Untranslated Region Interactions Research Articles

Epigallocatechin gallate activates miR-193a-3p and protects mice against glucocorticoid-induced osteoporosis by targeting NFATC1 expression

Purpose: To investigate the effect of epigallocatechin gallate (EGCG) on microRNAs in a mouse model of glucocorticoid-induced osteoporosis (GIOP), and the mechanism involved.
 Methods: Osteoclast-specific marker mRNA expressions, receptor activator of nuclear factor kappa-B ligand (RANKL), receptor activator of nuclear factor κ B (RANK), and miRNA expressions were determined using reverse transcription polymerase chain reaction (RT-qPCR) analysis. Western blotting was used to assay protein expressions, while miRNA and 3’UTR interaction studies were carried out with reporter assay.
 Results: Treatment with EGCG resulted in downregulation of glucocorticoid-induced expressions of RANKL, RANK and osteoclast-specific markers i.e. tumor necrosis factor receptor-associated factor 6, (TRAF6), nuclear factor of activated T cells 1 (NFATc1), cathepsin K, matrix metallopeptidase 9 (MMP9) and tartrate-resistant acid phosphatase (TRAP). Furthermore, EGCG treatment significantly reduced reactive oxygen species (ROS) levels and inflammatory cytokine expressions in GIOP mice. The expression of miRNA-targeting osteoclast marker mmu-mir-193-3p was significantly down-regulated in GIOP mice. However, EGCG treatment increased mmu-mir-193-3p expression and had specific interaction with NFATc1 3’UTR (3’-untranslated region). In vitro results showed that mmu-mir-193-3p mimics downregulated dexamethasone (DXM)-induced osteoclast-specific marker expressions.
 Conclusion: These results show that EGCG exerts a protective role against GIOP by upregulating miR- 193a-3p expressions.
 Keywords: Epigallocatechin gallate, Glucocorticoids, RANKL, Osteoporosis

APC gene 3'UTR SNPs and interactions with environmental factors are correlated with risk of colorectal cancer in Chinese Han population.

Objective: To study the correlation between adenomatous polyposis coli (APC) gene 3′ untranslated region (UTR) single nucleotide polymorphisms (SNPs) and their interactions with environmental factors and the risk of colorectal cancer (CRC) in a Chinese Han population.Methods: Genotypes of APC gene 3′UTR rs1804197, rs41116, rs448475, and rs397768 loci in 340 Chinese Han patients with CRC and 340 healthy controls were analyzed. All patients with CRC were analyzed for progression-free survival (PFS) during a 3-year follow-up.Results: The risk of CRC in subjects carrying the APC gene rs1804197 A allele was 2.95-times higher than for the C allele carriers. The interactions of the rs1804197 SNP with body mass index (BMI) and smoking were associated with the risk of CRC. The risk of CRC in the APC gene rs397768 G allele carriers was 1.68-times higher than in the A allele carriers. The interaction between the rs397768 locus SNP and gender was also associated with the risk of CRC. The 3-year PFS of patients with APC gene rs1804197 AA genotype, CA genotype, and CC genotype CRC decreased in this order, with significant difference. In addition, the 3-year PFS of rs397768 locus GG genotype, AG genotype, and AA genotype CRC patients decreased in this order, and the difference was significant.Conclusion: The rs1804197 locus in the 3′UTR region of the APC gene and its interactions with BMI and smoking are associated with the risk of CRC in a Chinese Han population. In addition, the interaction between rs397768 locus SNP and gender is related to the risk of CRC.

A Novel Method for Assessing the Statistical Significance of RNA-RNA Interactions Between Two Long RNAs.

RNA-RNA interactions are key mechanisms through which noncoding RNA (ncRNA) regions exert biological functions. Computational prediction of RNA-RNA interactions is an essential method for detecting novel RNA-RNA interactions because their comprehensive detection by biological experimentation is still quite difficult. Many RNA-RNA interaction prediction tools have been developed, but they tend to produce many false positives. Accordingly, assessment of the statistical significance of computationally predicted interactions is an important task. However, there is no method to evaluate the statistical significance of RNA-RNA interactions that is applicable to interactions between two long RNA sequences. We developed a method to calculate the p-value for the minimal interaction energy between two long RNA sequences. The developed method depends on the fact that minimum interaction energies of RNA-RNA interactions between long RNAs follow a Gumbel distribution when repeat sequences in RNAs are masked. To show the usefulness of the developed method, we applied it to whole human 5'-untranslated region (UTR) and 3'-UTR sequences to detect novel 5'-UTR-3'-UTR interactions. We thus identified two significant 5'-UTR-3'-UTR interactions. Specifically, the human small proline-rich repeat protein 3 shows conserved 5'-UTR-3'-UTR interactions with some nucleotide variations preserving base pairings among primates. Our developed method enables us to detect statistically significant RNA-RNA interactions between long RNAs such as long ncRNAs. Statistical significance estimates help in identification of interactions for experimental validation and provide novel insights into the function of ncRNA regions.

Phosphorylation of poly(rC) binding protein 1 (PCBP1) contributes to stabilization of mu opioid receptor (MOR) mRNA via interaction with AU-rich element RNA-binding protein 1 (AUF1) and poly A binding protein (PABP)

Gene regulation at the post-transcriptional level is frequently based on cis- and trans-acting factors on target mRNAs. We found a C-rich element (CRE) in mu-opioid receptor (MOR) 3′-untranslated region (UTR) to which poly (rC) binding protein 1 (PCBP1) binds, resulting in MOR mRNA stabilization. RNA immunoprecipitation and RNA EMSA revealed the formation of PCBP1-RNA complexes at the element. Knockdown of PCBP1 decreased MOR mRNA half-life and protein expression. Stimulation by forskolin increased cytoplasmic localization of PCBP1 and PCBP1/MOR 3′-UTR interactions via increased serine phosphorylation that was blocked by protein kinase A (PKA) or (phosphatidyl inositol-3) PI3-kinase inhibitors. The forskolin treatment also enhanced serine- and tyrosine-phosphorylation of AU-rich element binding protein (AUF1), concurrent with its increased binding to the CRE, and led to an increased interaction of poly A binding protein (PABP) with the CRE and poly(A) sites. AUF1 phosphorylation also led to an increased interaction with PCBP1. These findings suggest that a single co-regulator, PCBP1, plays a crucial role in stabilizing MOR mRNA, and is induced by PKA signaling by conforming to AUF1 and PABP.

MiR-17-92 represses PTPROt and PP2A phosphatases and amplifies tonic BCR signaling in DLBCL cells.

B-cell receptor (BCR) signaling plays a pivotal role in the pathogenesis of diffuse large B-cell lymphoma (DLBCL) and targeting the BCR pathway is a highly promising therapeutic strategy in this malignancy. The oncogenic microRNA miR-17-92 modulates multiple cellular processes such as survival, proliferation, apoptosis, angiogenesis, and BCR signaling. In the present study, we identified new targets of miR-17-92, PTPROt (protein phosphatase, receptor type O, truncated) and PP2A (protein phosphatase 2A) phosphatases, which regulate the activity of spleen tyrosine kinase (SYK) and AKT, critical components of BCR signal transduction in DLBCL cells. Introduction of miR-17-92 into DLBCL cells dampened the expression of the PTPROt and PP2A regulatory subunits PPP2R2A (protein phosphatase 2, regulatory subunit B, alpha) and PPP2R5E (protein phosphatase 2, regulatory subunit B, epsilon isoform) and increased the magnitude of SYK and AKT phosphorylation upon BCR ligation. Finally, we found that miR-17-92 expression modulates response to inhibitors of BCR signaling because downregulation of miR-17-92 increased SYK inhibitor-mediated toxicity in DLBCL cells. Our study reveals novel posttranscriptional regulatory pathways that contribute to the deregulation of BCR signaling and modulate SYK inhibitor activity in DLBCL.

Cellular Proteins Act as Bridge Between 5' and 3' Ends of the Coxsackievirus B3 Mediating Genome Circularization During RNA Translation.

The positive single-stranded RNA genome of the Coxsackievirus B3 (CVB3) contains a 5' untranslated region (UTR) which hosts the internal ribosome entry site (IRES) element that governs cap-independent translation initiation and a polyadenylated 3' UTR which is required for stimulating the IRES activity. Viral RNA genomes could circularize to regulate initiation of translation and RNA synthesis at 5' and 3' ends. Interactions could either take place by direct RNA-RNA contacts, through cellular protein bridges mediating RNA circularization or both. Accordingly, we aimed to assess the nature of molecular interactions between these two regions and to evaluate cellular factors required for mRNA 3' end-mediated stimulation of CVB3 IRES-driven translation. By gel shift assays, we have showed that combining, in vitro, 5' and 3' UTR fragments had no discernible effect on the structures of RNAs, arguing against the presence of specific canonical RNA-RNA cyclization sequences between these two regions. Competitive UV crosslinking assays using BHK-21 cell extract showed common cellular proteins eIF3b, PTB, and La binding to both 5'- and 3' end RNAs. PCBP 1-2 and PABP were shown to bind, respectively, to 5' and 3' UTR probes. Taking together, these data suggest that CVB3 5'-3' end bridging occurs through 5' UTR-protein-protein-3' UTR interactions and not through RNA-RNA direct contact. The dual involvement of the 3' and 5' UTRs in controlling viral translation and RNA synthesis highlights the relevance of these regions in the infectious virus life cycle, making them suitable candidates for targeted CVB3 antiviral therapy.

Recruitment of the 40S Ribosome Subunit to the 3′-Untranslated Region (UTR) of a Viral mRNA, via the eIF4 Complex, Facilitates Cap-independent Translation

Barley yellow dwarf virus mRNA, which lacks both cap and poly(A) tail, has a translation element (3'-BTE) in its 3'-UTR essential for efficient translation initiation at the 5'-proximal AUG. This mechanism requires eukaryotic initiation factor 4G (eIF4G), subunit of heterodimer eIF4F (plant eIF4F lacks eIF4A), and 3'-BTE-5'-UTR interaction. Using fluorescence anisotropy, SHAPE (selective 2'-hydroxyl acylation analyzed by primer extension) analysis, and toeprinting, we found that (i) 40S subunits bind to BTE (Kd = 350 ± 30 nm), (ii) the helicase complex eIF4F-eIF4A-eIF4B-ATP increases 40S subunit binding (Kd = 120 ± 10 nm) to the conserved stem-loop I of the 3'-BTE by exposing more unpaired bases, and (iii) long distance base pairing transfers this complex to the 5'-end of the mRNA, where translation initiates. Although 3'-5' interactions have been recognized as important in mRNA translation, barley yellow dwarf virus employs a novel mechanism utilizing the 3'-UTR as the primary site of ribosome recruitment.

Hallmarks of hepatitis C virus in equine hepacivirus.

Equine hepacivirus (EHcV) has been identified as a closely related homologue of hepatitis C virus (HCV) in the United States, the United Kingdom, and Germany, but not in Asian countries. In this study, we genetically and serologically screened 31 serum samples obtained from Japanese-born domestic horses for EHcV infection and subsequently identified 11 PCR-positive and 7 seropositive serum samples. We determined the full sequence of the EHcV genome, including the 3' untranslated region (UTR), which had previously not been completely revealed. The polyprotein of a Japanese EHcV strain showed approximately 95% homology to those of the reported strains. HCV-like cis-acting RNA elements, including the stem-loop structures of the 3' UTR and kissing-loop interaction were deduced from regions around both UTRs of the EHcV genome. A comparison of the EHcV and HCV core proteins revealed that Ile(190) and Phe(191) of the EHcV core protein could be important for cleavage of the core protein by signal peptide peptidase (SPP) and were replaced with Ala and Leu, respectively, which inhibited intramembrane cleavage of the EHcV core protein. The loss-of-function mutant of SPP abrogated intramembrane cleavage of the EHcV core protein and bound EHcV core protein, suggesting that the EHcV core protein may be cleaved by SPP to become a mature form. The wild-type EHcV core protein, but not the SPP-resistant mutant, was localized on lipid droplets and partially on the lipid raft-like membrane in a manner similar to that of the HCV core protein. These results suggest that EHcV may conserve the genetic and biological properties of HCV. EHcV, which shows the highest amino acid or nucleotide homology to HCV among hepaciviruses, was previously reported to infect horses from Western, but not Asian, countries. We herein report EHcV infection in Japanese-born horses. In this study, HCV-like RNA secondary structures around both UTRs were predicted by determining the whole-genome sequence of EHcV. Our results also suggest that the EHcV core protein is cleaved by SPP to become a mature form and then is localized on lipid droplets and partially on lipid raft-like membranes in a manner similar to that of the HCV core protein. Hence, EHcV was identified as a closely related homologue of HCV based on its genetic structure as well as its biological properties. A clearer understanding of the epidemiology, genetic structure, and infection mechanism of EHcV will assist in elucidating the evolution of hepaciviruses as well as the development of surrogate models for the study of HCV.

Susceptibility to chronic thromboembolic pulmonary hypertension may be conferred by miR-759 via its targeted interaction with polymorphic fibrinogen alpha gene

A deletion/insertion (Del/Ins) polymorphism of 28 base pairs (bp) in the 3' untranslated region (UTR) of fibrinogen alpha gene (FGA) was associated with thromboembolic diseases, but the underlying mechanisms remain unknown. Computational predication reveals that the 28 bp polymorphic fragment is complementary to the sequence of a microRNA, miR-759. In this study, we aim to investigate the association and implicated mechanisms between FGA polymorphisms and the susceptibility to chronic thromboembolic pulmonary hypertension (CTEPH). The Del/Ins polymorphism was analyzed in 190 patients with CTEPH and 628 controls. The FGA 3'UTR and miR-759 interaction was investigated using luciferase assay and quantitative RT-PCR method. Expression of miR-759 and FGA in human tissues was investigated by RT-PCR. The results reveal that the allele frequency of Ins was significantly higher in the patients than in the controls (55.8 vs. 47.1%, P=0.003, odds ratio=1.42, 95% confidence interval: 1.13-1.79). Both miR-759 and FGA were expressed in human liver. Co-transfection of miR-759 decreased the expression and mRNA stability of reporter gene containing the FGA 3'UTR. The effect of miR-759 was stronger on the Ins allele than on the Del allele. These observations suggest that the expression of FGA was regulated by miR-759 through its interaction at the polymorphic 3'UTR sequence, which was associated with the susceptibility to CTEPH.

Structural Lability in Stem–Loop 1 Drives a 5′ UTR–3′ UTR Interaction in Coronavirus Replication

The leader RNA of the 5′ untranslated region (UTR) of coronaviral genomes contains two stem–loop structures denoted SL1 and SL2. Herein, we show that SL1 is functionally and structurally bipartite. While the upper region of SL1 is required to be paired, we observe strong genetic selection against viruses that contain a deletion of A35, an extrahelical nucleotide that destabilizes SL1, in favor of genomes that contain a diverse panel of destabilizing second-site mutations, due to introduction of a noncanonical base pair near A35. Viruses containing destabilizing SL1-ΔA35 mutations also contain one of two specific mutations in the 3′ UTR. Thermal denaturation and imino proton solvent exchange experiments reveal that the lower half of SL1 is unstable and that second-site SL1-ΔA35 substitutions are characterized by one or more features of the wild-type SL1. We propose a “dynamic SL1” model, in which the base of SL1 has an optimized lability required to mediate a physical interaction between the 5′ UTR and the 3′ UTR that stimulates subgenomic RNA synthesis. Although not conserved at the nucleotide sequence level, these general structural characteristics of SL1 appear to be conserved in other coronaviral genomes.

Disruption of Ribosomal Scanning on the 5′-Untranslated Region, and Not Restriction of Translational Initiation per se, Modulates the Stability of Nonaberrant mRNAs in the Yeast Saccharomyces cerevisiae

Translation and mRNA decay constitute key players in the post-transcriptional control of gene expression. We examine the mechanisms by which the 5'-untranslated region (UTR) of nonaberrant mRNAs acts to modulate both these processes in Saccharomyces cerevisiae. Two classes of functional relationship between ribosome-5'-UTR interactions and mRNA decay are identifiable. In the first of these, elements in the main open reading frame (ORF) dictate how the decay process reacts to inhibitory structures in the 5'-UTR. The same types of stability modulation can be elicited by trans-regulation of translation via inducible binding of the iron-regulatory protein to an iron-responsive element located 9 nucleotides from the 5' cap. A eukaryotic translational repressor can therefore modulate mRNA decay via the 5'-UTR. In contrast, translational regulation mediated via changes in the activity of the cap-binding eukaryotic translation initiation factor eIF-4E bypasses translation-dependent pathways of mRNA degradation. Thus modulation of mRNA stability via the 5'-UTR depends on disruption of the scanning process, rather than changes in translational initiation efficiency per se. In the second class of pathway, an upstream ORF (uORF) functions as a powerful destabilizing element, inducing termination-dependent degradation that is apparently independent of any main ORF determinants but influenced by the efficiencies of ribosomal recognition of the uORF start and stop codons. This latter mechanism provides a regulatable means to modulate the stability of nonaberrant mRNAs via a UPF-dependent pathway.