MIR376A is a regulator of starvation-induced autophagy

Background: Autophagy is a vesicular trafficking process responsible for the degradation of long-lived, misfolded or abnormal proteins, as well as damaged or surplus organelles. Abnormalities of the autophagic activity may result in the accumulation of protein aggregates, organelle dysfunction, and autophagy disorders were associated with various diseases. Hence, mechanisms of autophagy regulation are under exploration. Methods: Over-expression of hsa-miR-376a1 (shortly MIR376A) was performed to evaluate its effects on autophagy. Autophagy-related targets of the miRNA were predicted using Microcosm Targets and MIRanda bioinformatics tools and experimentally validated. Endogenous miRNA was blocked using antagomirs and the effects on target expression and autophagy were analyzed. Luciferase tests were performed to confirm that 3’ UTR sequences in target genes were functional. Differential expression of MIR376A and the related MIR376B was compared using TaqMan quantitative PCR. Results: Here, we demonstrated that, a microRNA (miRNA) from the DlkI/Gtl2 gene cluster, MIR376A, played an important role in autophagy regulation. We showed that, amino acid and serum starvation-induced autophagy was blocked by MIR376A overexpression in MCF-7 and Huh-7 cells. MIR376A shared the same seed sequence and had overlapping targets with MIR376B, and similarly blocked the expression of key autophagy proteins ATG4C and BECN1 (Beclin 1). Indeed, 3’ UTR sequences in the mRNA of these autophagy proteins were responsive to MIR376A in luciferase assays. Antagomir tests showed that, endogenous MIR376A was participating to the control of ATG4C and BECN1 transcript and protein levels. Moreover, blockage of endogenous MIR376A accelerated starvation-induced autophagic activity. Interestingly, MIR376A and MIR376B levels were increased with different kinetics in response to starvation stress and tissue-specific level differences were also observed, pointing out to an overlapping but miRNA-specific biological role. Conclusions: Our findings underline the importance of miRNAs encoded by the DlkI/Gtl2 gene cluster in stress-response control mechanisms, and introduce MIR376A as a new regulator of autophagy

The miR-17~92 Cluster: A Key Player in the Control of Inflammation during Rheumatoid Arthritis.:

MicroRNAs (miRNAs) are now recognized as essential regulators of gene expression in plants and animals. They potentially modulate the expression of multiple genes thereby enabling homeostatic settings in physiological conditions. Their role is also increasingly considered in many diseases in which deregulated epigenetic mechanisms induce aberrant gene expression. Work conducted in our laboratory has recently led to the identification of miRNAs essential for the control of inflammatory reactions that occur during rheumatoid arthritis (RA). In this review, we describe two such miRNAs, members of the miR-17 ∼ 92 cluster, which has been previously implicated in cancer. Based on our data and on predicted miRNA:mRNA interactions, we will extrapolate a model whereby the miR-17 ∼ 92 cluster appears as a global regulator of the Apoptosis Signal-Regulating Kinase 1 signalosome, a central actor in the inflammatory pathways activated during RA. We will also discuss the potential therapeutic outcomes emerging from this model

Nucleic Acids Res

Micro (mi)RNAs are small non-coding RNAs with key regulatory functions. Recent advances in the field allowed researchers to identify their targets. However, much less is known regarding the regulation of miRNAs themselves. The accumulation of these tiny regulators can be modulated at various levels during their biogenesis from the transcription of the primary transcript (pri-miRNA) to the stability of the mature miRNA. Here, we studied the importance of the pri-miRNA secondary structure for the regulation of mature miRNA accumulation. To this end, we used the Kaposi's sarcoma herpesvirus, which encodes a cluster of 12 pre-miRNAs. Using small RNA profiling and quantitative northern blot analysis, we measured the absolute amount of each mature miRNAs in different cellular context. We found that the difference in expression between the least and most expressed viral miRNAs could be as high as 60-fold. Using high-throughput selective 2'-hydroxyl acylation analyzed by primer extension, we then determined the secondary structure of the long primary transcript. We found that highly expressed miRNAs derived from optimally structured regions within the pri-miRNA. Finally, we confirmed the importance of the local structure by swapping stem-loops or by targeted mutagenesis of selected miRNAs, which resulted in a perturbed accumulation of the mature miRNA

Cross-species comparative analysis of Dicer proteins during Sindbis virus infection

In plants and invertebrates RNA silencing is a major defense mechanism against virus infections. The first event in RNA silencing is dicing of long double stranded RNAs into small interfering RNAs (siRNAs). The Dicer proteins involved in this process are phylogenetically conserved and have the same domain organization. Accordingly, the production of viral derived siRNAs has also been observed in the mouse, but only in restricted cell types. To gain insight on this restriction, we compare the dicing activity of human Dicer and fly Dicer-2 in the context of Sindbis virus (SINV) infection. Expression of human Dicer in flies inefficiently rescues the production of viral siRNAs but confers some protection against SINV. Conversely, expression of Dicer-2 in human cells allows the production of viral 21 nt small RNAs. However, this does not confer resistance to viral infection, but on the contrary results in stronger accumulation of viral RNA. We further show that Dicer-2 expression in human cells perturbs interferon (IFN) signaling pathways and antagonizes protein kinase R (PKR)-mediated antiviral immunity. Overall, our data suggest that a functional incompatibility between the Dicer and IFN pathways explains the predominance of the IFN response in mammalian somatic cells

AU-Rich Element-Mediated mRNA Decay Can Occur Independently of the miRNA Machinery in Mouse Embryonic Fibroblasts and Drosophila S2-Cells

AU-rich elements (AREs) are regulatory sequences located in the 3′ untranslated region of many short-lived mRNAs. AREs are recognized by ARE-binding proteins and cause rapid mRNA degradation. Recent reports claimed that the function of AREs may be – at least in part – relayed through the miRNA pathway. We have revisited this hypothesis using dicer knock-out mouse embryonic fibroblasts and cultured Drosophila cells. In contrast to the published results, we find no evidence for a general requirement of the miRNA pathway in the function of AREs. Endogenous ier3 mRNA, which is known to contain a functional ARE, was degraded rapidly at indistinguishable rates in wild type and dicer knock-out mouse embryonic fibroblasts. In cultured Drosophila cells, both ARE-containing GFP reporter mRNAs and the endogenous cecA1 mRNA were resistant to depletion of the mi/siRNA factors dcr-1, dcr-2, ago1 and ago2. Furthermore, the Drosophila miRNA originally proposed to recognize AU-rich elements, miR-289, is not detectably expressed in flies or cultured S2 cells. Even our attempts to overexpress this miRNA from its genomic hairpin sequence failed. Thus, this sequence cannot serve as link between the miRNA and the AU-rich element mediated silencing pathways. Taken together, our studies in mammalian and Drosophila cells strongly argue that AREs can function independently of miRNAs

A Novel Pathway of TEF Regulation Mediated by MicroRNA-125b Contributes to the Control of Actin Distribution and Cell Shape in Fibroblasts

BACKGROUND: Thyrotroph embryonic factor (TEF), a member of the PAR bZIP family of transcriptional regulators, has been involved in neurotransmitter homeostasis, amino acid metabolism, and regulation of apoptotic proteins. In spite of its relevance, nothing is known about the regulation of TEF. PRINCIPAL FINDINGS: p53-dependent genotoxic agents have been shown to be much more harmful for PAR bZIP-deficient mice as compared to wild type animals. Here we demonstrate that TEF expression is controlled by p53 through upregulation of microRNA-125b, as determined by both regulating the activity of p53 and transfecting cells with microRNA-125b precursors. We also describe a novel role for TEF in controlling actin distribution and cell shape in mouse fibroblasts. Lack of TEF is accompanied by dramatic increase of cell area and decrease of elongation (bipolarity) and dispersion (multipolarity). Staining of actin cytoskeleton also showed that TEF (-/-) cells are characterized by appearance of circumferential actin bundles and disappearance of straight fibers. Interestingly, transfection of TEF (-/-) fibroblasts with TEF induced a wild type-like phenotype. Consistent with our previous findings, transfection of wild type fibroblasts with miR-125b promoted a TEF (-/-)-like phenotype, and a similar but weaker effect was observed following exogenous expression of p53. CONCLUSIONS/SIGNIFICANCE: These findings provide the first evidence of TEF regulation, through a miR-125b-mediated pathway, and describes a novel role of TEF in the maintenance of cell shape in fibroblasts

Optimal Use of Conservation and Accessibility Filters in MicroRNA Target Prediction

It is generally accepted that filtering microRNA (miRNA) target predictions by conservation or by accessibility can reduce the false discovery rate. However, these two strategies are usually not exploited in a combined and flexible manner. Here, we introduce PACCMIT, a flexible method that filters miRNA binding sites by their conservation, accessibility, or both. The improvement in performance obtained with each of these three filters is demonstrated on the prediction of targets for both i) highly and ii) weakly conserved miRNAs, i.e., in two scenarios in which the miRNA-target interactions are subjected to different evolutionary pressures. We show that in the first scenario conservation is a better filter than accessibility (as both sensitivity and precision are higher among the top predictions) and that the combined filter improves performance of PACCMIT even further. In the second scenario, on the other hand, the accessibility filter performs better than both the conservation and combined filters, suggesting that the site conservation is not equally effective in rejecting false positive predictions for all miRNAs. Regarding the quality of the ranking criterion proposed by Robins and Press and used in PACCMIT, it is shown that top ranking interactions correspond to more downregulated proteins than do the lower ranking interactions. Comparison with several other target prediction algorithms shows that the ranking of predictions provided by PACCMIT is at least as good as the ranking generated by other conservation-based methods and considerably better than the energy-based ranking used in other accessibility-based methods

Arbovirus-Derived piRNAs Exhibit a Ping-Pong Signature in Mosquito Cells

The siRNA pathway is an essential antiviral mechanism in insects. Whether other RNA interference pathways are involved in antiviral defense remains unclear. Here, we report in cells derived from the two main vectors for arboviruses, Aedes albopictus and Aedes aegypti, the production of viral small RNAs that exhibit the hallmarks of ping-pong derived piwi-associated RNAs (piRNAs) after infection with positive or negative sense RNA viruses. Furthermore, these cells produce endogenous piRNAs that mapped to transposable elements. Our results show that these mosquito cells can initiate de novo piRNA production and recapitulate the ping-pong dependent piRNA pathway upon viral infection. The mechanism of viral-piRNA production is discussed

siRNA-Like Double-Stranded RNAs Are Specifically Protected Against Degradation in Human Cell Extract

RNA interference (RNAi) is a set of intracellular pathways in eukaryotes that controls both exogenous and endogenous gene expression. The power of RNAi to knock down (silence) any gene of interest by the introduction of synthetic small-interfering (si)RNAs has afforded powerful insight into biological function through reverse genetic approaches and has borne a new field of gene therapeutics. A number of questions are outstanding concerning the potency of siRNAs, necessitating an understanding of how short double-stranded RNAs are processed by the cell. Recent work suggests unmodified siRNAs are protected in the intracellular environment, although the mechanism of protection still remains unclear. We have developed a set of doubly-fluorophore labeled RNAs (more precisely, RNA/DNA chimeras) to probe in real-time the stability of siRNAs and related molecules by fluorescence resonance energy transfer (FRET). We find that these RNA probes are substrates for relevant cellular degradative processes, including the RNase H1 mediated degradation of an DNA/RNA hybrid and Dicer-mediated cleavage of a 24-nucleotide (per strand) double-stranded RNA. In addition, we find that 21- and 24-nucleotide double-stranded RNAs are relatively protected in human cytosolic cell extract, but less so in blood serum, whereas an 18-nucleotide double-stranded RNA is less protected in both fluids. These results suggest that RNAi effector RNAs are specifically protected in the cellular environment and may provide an explanation for recent results showing that unmodified siRNAs in cells persist intact for extended periods of time

Small RNAs Targeting Transcription Start Site Induce Heparanase Silencing through Interference with Transcription Initiation in Human Cancer Cells

Heparanase (HPA), an endo-h-D-glucuronidase that cleaves the heparan sulfate chain of heparan sulfate proteoglycans, is overexpressed in majority of human cancers. Recent evidence suggests that small interfering RNA (siRNA) induces transcriptional gene silencing (TGS) in human cells. In this study, transfection of siRNA against −9/+10 bp (siH3), but not −174/−155 bp (siH1) or −134/−115 bp (siH2) region relative to transcription start site (TSS) locating at 101 bp upstream of the translation start site, resulted in TGS of heparanase in human prostate cancer, bladder cancer, and gastric cancer cells in a sequence-specific manner. Methylation-specific PCR and bisulfite sequencing revealed no DNA methylation of CpG islands within heparanase promoter in siH3-transfected cells. The TGS of heparanase did not involve changes of epigenetic markers histone H3 lysine 9 dimethylation (H3K9me2), histone H3 lysine 27 trimethylation (H3K27me3) or active chromatin marker acetylated histone H3 (AcH3). The regulation of alternative splicing was not involved in siH3-mediated TGS. Instead, siH3 interfered with transcription initiation via decreasing the binding of both RNA polymerase II and transcription factor II B (TFIIB), but not the binding of transcription factors Sp1 or early growth response 1, on the heparanase promoter. Moreover, Argonaute 1 and Argonaute 2 facilitated the decreased binding of RNA polymerase II and TFIIB on heparanase promoter, and were necessary in siH3-induced TGS of heparanase. Stable transfection of the short hairpin RNA construct targeting heparanase TSS (−9/+10 bp) into cancer cells, resulted in decreased proliferation, invasion, metastasis and angiogenesis of cancer cells in vitro and in athymic mice models. These results suggest that small RNAs targeting TSS can induce TGS of heparanase via interference with transcription initiation, and significantly suppress the tumor growth, invasion, metastasis and angiogenesis of cancer cells