The ribosomal protein RACK1 is required for microRNA function in both C. elegans and humans

Despite the importance of microRNAs (miRNAs) in gene regulation, it is unclear how the miRNA-Argonaute complex--or miRNA-induced silencing complex (miRISC)--can regulate the translation of their targets in such diverse ways. We demonstrate here a direct interaction between the miRISC and the ribosome by showing that a constituent of the eukaryotic 40S subunit, receptor for activated C-kinase (RACK1), is important for miRNA-mediated gene regulation in animals. In vivo studies demonstrate that RACK1 interacts with components of the miRISC in nematodes and mammals. In both systems, the alteration of RACK1 expression alters miRNA function and impairs the association of the miRNA complex with the translating ribosomes. Our data indicate that RACK1 can contribute to the recruitment of miRISC to the site of translation, and support a post-initiation mode of miRNA-mediated gene repression.     

The slicing activity of miRNA-specific Argonautes is essential for the miRNA pathway in C. elegans

Among the set of Argonautes proteins encoded by metazoan genomes, some have conserved amino acids important for catalytic or slicing activity. The functional significance of these residues in microRNA (miRNA)-specific Argonautes in animals is still unclear since miRNAs do not induce site-specific cleavage of targeted messenger RNAs (mRNAs), unlike small interfering RNAs (siRNAs). Here, we report that miRNA-specific ALG-1 and ALG-2 Argonautes from Caenorhabditis elegans possess the slicing activity normally implicated in the siRNA-silencing pathway. We also find that ALG-1/2 can bind and use a Dicer-processed miRNA duplex to target mRNAs, suggesting an ability to displace RNA strands. Importantly, the slicing activity of ALG-1 or ALG-2 is essential for the miRNA pathway in vivo, as shown by the accumulation of truncated miRNA precursors and altered miRNA-induced silencing complex (miRISC) formation. Taken together, our data demonstrate that the slicing activity of Argonautes contributes to a new and unexpected step in the canonical miRNA pathway that occurs prior to miRISC loading in animals.     

Dicer-1, but not Loquacious, is critical for assembly of miRNA-induced silencing complexes

Double-stranded RNA-binding proteins (dsRBPs), such as R2D2 and Loquacious (Loqs), function in tandem with Dicer (Dcr) enzymes in RNA interference (RNAi). In Drosophila, Dcr-1/Loqs and Dcr-2/R2D2 complexes generate microRNAs (miRNAs) and small interfering RNAs (siRNAs), respectively. Although R2D2 does not regulate siRNA production, R2D2 and Dcr-2 coordinately bind siRNAs to promote assembly of the siRNA-induced silencing (siRISC) complexes. Conversely, Loqs enhances miRNA production. It is uncertain if Dcr-1 and Loqs facilitate miRNA loading onto the miRISC complexes. Here we used loqs knockout (KO) flies to characterize the physiological functions of Loqs in the miRNA pathway. Northern analysis revealed consistent accumulation of precursor (pre)-miRNAs in loqs(KO) flies. However, the lack of Loqs had differential effects on mature miRNAs: some are diminished, whereas others maintain wild-type levels. Importantly, the data suggest that miRNA production is not the rate-limiting step of the miRNA pathway. We show that Dcr-1, but not Loqs, is critical for assembly of miRISCs by using dcr-1 or loqs null egg extract. Consistent with this, recombinant Dcr-1 could efficiently interact with miRNA duplex in the absence of Loqs. Together, our results indicate that Loqs plays a prominent role in miRNA biogenesis, but is largely dispensable for miRISC assembly. Thus, Loqs and R2D2 represent two distinct functional modes for dsRBPs in the RNAi pathways.     

Big roles of microRNAs in tumorigenesis and tumor development

MicroRNAs (miRNAs) are a class of endogenous non-protein-coding small RNAs that are evolutionarily conserved and widely distributed among species. Their major function is to negatively regulate target gene expression. A single miRNA can regulate multiple target genes, indicating that miRNAs may regulate multiple signaling pathways and participate in a variety of physiological and pathological processes. Currently, approximately 50% of identified human miRNA-coding genes are located at tumor-related fragile chromosome regions. Abnormal miRNA expression and/or mutations have been found in almost all types of malignancies. These abnormally expressed miRNAs play roles similar to tumor suppressor genes or oncogenes by regulating the expression and/or function of tumor-related genes. Therefore, miRNAs, miRNA target genes, and the genes regulating miRNAs form a regulatory network with miRNAs in the hub. This network plays a pivotal role in tumorigenesis and tumor development.     

Cloning and characterization of miRNAs from maize seedling roots under low phosphorus stress

MicroRNAs (miRNAs) are a class of small, non-coding regulatory RNAs that regulate gene expression by guiding target mRNA cleavage or translational inhibition in plants and animals. In this study, a small RNA library was constructed to identify conserved miRNAs as well as novel miRNAs in maize seedling roots under low level phosphorus stress. Twelve miRNAs were identified by high throughput sequencing of the library and subsequent analysis, two belong to conserved miRNA families (miRNA399b and miRNA156), and the remaining ten are novel and one of latter is conserved in gramineous species. Based on sequence homology, we predicted 125 potential target genes of these miRNAs and then expression patterns of 7 miRNAs were validated by semi-RT-PCR analysis. MiRNA399b, Zma-miR3, and their target genes (Zmpt1 and Zmpt2) were analyzed by real-time PCR. It is shown that both miRNA399b and Zma-miR3 are induced by low phosphorus stress and regulated by their target genes (Zmpt1 and Zmpt2). Moreover, Zma-miR3, regulated by two maize inorganic phosphate transporters as a newly identified miRNAs, would likely be directly involved in phosphate homeostasis, so was miRNA399b in Arabidopsis and rice. These results indicate that both conserved and maize-specific miRNAs play important roles in stress responses and other physiological processes correlated with phosphate starvation, regulated by their target genes. Identification of these differentially expressed miRNAs will facilitate us to uncover the molecular mechanisms underlying the progression of maize seedling roots development under low level phosphorus stress.     

miRNAs got rhythm

Despite significant advances in treatments, cardiovascular disease (CVD) remains the leading cause of human morbidity and mortality in developed countries. The development of novel and efficient treatment strategies requires an understanding of the basic molecular mechanisms underlying cardiac function. MicroRNAs (miRNAs) are a family of small nonprotein-coding RNAs that have emerged as important regulators in cardiac and vascular developmental and pathological processes, including cardiac arrhythmia, fibrosis, hypertrophy and ischemia, heart failure and vascular atherosclerosis. The miRNA acts as an adaptor for the miRNA-induced silencing complex (miRISC) to specifically recognize and regulate particular mRNAs. Mature miRNAs recognize their target mRNAs by base-pairing interactions between nucleotides 2 and 8 of the miRNA (the seed region) and complementary nucleotides in the 3'-untranslated region (3'-UTR) of mRNAs and miRISCs subsequently inhibit gene expression by targeting mRNAs for translational repression or cleavage. In this review we summarize the basic mechanisms of action of miRNAs as they are related to cardiac arrhythmia and address the potential for miRNAs to be therapeutically manipulated in the treatment of arrhythmias.     

On the availability of microRNA-induced silencing complexes,saturation of microRNA-binding sites and stoichiometry

Several authors have suggested or inferred that modest changes in microRNA expression can potentiate or impinge on their capacity to mediate gene repression, and that doing so could play a significant role in diseases. Such interpretations are based on several assumptions, namely: (i) changes in microRNA expression correlate with changes in the availability of mature, functional miRISC, (ii) changes in microRNA expression can significantly alter the stoichiometry of miRISC populations with their cognate targets, (iii) and this, in turn, can result in changes in miRISC silencing output. Here, we experimentally challenge those assumptions by quantifying and altering the availability of miRISC across several families of microRNAs. Doing so revealed a surprising fragmentation in the miRISC functional pool, striking differences in the availability of miRNA families and saturability of miRNA-mediated silencing. Furthermore, we provide direct experimental evidence that only a limited subset of miRNAs, defined by a conjuncture of expression threshold, miRISC availability and low target site abundance, is susceptible to competitive effects through microRNA-binding sites.     

MicroRNA Profiling in Human Colon Cancer Cells during 5-Fluorouracil-Induced Autophagy

Autophagy modulation is now recognized as a potential therapeutic approach for cancer (including colorectal cancer), yet the molecular mechanisms regulating autophagy in response to cellular stress are still not well understood. MicroRNAs (miRNAs) have been found to play important roles in controlling many cellular functions, including growth, metabolism and stress response. The physiological importance of the miRNA-autophagy interconnection is only beginning to be elucidated. MiRNA microarray technology facilitates analysis of global miRNA expression in certain situations. In this study, we explored the expression profile of miRNAs during the response of human colon cancer cells (HT29s) to 5-FU treatment and nutrient starvation using miRNA microarray analysis. The alteration of miRNA expression showed the same pattern under both conditions was further testified by qRT-PCR in three human colon cancer cell lines. In addition, bioinformatic prediction of target genes, pathway analysis and gene network analysis were performed to better understand the roles of these miRNAs in the regulation of autophagy. We identified and selected four downregulated miRNAs including hsa-miR-302a-3p and 27 upregulated miRNAs under these two conditions as having the potential to target genes involved in the regulation of autophagy in human colon cancer cells. They have the potential to modulate autophagy in 5-FU-based chemotherapy in colorectal cancer.