Posttranslational modification of Argonautes and their role in small RNA-mediated gene regulation

Shortly after their discovery, repertoires of miRNA were identified, together with proteins involved in their biogenesis and action. It is now obvious that miRNA-mediated gene regulation itself is regulated at multiple levels. Identifying the regulatory mechanisms that underpin small RNA homeostasis by modulation of their biogenesis and action has become a key issue, which can be partly resolved by identifying mediators of Argonautes turnover. An emerging theme in the control of Argonaute stability and activity is through posttranslational modifications, which are the focus of this review

The miRNA biogenesis factors, p72/DDX17 and KHSRP regulate the protein level of Ago2 in human cells

© 2016 Elsevier B.V. MicroRNAs (miRNAs) are short (21–23 nt long) RNAs that post-transcriptionally regulate gene expression in plants and animals. They are key regulators in all biological processes. In mammalian cells miRNAs are loaded into one of the four members of the Argonaute (Ago) protein family to form the RNA-induced silencing complex (RISC). RISCs inhibit the translation of mRNAs that share sequence complementarity with their loaded miRNAs. miRNA processing and miRNA-mediated gene regulation are highly regulated processes and involve many RNA-binding proteins as auxiliary factors. Here we show that the two RNA-binding proteins, p72 and KHSRP, both with known roles in promoting miRNA biogenesis, regulate the protein level of human Ago2 in transformed human cells. We determined that p72 and KHSRP influence Ago2 stability by regulating miRNA levels in the cell and that loss of p72/KHSRP results in a decrease of unloaded Ago2

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. © 2011 European Molecular Biology Organization

Integration of microRNA changes in vivo identifies novel molecular features of muscle insulin resistance in type 2 diabetes

Skeletal muscle insulin resistance (IR) is considered a critical component of type II diabetes, yet to date IR has evaded characterization at the global gene expression level in humans. MicroRNAs (miRNAs) are considered fine-scale rheostats of protein-coding gene product abundance. The relative importance and mode of action of miRNAs in human complex diseases remains to be fully elucidated. We produce a global map of coding and non-coding RNAs in human muscle IR with the aim of identifying novel disease biomarkers. We profiled >47,000 mRNA sequences and >500 human miRNAs using gene-chips and 118 subjects (n = 71 patients versus n = 47 controls). A tissue-specific gene-ranking system was developed to stratify thousands of miRNA target-genes, removing false positives, yielding a weighted inhibitor score, which integrated the net impact of both up- and down-regulated miRNAs. Both informatic and protein detection validation was used to verify the predictions of in vivo changes. The muscle mRNA transcriptome is invariant with respect to insulin or glucose homeostasis. In contrast, a third of miRNAs detected in muscle were altered in disease (n = 62), many changing prior to the onset of clinical diabetes. The novel ranking metric identified six canonical pathways with proven links to metabolic disease while the control data demonstrated no enrichment. The Benjamini-Hochberg adjusted Gene Ontology profile of the highest ranked targets was metabolic (P < 7.4 × 10-8), post-translational modification (P < 9.7 × 10-5) and developmental (P < 1.3 × 10-6) processes. Protein profiling of six development-related genes validated the predictions. Brain-derived neurotrophic factor protein was detectable only in muscle satellite cells and was increased in diabetes patients compared with controls, consistent with the observation that global miRNA changes were opposite from those found during myogenic differentiation. We provide evidence that IR in humans may be related to coordinated changes in multiple microRNAs, which act to target relevant signaling pathways. It would appear that miRNAs can produce marked changes in target protein abundance in vivo by working in a combinatorial manner. Thus, miRNA detection represents a new molecular biomarker strategy for insulin resistance, where micrograms of patient material is needed to monitor efficacy during drug or life-style interventions

Loss of miRNA biogenesis induces p19Arf-p53 signaling and senescence in primary cells

Dicer, an enzyme involved in microRNA (miRNA) maturation, is required for proper cell differentiation and embryogenesis in mammals. Recent evidence indicates that Dicer and miRNA may also regulate tumorigenesis. To better characterize the role of miRNA in primary cell growth, we generated Dicer-conditional mice. Ablation of Dicer and loss of mature miRNAs in embryonic fibroblasts up-regulated p19Arf and p53 levels, inhibited cell proliferation, and induced a premature senescence phenotype that was also observed in vivo after Dicer ablation in the developing limb and in adult skin. Furthermore, deletion of the Ink4a/Arf or p53 locus could rescue fibroblasts from premature senescence induced by Dicer ablation. Although levels of Ras and Myc oncoproteins appeared unaltered, loss of Dicer resulted in increased DNA damage and p53 activity in these cells. These results reveal that loss of miRNA biogenesis activates a DNA damage checkpoint, up-regulates p19Arf-p53 signaling, and induces senescence in primary cells

miR-132/212 knockout mice reveal roles for these miRNAs in regulating cortical synaptic transmission and plasticity

miR-132 and miR-212 are two closely related miRNAs encoded in the same intron of a small non-coding gene, which have been suggested to play roles in both immune and neuronal function. We describe here the generation and initial characterisation of a miR-132/212 double knockout mouse. These mice were viable and fertile with no overt adverse phenotype. Analysis of innate immune responses, including TLR-induced cytokine production and IFNβ induction in response to viral infection of primary fibroblasts did not reveal any phenotype in the knockouts. In contrast, the loss of miR-132 and miR-212, while not overtly affecting neuronal morphology, did affect synaptic function. In both hippocampal and neocortical slices miR-132/212 knockout reduced basal synaptic transmission, without affecting paired-pulse facilitation. Hippocampal long-term potentiation (LTP) induced by tetanic stimulation was not affected by miR-132/212 deletion, whilst theta burst LTP was enhanced. In contrast, neocortical theta burst-induced LTP was inhibited by loss of miR-132/212. Together these results indicate that miR-132 and/or miR-212 play a significant role in synaptic function, possibly by regulating the number of postsynaptic AMPA receptors under basal conditions and during activity-dependent synaptic plasticity

A microRNA in a multiple-turnover RNAi enzyme complex

In animals, the double-stranded RNA-specific endonuclease Dicer produces two classes of functionally distinct, tiny RNAs: microRNAs (miRNAs) and small interfering RNAs (siRNAs). miRNAs regulate mRNA translation, whereas siRNAs direct RNA destruction via the RNA interference (RNAi) pathway. Here we show that, in human cell extracts, the miRNA let-7 naturally enters the RNAi pathway, which suggests that only the degree of complementarity between a miRNA and its RNA target determines its function. Human let-7 is a component of a previously identified, miRNA-containing ribonucleoprotein particle, which we show is an RNAi enzyme complex. Each let-7-containing complex directs multiple rounds of RNA cleavage, which explains the remarkable efficiency of the RNAi pathway in human cells

Small RNAs derived from the 5' end of tRNA can inhibit protein translation in human cells

Recently, it has been shown that tRNA molecules can be processed into small RNAs that are derived from both the 5′ and 3′ termini. To date, the function of these tRNA fragments (tRFs) derived from the 5′ end of tRNAs has not been investigated in depth. We present evidence that conserved residues in tRNAs, present in all 5′ tRFs, can inhibit the process of protein translation without the need for complementary target sites in the mRNA. These results implicate 5′ tRFs in a new mechanism of gene regulation by small RNAs in human cells. © 2013 Landes Bioscience