Purification of an Arabidopsis chloroplast extract with in vitro RNA processing activity on psbA and petD 3'-untranslated regions

The mature 3′-end of many chloroplast mRNAs is generated by the processing of the 3′-untranslated region (3′-UTR), which is a mechanism that involves the removal of a segment located downstream an inverted repeat sequence that forms a stem-loop structure. Nuclear-encoded chloroplast RNA binding proteins associate with the stem-loop to process the 3′-UTR or to influence mRNA stability. A spinach chloroplast processing extract (CPE) has been previously generated and used to in vitro dissect the biochemical mechanism underlying 3′-UTR processing. Being Arabidopsis thaliana an important genetic model, the development of a CPE allowing to correlate 3′-UTR processing activity with genes encoding proteins involved in this process, would be of great relevance. Here, we developed a purification protocol that generated an Arabidopsis CPE able to correctly process a psbA 3′-UTR precursor. By UV crosslinking, we characterized the protein patterns generated by the interaction of RNA binding proteins with Arabidopsis psbA and petD 3′-UTRs, finding that each 3′-UTR bound specific proteins. By testing whether Arabidopsis CPE proteins were able to bind spinach ortholog 3′-UTRs, we also found they were bound by specific proteins. When Arabidopsis CPE 3′-UTR processing activity on ortholog spinach 3′-UTRs was assessed, stable products appeared: for psbA, a smaller size product than the expected mature 3′-end, and for petD, low amounts of the expected product plus several others of smaller sizes. These results suggest that the 3′-UTR processing mechanism of these chloroplast mRNAs might be partially conserved in Arabidopsis and spinach.     

MicroRNA-101 downregulates Alzheimer's amyloid-β precursor protein levels in human cell cultures and is differentially expressed

The full repertoire of regulatory interactions utilized by human cells to control expression of amyloid-β precursor protein (APP) is still undefined. We investigated here the contribution of microRNA (miRNA) to this regulatory network. Several bioinformatic algorithms predicted miR-101 target sites within the APP 3′-untranslated region (3′-UTR). Using reporter assays, we confirmed that, in human cell cultures, miR-101 significantly reduced the expression of a reporter under control of APP 3′-UTR. Mutation of predicted site 1, but not site 2, eliminated this reporter response. Delivery of miR-101 directly to human HeLa cells significantly reduced APP levels and this effect was eliminated by co-transfection with a miR-101 antisense inhibitor. Delivery of a specific target protector designed to blockade the interaction between miR-101 and its functional target site within APP 3′-UTR enhanced APP levels in HeLa. Therefore, endogenous miR-101 regulates expression of APP in human cells via a specific site located within its 3′-UTR. Finally, we demonstrate that, across a series of human cell lines, highest expression of miR-101 levels was observed in model NT2 neurons.     

MicroRNAs function as cis- and trans-acting modulators of peripheral circadian clocks

Based on their extracellular expression and targeting of the clock gene Bmal1, miR-142-3p and miR-494 were analyzed for evidence of vesicle-mediated communication between cells and intracellular functional activity. Our studies demonstrate that: miR-142-3p + miR-494 overexpression decreases endogenous BMAL1 levels, increases the period of Per2 oscillations, and increases extracellular miR-142-3p/miR-494 abundance in conditioned medium; miRNA-enriched medium increases intracellular expression of miR-142-3p and represses Bmal1 3′-UTR activity in naïve cells; and inhibitors of vesicular trafficking modulate intercellular communication of these miRNAs and ensemble Per2 rhythms. Thus, miR-142-3p and miR-494 may function as cis- and trans-acting signals contributing to local temporal coordination of cell-autonomous circadian clocks.     

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.     

Identification of miR-200a as a novel suppressor of connexin 43?in breast cancer cells

Both miRNAs (miRs) and connexin 43 (Cx43) were important regulators of the metastasis of breast cancer, whereas the miRs regulating Cx43 expression in breast cancer cells were still obscure. In the present study, we scanned and found miR-1, miR-206, miR-200a, miR-381, miR-23a/b and miR-186 were functional suppressors of human Cx43 mRNA and protein expression. Specially, we demonstrated that only miR-200a could directly target the 3′-untranslated region (3′-UTR) of human Cx43 gene. Functionally, overexpression of Cx43 in MCF cells potentiated the migration activity, whereas additional miR-200a treatment notably prevented this effect. Finally, we demonstrated that decreased levels of miR-200a and elevated expression of Cx43 in the metastatic breast cancer tissues compared with the primary ones. Thus, we are the first to identify miR-200a as a novel and direct suppressor of human Cx43, indicating that miR200a/Cx43 axis might be a useful diagnostic and therapeutic target of metastatic breast cancer.     

miR-17 inhibitor suppressed osteosarcoma tumor growth and metastasis via increasing PTEN expression

MicroRNAs (miRNAs) play essential roles in cancer development and progression. Here, we investigated the role of miR-17 in the progression and metastasis of osteosarcoma (OS). miR-17 was frequently increased in OS tissues and cell lines. Inhibition of miR-17 in OS cell lines substantially suppressed cell proliferation, migration, and invasion. Phosphatase and tensin homolog (PTEN) was identified as a target of miR-17, and ectopic expression of miR-17 inhibited PTEN by direct binding to its 3′-untranslated region (3′-UTR). Expression of miR-17 was negatively correlated with PTEN in OS tissues. Together, these findings indicate that miR-17 acts as an oncogenic miRNA and may contribute to the progression and metastasis of OS, suggesting miR-17 as a potential novel diagnostic and therapeutic target of OS.     

Differential stimulation of hepatitis C virus RNA translation by microRNA-122 in different cell cycle phases

Hepatitis C virus (HCV) replicates preferentially in the liver, and in most cases, the HCV infection becomes chronic and often results in hepatocellular carcinoma. When the HCV plus-strand RNA genome has been delivered to the cytosol of the infected cell, its translation is directed by the internal ribosome entry site (IRES) in the 5′-untranslated region (5′-UTR) of the viral RNA. Thereby, IRES activity is modulated by several host factors. In particular, the liver-specific microRNA-122 (miR-122) interacts with two target sites in the HCV 5′-UTR and stimulates HCV translation, thereby most likely contributing to HCV liver tropism. Here, we show that HCV IRES-dependent translation efficiency in the hepatoma cell line Huh7 is highest during the G₀ and G₁ phases of the cell cycle but significantly drops during S phase and even more in the G₂/M phase. The superimposed stimulation of HCV translation by ectopic miR-122 works best during G₀, G₁ and G₂/M phases but is lower during S phase. However, the levels of Ago2 protein do not substantially change during cell cycle phases, indicating that other cellular factors involved in HCV translation stimulation by miR-122 may be differentially expressed in different cell cycle phases. Moreover, the levels of endogenously expressed miR-122 in Huh7 cells are lowest in S phase, indicating that the predominant G₀/G₁ state of non-dividing hepatocytes in the liver facilitates high expression of the HCV genome and stimulation by miR-122, with yet-unknown factors involved in the differential extent of stimulation by miR-122.Key words: HCV, translation, miR-122, microRNA, miRNA, Ago, Ago2     

Genetic variations of mitochondrial antiviral signaling gene (MAVS) in domestic chickens

Mitochondrial antiviral signaling (MAVS) gene plays a key role in antiviral regulation in mammals potentially by activating IRF3/7 and NF-κB and leading to the induction of type I interferon (IFN)-mediated antiviral and inflammatory responses. In this study, we screened genetic polymorphisms of the MAVS gene in various Chinese domestic chicken breeds/populations and evaluated its potential effect on gene expression. Among the sequenced fragment (4678 bp), a total of 75 single nucleotide polymorphisms (SNPs) were identified in 46 chickens from 10 breeds/populations, including 30 coding SNPs and 45 non-coding SNPs. Extremely high haplotype diversity (37 nucleotide haplotypes, 18 amino acid haplotypes) was observed in the coding region (CDS), and a similar pattern of high polymorphisms was also observed for the 3′-untranslated region (3′-UTR). Luciferase assays of two representative 3′-UTR haplotypes were performed in both HEK293 cells and DF-1 chicken fibroblast cells, and we found that they were differentially associated with different abilities on regulating mRNA expression level (P < 0.05). Collectively, we observed a considerably high genetic variability of the MAVS gene, and the 3′-UTR variants had an ability to regulate mRNA expression. These results would cast some clues on understanding the potential role of MAVS on viral resistance in chicken.     

Cell type-specific reciprocal regulation of HIF1A gene expression is dependent on 5′- and 3′-UTRs

In the present study, we demonstrated the reciprocal regulation of hypoxia-inducible factor 1 alpha (HIF1A) gene expression via untranslated region-(UTR) dependent mechanisms. A 151 nucleotide sequence found in the HIF1A 5′-UTR is sufficient for significant translational up-regulation. On the other hand, the 3′-UTR of HIF1A has been implicated in mRNA degradation. In the non-metastatic breast cancer cell line MCF7, the 3′-UTR-dependent down-regulatory machinery predominates over the 5′-UTR-dependent up-regulation of HIF1A. However, 5′-UTR-dependent up-regulation is dominant among metastatic cell lines (MDA-MB453, U87MG). It is therefore likely that the predominance of 5′-UTR-dependent translational enhancement of HIF1A is critical for the malignant phenotype of cancer cells. PTBP-1, but not HuR, is a candidate RNA binding protein for the translational control of HIF1A.     

An RNA electrophoretic mobility shift and mutational analysis of rnp-4f 5′-UTR intron splicing regulatory proteins in Drosophila reveals a novel new role for a dADAR protein isoform

Alternative splicing greatly enhances the diversity of proteins encoded by eukaryotic genomes, and is also important in gene expression control. In contrast to the great depth of knowledge as to molecular mechanisms in the splicing pathway itself, relatively little is known about the regulatory events behind this process. The 5′-UTR and 3′-UTR in pre-mRNAs play a variety of roles in controlling eukaryotic gene expression, including translational modulation, and nearly 4000 of the roughly 14,000 protein coding genes in Drosophila contain introns of unknown functional significance in their 5′-UTR. Here we report the results of an RNA electrophoretic mobility shift analysis of Drosophila rnp-4f 5′-UTR intron 0 splicing regulatory proteins. The pre-mRNA potential regulatory element consists of an evolutionarily-conserved 177-nt stem-loop arising from pairing of intron 0 with part of adjacent exon 2. Incubation of in vitro transcribed probe with embryo protein extract is shown to result in two shifted RNA–protein bands, and protein extract from a dADAR null mutant fly line results in only one shifted band. A mutated stem-loop in which the conserved exon 2 primary sequence is changed but secondary structure maintained by introducing compensatory base changes results in diminished band shifts. To test the hypothesis that dADAR plays a role in intron splicing regulation in vivo, levels of unspliced rnp-4f mRNA in dADAR mutant were compared to wild-type via real-time qRT-PCR. The results show that during embryogenesis unspliced rnp-4f mRNA levels fall by up to 85% in the mutant, in support of the hypothesis. Taken together, these results demonstrate a novel role for dADAR protein in rnp-4f 5′-UTR alternative intron splicing regulation which is consistent with a previously proposed model.     

Cytosine methylation in miR-153 gene promoters increases the expression of holocarboxylase synthetase,thereby increasing the abundance of histone H4 biotinylation marks in HEK-293 human kidney cells

Holocarboxylase synthetase (HCS) plays an essential role in catalyzing the biotinylation of carboxylases and histones. Biotinylated carboxylases are important for the metabolism of glucose, lipids and leucine; biotinylation of histones plays important roles in gene regulation and genome stability. Recently, we reported that HCS activity is partly regulated by subcellular translocation events and by miR-539. Here we tested the hypothesis that the HCS 3′-untranslated region (3′-UTR) contains binding sites for miR other than miR-539. A binding site for miR-153 was predicted to reside in the HCS 3′-UTR by using in silico analyses. When miR-153 site was overexpressed in transgenic HEK-293 human embryonic kidney cells, the abundance of HCS mRNA decreased by 77% compared with controls. In silico analyses also predicted three putative cytosine methylation sites in two miR-153 genes; the existence of these sites was confirmed by methylation-sensitive polymerase chain reaction. When cytosines were demethylated by treatment with 5-aza-2′-deoxycytidine, the abundance of miR-153 increased by more than 25 times compared with untreated controls, and this increase coincided with low levels of HCS and histone biotinylation. Together, this study provides novel insights into the mechanisms of novel epigenetic synergies among folate-dependent methylation events, miR and histone biotinylation.     

Downregulation of adenomatous polyposis coli by microRNA-663 promotes odontogenic differentiation through activation of Wnt/beta-catenin signaling

MicroRNAs (miRNAs) regulate cell differentiation by inhibiting mRNA translation or by inducing its degradation. However, the role of miRNAs in odontogenic differentiation is largely unknown. In this present study, we observed that the expression of miR-663 increased significantly during differentiation of MDPC-23 cells to odontoblasts. Furthermore, up-regulation of miR-663 expression promoted odontogenic differentiation and accelerated mineralization without proliferation in MDPC-23 cells. In addition, target gene prediction for miR-663 revealed that the mRNA of the adenomatous polyposis coli (APC) gene, which is associated with the Wnt/β-catenin signaling pathway, has a miR-663 binding site in its 3′-untranslated region (3′UTR). Furthermore, APC expressional was suppressed significantly by miR-663, and this down-regulation of APC expression triggered activation of Wnt/β-catenin signaling through accumulation of β-catenin in the nucleus. Taken together, these findings suggest that miR-663 promotes differentiation of MDPC-23 cells to odontoblasts by targeting APC-mediated activation of Wnt/β-catenin signaling. Therefore, miR-663 can be considered a critical regulator of odontoblast differentiation and can be utilized for developing miRNA-based therapeutic agents.     

Far upstream element binding protein 1 activates translation of p27Kip1 mRNA through its internal ribosomal entry site

The cyclin dependent kinase inhibitor p27 plays an important role in controlling the eukaryotic cell cycle by regulating progression through G1 and entry into S phase. It is often elevated during differentiation and under conditions of cellular stress. In contrast, it is commonly downregulated in cancer cells and its levels are generally inversely correlated with favorable prognosis. The cellular levels of p27 are regulated, in part, by translational control mechanisms. The 5′-untranslated region (5′-UTR) of the p27 mRNA harbors an internal ribosome entry site (IRES) which may facilitate synthesis of p27 in certain conditions. In this study, Far Upstream Element (FUSE) Binding Protein 1 (FBP1) was shown to directly bind to the human p27 5′-UTR and to promote IRES activity. An eight-nucleotide element downstream of a U-rich region within the 5′-UTR was important for FBP1 binding and p27 IRES activity. Overexpression of FBP1 enhanced endogenous p27 levels and stimulated translation initiation. In contrast, repression of FBP1 by siRNA transfection downregulated endogenous p27 protein levels. Using rabbit reticulocyte lysates, FBP1 stimulated p27 mRNA translation in vitro. The central domain of FBP1, containing four K homology motifs, was required for p27 5′-UTR RNA binding and the N terminal domain was important for translational activation. These findings indicate that FBP1 is a novel activator of p27 translation upon binding to the 5′-UTR.