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Long noncoding RNAs (lncRNAs) are emerging as regulators of fundamental biological processes. Here we report on the characterization of an intergenic lncRNA expressed in epithelial tissues which we termed EPR (Epithelial cell Program Regulator). EPR is rapidly downregulated by TGF-β and its sustained expression largely reshapes the transcriptome, favors the acquisition of epithelial traits, and reduces cell proliferation in cultured mammary gland cells as well as in an animal model of orthotopic transplantation. EPR generates a small peptide that localizes at epithelial cell junctions but the RNA molecule per se accounts for the vast majority of EPR-induced gene expression changes. Mechanistically, EPR interacts with chromatin and regulates Cdkn1a gene expression by affecting both its transcription and mRNA decay through its association with SMAD3 and the mRNA decay-promoting factor KHSRP, respectively. We propose that EPR enables epithelial cells to control proliferation by modulating waves of gene expression in response to TGF-β. Several lncRNAs are regulated by TGF-β. Here the authors report that an intergenic lncRNA —EPR— is a component of the TGF-β signaling pathway and controls epithelial cell proliferation by altering transcription and mRNA decay of Cdkn1a. EPR overexpression restrains tumor growth of orthotopically transplanted mice.

The Golgi of goblet cells represents a specialized machine for mucin glycosylation. This process occurs in a specialized form of the secretory pathway, which remains poorly examined. Here, using high-resolution three-dimensional electron microscopy (EM), EM tomography, serial block face scanning EM (SBF-SEM) and immune EM we analyzed the secretory pathway in goblet cells and revealed that COPII-coated buds on the endoplasmic reticulum (ER) are extremely rare. The ERES vesicles with dimensions typical for the COPII-dependent vesicles were not found. The Golgi is formed by a single cisterna organized in a spiral with characteristics of the cycloid surface. This ribbon has a shape of a cup with irregular perforations. The Golgi cup is filled with secretory granules (SGs) containing glycosylated mucins. Their diameter is close to 1 µm. The cup is connected with ER exit sites (ERESs) with temporal bead-like connections, which are observed mostly near the craters observed at the externally located cis surface of the cup. The craters represent conus-like cavities formed by aligned holes of gradually decreasing diameters through the first three Golgi cisternae. These craters are localized directly opposite the ERES. Clusters of the 52 nm vesicles are visible between Golgi cisternae and between SGs. The accumulation of mucin, started in the fourth cisternal layer, induces distensions of the cisternal lumen. The thickness of these distensions gradually increases in size through the next cisternal layers. The spherical distensions are observed at the edges of the Golgi cup, where they fuse with SGs and detach from the cisternae. After the fusion of SGs located just below the apical plasma membrane (APM) with APM, mucus is secreted. The content of this SG becomes less osmiophilic and the excessive surface area of the APM is formed. This membrane is eliminated through the detachment of bubbles filled with another SG and surrounded with a double membrane or by collapse of the empty SG and transformation of the double membrane lacking a visible lumen into multilayered organelles, which move to the cell basis and are secreted into the intercellular space where the processes of dendritic cells are localized. These data are evaluated from the point of view of existing models of intracellular transport.

Although mammals possess roughly the same number of protein-coding genes as worms, it is evident that the non-coding transcriptome content has become far broader and more sophisticated during evolution. Indeed, the vital regulatory importance of both short and long non-coding RNAs (lncRNAs) has been demonstrated during the last two decades. RNA binding proteins (RBPs) represent approximately 7.5% of all proteins and regulate the fate and function of a huge number of transcripts thus contributing to ensure cellular homeostasis. Transcriptomic and proteomic studies revealed that RBP-based complexes often include lncRNAs. This review will describe examples of how lncRNA-RBP networks can virtually control all the post-transcriptional events in the cell.

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. Significance Long noncoding RNAs (lncRNAs) provide new layers of complexity to gene expression control. We report on the functional consequences of the interaction between the ssRNA-binding protein K homology-type splicing regulatory protein (KSRP) with H19 lncRNA (H19) in multipotent C2C12 cells able to differentiate in culture toward myotubes in response to activation of cell signaling pathways, including AKT. KSRP and H19 interact exclusively in undifferentiated C2C12 cells, and this favors KSRP’s ability to interact with the promyogenic transcript myogenin and to favor its degradation. AKT activation induces KSRP dissociation from H19 and, as a consequence, from myogenin mRNA that is stabilized. H19 likely acts as a scaffold that favors KSRP-mediated degradation of myogenin to contribute to the maintenance of the undifferentiated state of C2C12 cells.

KSRP is involved in mRNA instability, a role that is repressed upon AKT kinase–mediated phosphorylation, which promotes 14-3-3 interaction. This modification site is now shown to be exposed upon AKT phosphorylation through unfolding of the KH1 domain of KSRP, an event that allows 14-3-3 interaction, which in turn affects nuclear cytoplasmic partitioning. The AU-rich element (ARE)-mediated mRNA-degradation activity of the RNA binding K-homology splicing regulator protein (KSRP) is regulated by phosphorylation of a serine within its N-terminal KH domain (KH1). In the cell, phosphorylation promotes the interaction of KSRP and 14-3-3ζ protein and impairs the ability of KSRP to promote the degradation of its RNA targets. Here we examine the molecular details of this mechanism. We report that phosphorylation leads to the unfolding of the structurally atypical and unstable KH1, creating a site for 14-3-3ζ binding. Using this site, 14-3-3ζ discriminates between phosphorylated and unphosphorylated KH1, driving the nuclear localization of KSRP. 14-3-3ζ –KH1 interaction regulates the mRNA-decay activity of KSRP by sequestering the protein in a separate functional pool. This study demonstrates how an mRNA-degradation pathway is connected to extracellular signaling networks through the reversible unfolding of a protein domain.

Beta-catenin plays an essential role in several biological events including cell fate determination, cell proliferation, and transformation. Here we report that beta-catenin is encoded by a labile transcript whose half-life is prolonged by Wnt and phosphatidylinositol 3-kinase-AKT signaling. AKT phosphorylates the mRNA decay-promoting factor KSRP at a unique serine residue, induces its association with the multifunctional protein 14-3-3, and prevents KSRP interaction with the exoribonucleolytic complex exosome. This impairs KSRP's ability to promote rapid mRNA decay. Our results uncover an unanticipated level of control of beta-catenin expression pointing to KSRP as a required factor to ensure rapid degradation of beta-catenin in unstimulated cells. We propose KSRP phosphorylation as a link between phosphatidylinositol 3-kinase-AKT signaling and beta-catenin accumulation.

Pitx1 (refs 1 , 2 , 3 ) and Pitx2 (refs 4 , 5 ) are highly homologous, bicoid-related transcription factors. Pitx2 was initially identified as the gene responsible for the human Rieger syndrome 4 , an autosomal dominant condition that causes developmental abnormalities. Pitx2 is asymmetrically expressed in the left lateral-plate mesoderm 5 , 6 , 7 , 8 , 9 , 10 , 11 , and mutant mice with laterality defects show altered patterns of Pitx2 expression that correlate with changes in the visceral symmetry ( situs ). Ectopic expression of Pitx2 in the right lateral-plate mesoderm alters looping of the heart and gut and reverses body rotation in chick and Xenopus embryos 6 , 7 , 8 , 9 , 10 , 11 . Here we describe the phenotype of Pitx2 gene-deleted mice, characterized by defective body-wall closure, right pulmonary isomerism, altered cardiac position, arrest in turning and, subsequently, a block in the determination and proliferation events of anterior pituitary gland and tooth organogenesis. Thus, Pitx2 is a transcription factor that encodes ‘leftness’ of the lung.

Gene silencing mediated by either microRNAs (miRNAs) or Adenylate/uridylate-rich elements Mediated mRNA Degradation (AMD) is a powerful way to post-transcriptionally modulate gene expression. We and others have reported that the RNA-binding protein KSRP favors the biogenesis of select miRNAs (including let-7 family) and activates AMD promoting the decay of inherently labile mRNAs. Different layers of interplay between miRNA- and AMD-mediated gene silencing have been proposed in cultured cells, but the relationship between the two pathways in living organisms is still elusive. We conditionally deleted Dicer in mouse pituitary from embryonic day (E) 9.5 through Cre-mediated recombination. In situ hybridization, immunohistochemistry, and quantitative reverse transcriptase-PCR revealed that Dicer is essential for pituitary morphogenesis and correct expression of hormones. Strikingly, αGSU (alpha glycoprotein subunit, common to three pituitary hormones) was absent in Dicer-deleted pituitaries. αGSU mRNA is unstable and its half-life increases during pituitary development. A transcriptome-wide analysis of microdissected E12.5 pituitaries revealed a significant increment of KSRP expression in conditional Dicer-deleted mice. We found that KSRP directly binds to αGSU mRNA, promoting its rapid decay; and, during pituitary development, αGSU expression displays an inverse temporal relationship to KSRP. Further, let-7b/c downregulated KSRP expression, promoting the degradation of its mRNA by directly binding to the 3'UTR. Therefore, we propose a model in which let-7b/c and KSRP operate within a negative feedback loop. Starting from E12.5, KSRP induces the maturation of let-7b/c that, in turn, post-transcriptionally downregulates the expression of KSRP itself. This event leads to stabilization of αGSU mRNA, which ultimately enhances the steady-state expression levels. We have identified a post-transcriptional regulatory network active during mouse pituitary development in which the expression of the hormone αGSU is increased by let7b/c through downregulation of KSRP. Our study unveils a functional crosstalk between miRNA- and AMD-dependent gene regulation during mammalian organogenesis events.

Pitx2 is a bicoid-related homeodomain factor that is required for effective cell type-specific proliferation directly activating a specific growth-regulating gene cyclin D2. Here, we report that Pitx2, in response to the Wnt/β-catenin pathway and growth signals, also can regulate c-Myc and cyclin D1. Investigation of molecular mechanisms required for Pitx2-dependent proliferation, in these cases, further supports a nuclear role for β-catenin in preventing the histone deacetylase 1-dependent inhibitory functions of several DNA-binding transcriptional repressors, potentially including E2F4/p130 pocket protein inhibitory complex, as well as lymphoid enhancer factor 1 and Pitx2, by dismissal of histone deacetylase 1 and loss of its enzymatic activity. Thus, β-catenin plays a signal-integrating role in Wnt- and growth factor-dependent proliferation events in mammalian development by both derepressing several classes of repressors and by activating Pitx2, regulating the activity of several growth control genes.