The P body protein LSm1 contributes to stimulation of hepatitis C virus translation,but not replication,by microRNA-122

The P body protein LSm1 stimulates translation and replication of hepatitis C virus (HCV). As the liver-specific microRNA-122 (miR-122) is required for HCV replication and is associated with P bodies, we investigated whether regulation of HCV by LSm1 involves miR-122. Here, we demonstrate that LSm1 contributes to activation of HCV internal ribosome entry site (IRES)-driven translation by miR-122. This role for LSm1 is specialized for miR-122 translation activation, as LSm1 depletion does not affect the repressive function of miR-122 at 3′ untranslated region (UTR) sites, or miR-122–mediated cleavage at a perfectly complementary site. We find that LSm1 does not influence recruitment of the microRNA (miRNA)-induced silencing complex to the HCV 5′UTR, implying that it regulates miR-122 function subsequent to target binding. In contrast to the interplay between miR-122 and LSm1 in translation, we find that LSm1 is not required for miR-122 to stimulate HCV replication, suggesting that miR-122 regulation of HCV translation and replication have different requirements. For the first time, we have identified a protein factor that specifically contributes to activation of HCV IRES-driven translation by miR-122, but not to other activities of the miRNA. Our results enhance understanding of the mechanisms by which miR-122 and LSm1 regulate HCV.     

MicroRNA-1291-mediated silencing of IRE1α enhances Glypican-3 expression

MicroRNAs (miRNA) are generally described as negative regulators of gene expression. However, some evidence suggests that they may also play positive roles. As such, we reported that miR-1291 leads to a GPC3 mRNA expression increase in hepatoma cells through a 3′ untranslated region (UTR)-dependent mechanism. In the absence of any direct interaction between miR-1291 and GPC3 mRNA, we hypothesized that miR-1291 could act by silencing a negative regulator of GPC3 mRNA expression. Based on in silico predictions and experimental validation, we demonstrate herein that miR-1291 represses the expression of the mRNA encoding the endoplasmic reticulum (ER)-resident stress sensor IRE1α by interacting with a specific site located in the 5′ UTR. Moreover, we show, in vitro and in cultured cells, that IRE1α cleaves GPC3 mRNA at a 3′ UTR consensus site independently of ER stress, thereby prompting GPC3 mRNA degradation. Finally, we show that the expression of a miR-1291-resistant form of IRE1α abrogates the positive effects of miR-1291 on GPC3 mRNA expression. Collectively, our data demonstrate that miR-1291 is a biologically relevant regulator of GPC3 expression in hepatoma cells and acts through silencing of the ER stress sensor IRE1α.     

PARN deadenylase is involved in miRNA-dependent degradation of TP53 mRNA in mammalian cells

mRNA deadenylation is under the control of cis-acting regulatory elements, which include AU-rich elements (AREs) and microRNA (miRNA) targeting sites, within the 3′ untranslated region (3′ UTRs) of eukaryotic mRNAs. Deadenylases promote miRNA-induced mRNA decay through their interaction with miRNA-induced silencing complex (miRISC). However, the role of poly(A) specific ribonuclease (PARN) deadenylase in miRNA-dependent mRNA degradation has not been elucidated. Here, we present evidence that not only ARE- but also miRNA-mediated pathways are involved in PARN-mediated regulation of the steady state levels of TP53 mRNA, which encodes the tumor suppressor p53. Supporting this, Argonaute-2 (Ago-2), the core component of miRISC, can coexist in complexes with PARN resulting in the activation of its deadenylase activity. PARN regulates TP53 mRNA stability through not only an ARE but also an adjacent miR-504/miR-125b-targeting site in the 3′ UTR. More importantly, we found that miR-125b-loaded miRISC contributes to the specific recruitment of PARN to TP53 mRNA, and that can be reverted by the ARE-binding protein HuR. Together, our studies provide new insights into the role of PARN in miRNA-dependent control of mRNA decay and into the mechanisms behind the regulation of p53 expression.     

MicroRNA 181 Suppresses Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) Infection by Targeting PRRSV Receptor CD163

We previously showed that microRNA 181 (miR-181) can inhibit PRRSV replication by directly targeting its genomic RNA. Here, we report that miR-181 can downregulate the PRRSV receptor CD163 in blood monocytes and porcine alveolar macrophages (PAMs) through targeting the 3′ untranslated region (UTR) of CD163 mRNA. Downregulation of CD163 leads to the inhibition of PRRSV entry into PAMs and subsequently suppresses PRRSV infection. Our findings indicate that delivery of miR-181 can be used as antiviral therapy against PRRSV infection.     

In vitro characterization of a miR-122-sensitive double-helical switch element in the 5′ region of hepatitis C virus RNA

It has been proposed that the hepatitis C virus (HCV) internal ribosome entry site (IRES) resides within a locked conformation, owing to annealing of its immediate flanking sequences. In this study, structure probing using Escherichia coli dsRNA-specific RNase III and other classical tools showed that this region switches to an open conformation triggered by the liver-specific microRNA, miR-122. This structural transition, observed in vitro, may be the mechanistic basis for the involvement of downstream IRES structural domain VI in translation, as well as providing a role of liver-specific miR-122 in HCV infection. In addition, the induced RNA switching at the 5′ untranslated region could ultimately represent a new mechanism of action of micro-RNAs.