Competition between RNA-binding proteins CELF1 and HuR modulates MYC translation and intestinal epithelium renewal

The mammalian intestinal epithelium is one of the most rapidly self-renewing tissues in the body, and its integrity is preserved through strict regulation. The RNA-binding protein (RBP) ELAV-like family member 1 (CELF1), also referred to as CUG-binding protein 1 (CUGBP1), regulates the stability and translation of target mRNAs and is implicated in many aspects of cellular physiology. We show that CELF1 competes with the RBP HuR to modulate MYC translation and regulates intestinal epithelial homeostasis. Growth inhibition of the small intestinal mucosa by fasting in mice was associated with increased CELF1/Myc mRNA association and decreased MYC expression. At the molecular level, CELF1 was found to bind the 3′-untranslated region (UTR) of Myc mRNA and repressed MYC translation without affecting total Myc mRNA levels. HuR interacted with the same Myc 3′-UTR element, and increasing the levels of HuR decreased CELF1 binding to Myc mRNA. In contrast, increasing the concentrations of CELF1 inhibited formation of the [HuR/Myc mRNA] complex. Depletion of cellular polyamines also increased CELF1 and enhanced CELF1 association with Myc mRNA, thus suppressing MYC translation. Moreover, ectopic CELF1 overexpression caused G1-phase growth arrest, whereas CELF1 silencing promoted cell proliferation. These results indicate that CELF1 represses MYC translation by decreasing Myc mRNA association with HuR and provide new insight into the molecular functions of RBPs in the regulation of intestinal mucosal growth.     

Human miR-3145 inhibits influenza A viruses replication by targeting and silencing viral PB1 gene

MicroRNAs (miRNAs) play an important role in the regulation of gene expression and are involved in many cellular processes including inhibition of viral replication in infected cells. In this study, three subtypes of influenza A viruses (pH1N1, H5N1 and H3N2) were analyzed to identify candidate human miRNAs targeting and silencing viral genes expression. Candidate human miRNAs were predicted by miRBase and RNAhybrid based on minimum free energy (MFE) and hybridization patterns between human miRNAs and viral target genes. In silico analysis presented 76 miRNAs targeting influenza A viruses, including 70 miRNAs that targeted specific subtypes (21 for pH1N1, 27 for H5N1 and 22 for H3N2) and 6 miRNAs (miR-216b, miR-3145, miR-3682, miR-4513, miR-4753 and miR-5693) that targeted multiple subtypes of influenza A viruses. Interestingly, miR-3145 is the only candidate miRNA targeting all three subtypes of influenza A viruses. The miR-3145 targets to PB1 encoding polymerase basic protein 1, which is the main component of the viral polymerase complex. The silencing effect of miR-3145 was validated by 3′-UTR reporter assay and inhibition of influenza viral replication in A549 cells. In 3′-UTR reporter assay, results revealed that miR-3145 triggered significant reduction of the luciferase activity. Moreover, expression of viral PB1 genes was also inhibited considerably (P value < 0.05) in viral infected cells expressing mimic miR-3145. In conclusion, this study demonstrated that human miR-3145 triggered silencing of viral PB1 genes and lead to inhibition of multiple subtypes of influenza viral replication. Therefore, hsa-miR-3145 might be useful for alternative treatment of influenza A viruses in the future.     

RNA-binding protein HuR promotes growth of small intestinal mucosa by activating the Wnt signaling pathway

Inhibition of growth of the intestinal epithelium, a rapidly self-renewing tissue, is commonly found in various critical disorders. The RNA-binding protein HuR is highly expressed in the gut mucosa and modulates the stability and translation of target mRNAs, but its exact biological function in the intestinal epithelium remains unclear. Here, we investigated the role of HuR in intestinal homeostasis using a genetic model and further defined its target mRNAs. Targeted deletion of HuR in intestinal epithelial cells caused significant mucosal atrophy in the small intestine, as indicated by decreased cell proliferation within the crypts and subsequent shrinkages of crypts and villi. In addition, the HuR-deficient intestinal epithelium also displayed decreased regenerative potential of crypt progenitors after exposure to irradiation. HuR deficiency decreased expression of the Wnt coreceptor LDL receptor–related protein 6 (LRP6) in the mucosal tissues. At the molecular level, HuR was found to bind the Lrp6 mRNA via its 3′-untranslated region and enhanced LRP6 expression by stabilizing Lrp6 mRNA and stimulating its translation. These results indicate that HuR is essential for normal mucosal growth in the small intestine by altering Wnt signals through up-regulation of LRP6 expression and highlight a novel role of HuR deficiency in the pathogenesis of intestinal mucosal atrophy under pathological conditions.     

MicroRNAs Regulate Pituitary Development,and MicroRNA 26b Specifically Targets Lymphoid Enhancer Factor 1 (Lef-1), Which Modulates Pituitary Transcription Factor 1 (Pit-1) Expression

To understand the role of microRNAs (miRNAs) in pituitary development, a group of pituitary-specific miRNAs were identified, and Dicer1 was then conditionally knocked out using the Pitx2-Cre mouse, resulting in the loss of mature miRNAs in the anterior pituitary. The Pitx2-Cre/Dicer1 mutant mice demonstrate growth retardation, and the pituitaries are hypoplastic with an abnormal branching of the anterior lobe, revealing a role for microRNAs in pituitary development. Growth hormone, prolactin, and thyroid-stimulating hormone β-subunit expression were decreased in the Dicer1 mutant mouse, whereas proopiomelanocortin and luteinizing hormone β-subunit expression were normal in the mutant pituitary. Further analyses revealed decreased Pit-1 and increased Lef-1 expression in the mutant mouse pituitary, consistent with the repression of the Pit-1 promoter by Lef-1. Lef-1 directly targets and represses the Pit-1 promoter. miRNA-26b (miR-26b) was identified as targeting Lef-1 expression, and miR-26b represses Lef-1 in pituitary and non-pituitary cell lines. Furthermore, miR-26b up-regulates Pit-1 and growth hormone expression by attenuating Lef-1 expression in GH3 cells. This study demonstrates that microRNAs are critical for anterior pituitary development and that miR-26b regulates Pit-1 expression by inhibiting Lef-1 expression and may promote Pit-1 lineage differentiation during pituitary development.     

microRNA-122 Dependent Binding of Ago2 Protein to Hepatitis C Virus RNA Is Associated with Enhanced RNA Stability and Translation Stimulation

Translation of Hepatitis C Virus (HCV) RNA is directed by an internal ribosome entry site (IRES) in the 5′-untranslated region (5′-UTR). HCV translation is stimulated by the liver-specific microRNA-122 (miR-122) that binds to two binding sites between the stem-loops I and II near the 5′-end of the 5′-UTR. Here, we show that Argonaute (Ago) 2 protein binds to the HCV 5′-UTR in a miR-122-dependent manner, whereas the HCV 3′-UTR does not bind Ago2. miR-122 also recruits Ago1 to the HCV 5’-UTR. Only miRNA duplex precursors of the correct length stimulate HCV translation, indicating that the duplex miR-122 precursors are unwound by a complex that measures their length. Insertions in the 5′-UTR between the miR-122 binding sites and the IRES only slightly decrease translation stimulation by miR-122. In contrast, partially masking the miR-122 binding sites in a stem-loop structure impairs Ago2 binding and translation stimulation by miR-122. In an RNA decay assay, also miR-122-mediated RNA stability contributes to HCV translation stimulation. These results suggest that Ago2 protein is directly involved in loading miR-122 to the HCV RNA and mediating RNA stability and translation stimulation.     

Transgenic Expression of miR-222 Disrupts Intestinal Epithelial Regeneration by Targeting Multiple Genes Including Frizzled-7

Defects in intestinal epithelial integrity occur commonly in various pathologies. miR-222 is implicated in many aspects of cellular function and plays an important role in several diseases, but its exact biological function in the intestinal epithelium is underexplored. We generated mice with intestinal epithelial tissue-specific overexpression of miR-222 to investigate the function of miR-222 in intestinal physiology and diseases in vivo. Transgenic expression of miR-222 inhibited mucosal growth and increased susceptibility to apoptosis in the small intestine, thus leading to mucosal atrophy. The miR-222–elevated intestinal epithelium was vulnerable to pathological stress, since local overexpression of miR-222 not only delayed mucosal repair after ischemia/reperfusion-induced injury, but also exacerbated gut barrier dysfunction induced by exposure to cecal ligation and puncture. miR-222 overexpression also decreased expression of the Wnt receptor Frizzled-7 (FZD7), cyclin-dependent kinase 4 and tight junctions in the mucosal tissue. Mechanistically, we identified the Fzd7 messenger ribonucleic acid (mRNA) as a novel target of miR-222 and found that [miR-222/Fzd7 mRNA] association repressed Fzd7 mRNA translation. These results implicate miR-222 as a negative regulator of normal intestinal epithelial regeneration and protection by downregulating expression of multiple genes including the Fzd7. Our findings also suggest a novel role of increased miR-222 in the pathogenesis of mucosal growth inhibition, delayed healing and barrier dysfunction.     

MicroRNA 218 Acts as a Tumor Suppressor by Targeting Multiple Cancer Phenotype-associated Genes in Medulloblastoma

Aberrant expression of microRNAs has been implicated in many cancers. We recently demonstrated differential expression of several microRNAs in medulloblastoma. In this study, the regulation and function of microRNA 218 (miR-218), which is significantly underexpressed in medulloblastoma, was evaluated. Re-expression of miR-218 resulted in a significant decrease in medulloblastoma cell growth, cell colony formation, cell migration, invasion, and tumor sphere size. We used C17.2 neural stem cells as a model to show that increased miR-218 expression results in increased cell differentiation and also decreased malignant transformation when transfected with the oncogene REST. These results suggest that miR-218 acts as a tumor suppressor in medulloblastoma. MicroRNAs function by down-regulating translation of target mRNAs. Targets are determined by imperfect base pairing of the microRNA to the 3′-UTR of the mRNA. To comprehensively identify actual miR-218 targets, medulloblastoma cells overexpressing miR-218 and control cells were subjected to high throughput sequencing of RNA isolated by cross-linking immunoprecipitation, a technique that identifies the mRNAs bound to the RNA-induced silencing complex component protein Argonaute 2. High throughput sequencing of mRNAs identified 618 genes as targets of miR-218 and included both previously validated targets and many targets not predicted computationally. Additional work further confirmed CDK6, RICTOR, and CTSB (cathepsin B) as targets of miR-218 and examined the functional role of one of these targets, CDK6, in medulloblastoma.