesis and suggest that exonucleolytic degradation from Drosha cleavage sites in pre-mRNAs may influence the splicing and maturation of numerous mRNAs.

Pathological molecular mechanisms involved in myocardial remodeling contribute to alter the existing structure of the heart, leading to cardiac dysfunction. Among the complex signaling network that characterizes myocardial remodeling, the distinct processes are myocyte loss, cardiac hypertrophy, alteration of extracellular matrix homeostasis, fibrosis, defective autophagy, metabolic abnormalities, and mitochondrial dysfunction. Several pathophysiological stimuli, such as pressure and volume overload, trigger the remodeling cascade, a process that initially confers protection to the heart as a compensatory mechanism. Yet chronic inflammation after myocardial infarction also leads to cardiac remodeling that, when prolonged, leads to heart failure progression. Here, we review the molecular pathways involved in cardiac remodeling, with particular emphasis on those associated with myocardial infarction. A better understanding of cell signaling involved in cardiac remodeling may support the development of new therapeutic strategies towards the treatment of heart failure and reduction of cardiac complications. We will also discuss data derived from gene therapy approaches for modulating key mediators of cardiac remodeling.

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Subclones homozygous for JAK2V617F are more common in polycythemia vera (PV) than essential thrombocythemia (ET), but their prevalence and significance remain unclear. The JAK2 mutation status of 6495 BFU-E, grown in low erythropoietin conditions, was determined in 77 patients with PV or ET. Homozygousmutant colonies were common in patients with JAK2V617F-positive PV and were surprisingly prevalent in JAK2V617F-positive ET and JAK2 exon 12-mutated PV. Using microsatellite PCR to map loss-of-heterozygosity breakpoints within individual colonies, we demonstrate that recurrent acquisition of JAK2V617F homozygosity occurs frequently in both PV and ET. PV was distinguished from ET by expansion of a dominant homozygous subclone, the selective advantage of which is likely to reflect additional genetic or epigenetic lesions. Our results suggest a model in which development of a dominant JAK2V617F-homzygous subclone drives erythrocytosis in many PV patients, with alternative mechanisms operating in those with small or undetectable homozygous-mutant clones. (Blood. 2012;120(13):2704-2707) IntroductionThe JAK2V617F mutation is found in ϳ 95% of patients with polycythemia vera (PV) and 60% of those with essential thrombocythemia (ET), 1-4 but the mechanisms responsible for the different disease phenotypes remain unclear. Several lines of circumstantial evidence suggest that increased signaling through mutant JAK2 is important: (1) A "homozygous" sequence pattern in granulocytes was identified in ϳ 30% of patients with PV but was rare in ET 1-4 ; (2) in PV patients, JAK2V617F allele burden correlates with higher hemoglobin levels and white cell counts but lower platelet counts 5,6 ; (3) the copy number of mutant JAK2 influences phenotype in mouse models 7,8 ; and (4) JAK2 exon 12 mutations are reported to signal more strongly than JAK2V617F and are associated with PV but not ET. 9 Homozygosity for JAK2V617F results from mitotic recombination, 1-4 and homozygous-mutant BFU-E were present in most patients with PV but not in those with ET. 10 This observation raised the possibilities that patients with PV are more prone to develop a homozygous subclone or that homozygous-mutant cells have a selective advantage in patients with PV but not in those with ET. In either case, it has been widely assumed that homozygousmutant cells in a given patient are usually members of a single clone with a selective advantage and that JAK2V617F homozygosity plays a causal role in PV pathogenesis. However, this model is complicated by several observations: some patients with PV have very small homozygous-mutant clones 10 ; 2 individual patients have been reported to harbor 2 distinct homozygous clones 11,12 ; a defect in STAT1 phosphorylation has been identified in PV patients 13 ; and reports of small numbers of ET patients who harbor homozygousmutant BFU-E 14-16 and of PV patients with none. 14, 15 We have therefore systematically assessed the prevalence and clonal relationship of homozygous-mutant BFU-E precursors in patients with JAK2-mutated...

The first step in microRNA (miRNA) biogenesis occurs in the nucleus and is mediated by the Microprocessor complex containing the RNase III-like enzyme Drosha and its cofactor DGCR8. Here we show that the 533 exonuclease Xrn2 associates with independently transcribed miRNAs and, in combination with Drosha processing, attenuates transcription in downstream regions. We suggest that, after Drosha cleavage, a torpedo-like mechanism acts on nascent long precursor miRNAs, whereby Xrn2 exonuclease degrades the RNA polymerase II-associated transcripts inducing its release from the template. While involved in primary transcript termination, this attenuation effect does not restrict clustered miRNA expression, which, in the majority of cases, is separated by short spacers. We also show that transcripts originating from a miRNA promoter are retained on the chromatin template and are more efficiently processed than those produced from mRNA or snRNA Pol II-dependent promoters. These data imply that coupling between transcription and processing promotes efficient expression of independently transcribed miRNAs.It is well documented that efficient and regulated mRNA biogenesis is ensured by coupling processing to transcription within the framework of an "mRNA factory" comprising the elongating RNA polymerase II (Pol II) and the associated processing factors. This machinery relies on a complex network of protein interactions leading to the release from chromatin of properly modified and "marked" mRNAs (19,24,27). Notably, this complex apparatus also provides a quality control mechanism that prevents "incorrect" molecules from progressing along the maturation pathway (1). It has been previously shown in yeast (Saccharomyces cerevisiae) that snoRNA biogenesis also relies on a specific "factory" in which transcription and termination events are intimately coupled to processing and release of correctly assembled snoRNP particles (2, 22). In eukaryotes, Pol II is also responsible for the transcription of a different class of transcripts, the microRNAs (miRNAs). The tiny single-stranded miRNA molecules are transcribed as long precursors, pri-miRNAs. More than half of the human miRNA genes are carried within introns of both coding and noncoding genes, while others are transcribed as independent monocistronic or polycistronic units (16). The Microprocessor complex, containing the RNase III-like enzyme Drosha and its cofactor DGCR8, converts the nascent pri-miRNAs into small 60-to 90-nucleotide-long hairpins. For intron-encoded miRNAs, cotranscriptional Drosha cleavage was shown to occur before splicing without affecting the release of spliced mRNA (16, 23). Moreover, the exosome and Xrn2 exonuclease activities were shown to mediate intronic clearance, while the exonic regions, protected from degradation by tethering to the elongating Pol II complex, could undergo efficient splicing (9, 23). Here we show that the 5Ј33Ј Xrn2 exonuclease also associates with chromatin of intergenic miRNA genes. In this situation, the action of the Xrn2 exon...

The carboxy-terminal domain (CTD) of RNA polymerase II large subunit acts as a platform to assemble the RNA processing machinery in a controlled way throughout the transcription cycle. In yeast, recent findings revealed a physical connection between phospho-CTD, generated by the Ctk1p kinase, and protein factors having a function in small nucleolar RNA (snoRNA) biogenesis. The snoRNAs represent a large family of polymerase II noncoding transcripts that are associated with highly conserved polypeptides to form stable ribonucleoprotein particles (snoRNPs). In this work, we have studied the biogenesis of the snoRNPs belonging to the box H/ACA class. We report that the assembly factor Naf1p and the core components Cbf5p and Nhp2p are recruited on H/ACA snoRNA genes very early during transcription. We also show that the cotranscriptional recruitment of Naf1p and Cbf5p is Ctk1p dependent and that Ctk1p and Cbf5p are required for preventing the readthrough into the snoRNA downstream genes. All these data suggest that proper cotranscriptional snoRNP assembly controls 3-end formation of snoRNAs and, consequently, the release of a functional particle.

The epithelial-to-mesenchymal transition (EMT) is an essential trans-differentiation process, which plays a critical role in embryonic development, wound healing, tissue regeneration, organ fibrosis, and cancer progression. It is the fundamental mechanism by which epithelial cells lose many of their characteristics while acquiring features typical of mesenchymal cells, such as migratory capacity and invasiveness. Depending on the contest, EMT is complemented and balanced by the reverse process, the mesenchymal-to-epithelial transition (MET). In the saving economy of the living organisms, the same (Ying-Yang) tool is integrated as a physiological strategy in embryonic development, as well as in the course of reparative or disease processes, prominently fibrosis, tumor invasion and metastasis. These mechanisms and their related signaling (e.g., TGF-β and BMPs) have been effectively studied in vitro by tissue-derived cell spheroids models. These three-dimensional (3D) cell culture systems, whose phenotype has been shown to be strongly dependent on TGF-β-regulated EMT/MET processes, present the advantage of recapitulating in vitro the hypoxic in vivo micro-environment of tissue stem cell niches and their formation. These spheroids, therefore, nicely reproduce the finely regulated Ying-Yang equilibrium, which, together with other mechanisms, can be determinant in cell fate decisions in many pathophysiological scenarios, such as differentiation, fibrosis, regeneration, and oncogenesis. In this review, current progress in the knowledge of signaling pathways affecting EMT/MET and stemness regulation will be outlined by comparing data obtained from cellular spheroids systems, as ex vivo niches of stem cells derived from normal and tumoral tissues. The mechanistic correspondence in vivo and the possible pharmacological perspective will be also explored, focusing especially on the TGF-β-related networks, as well as others, such as SNAI1, PTEN, and EGR1. This latter, in particular, for its ability to convey multiple types of stimuli into relevant changes of the cell transcriptional program, can be regarded as a heterogeneous "stress-sensor" for EMT-related inducers (growth factor, hypoxia, mechano-stress), and thus as a therapeutic target.

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