microRNA-Mediated Messenger RNA Deadenylation Contributes to Translational Repression in Mammalian Cells

Animal microRNAs (miRNAs) typically regulate gene expression by binding to partially complementary target sites in the 3′ untranslated region (UTR) of messenger RNA (mRNA) reducing its translation and stability. They also commonly induce shortening of the mRNA 3′ poly(A) tail, which contributes to their mRNA decay promoting function. The relationship between miRNA-mediated deadenylation and translational repression has been less clear. Using transfection of reporter constructs carrying three imperfectly matching let-7 target sites in the 3′ UTR into mammalian cells we observe rapid target mRNA deadenylation that precedes measureable translational repression by endogenous let-7 miRNA. Depleting cells of the argonaute co-factors RCK or TNRC6A can impair let-7-mediated repression despite ongoing mRNA deadenylation, indicating that deadenylation alone is not sufficient to effect full repression. Nevertheless, the magnitude of translational repression by let-7 is diminished when the target reporter lacks a poly(A) tail. Employing an antisense strategy to block deadenylation of target mRNA with poly(A) tail also partially impairs translational repression. On the one hand, these experiments confirm that tail removal by deadenylation is not strictly required for translational repression. On the other hand they show directly that deadenylation can augment miRNA-mediated translational repression in mammalian cells beyond stimulating mRNA decay. Taken together with published work, these results suggest a dual role of deadenylation in miRNA function: it contributes to translational repression as well as mRNA decay and is thus critically involved in establishing the quantitatively appropriate physiological response to miRNAs

Mathematical modeling of microRNA-mediated mechanisms of translation repression

MicroRNAs can affect the protein translation using nine mechanistically different mechanisms, including repression of initiation and degradation of the transcript. There is a hot debate in the current literature about which mechanism and in which situations has a dominant role in living cells. The worst, same experimental systems dealing with the same pairs of mRNA and miRNA can provide ambiguous evidences about which is the actual mechanism of translation repression observed in the experiment. We start with reviewing the current knowledge of various mechanisms of miRNA action and suggest that mathematical modeling can help resolving some of the controversial interpretations. We describe three simple mathematical models of miRNA translation that can be used as tools in interpreting the experimental data on the dynamics of protein synthesis. The most complex model developed by us includes all known mechanisms of miRNA action. It allowed us to study possible dynamical patterns corresponding to different miRNA-mediated mechanisms of translation repression and to suggest concrete recipes on determining the dominant mechanism of miRNA action in the form of kinetic signatures. Using computational experiments and systematizing existing evidences from the literature, we justify a hypothesis about co-existence of distinct miRNA-mediated mechanisms of translation repression. The actually observed mechanism will be that acting on or changing the limiting "place" of the translation process. The limiting place can vary from one experimental setting to another. This model explains the majority of existing controversies reported.Comment: 40 pages, 9 figures, 4 tables, 91 cited reference. The analysis of kinetic signatures is updated according to the new model of coupled transcription, translation and degradation, and of miRNA-based regulation of this process published recently (arXiv:1204.5941). arXiv admin note: text overlap with arXiv:0911.179

Why mouse oocytes and early embryos ignore miRNAs?

Small RNA molecules regulating gene expression received a status of omnipresent master regulators of eukaryotic lives with almost supernatural powers. Mammals hold at least three mechanisms employing small RNA molecules for regulating gene expression. One of these mechanisms, the microRNA (miRNA) pathway, currently involves over a thousand of genome-encoded different miRNAs that are claimed to extend their control over more than a half of a genome. Here, I discuss how and why mouse oocytes and early embryos ignore the regulatory power of miRNAs, adding another surprising feature to the field of small RNAs

miRNA repression involves GW182-mediated recruitment of CCR4-NOT through conserved W-containing motifs

miRNA-mediated repression in animals is dependent on the GW182 protein family. GW182 proteins are recruited to the miRNA repression complex through direct interaction with Argonaute proteins, and they function downstream to repress target mRNA. Here we demonstrate that in human and Drosophila melanogaster cells, the critical repressive features of both the N-terminal and C-terminal effector domains of GW182 proteins are Gly/Ser/Thr-Trp (G/S/TW) or Trp-Gly/Ser/Thr (WG/S/T) motifs. These motifs, which are dispersed across both domains and act in an additive manner, function by recruiting components of the CCR4-NOT deadenylation complex. A heterologous yeast polypeptide with engineered WG/S/T motifs acquired the ability to repress tethered mRNA and to interact with the CCR4-NOT complex. These results identify previously unknown effector motifs functioning as important mediators of miRNA-induced silencing in both species, and they reveal that recruitment of the CCR4-NOT complex by tryptophan-containing motifs acts downstream of GW182 to repress mRNAs, including inhibiting translation independently of deadenylation

Structural insights into the human GW182-PABC interaction in microRNA-mediated deadenylation

GW182-family proteins are essential for microRNA-mediated translational repression and deadenylation in animal cells. Here we show that a conserved motif in the human GW182 paralog TNRC6C interacts with the C-terminal domain of polyadenylate binding protein 1 (PABC) and present the crystal structure of the complex. Mutations at the complex interface impair mRNA deadenylation in mammalian cell extracts, suggesting that the GW182-PABC interaction contributes to microRNA-mediated gene silencing. MicroRNAs (miRNAs) are short endogenous RNAs that regulate a variety of eukaryotic cell processes, including cell growth, proliferation and differentiation. In Metazoa, miRNAs associate with Argonaute (Ago) proteins in miRNA-induced silencing complexes (miRISCs) to silence target mRNAs by a mechanism involving translational repression and concurrent stimulation of mRNA deadenylation and subsequent degradation1, 2. In addition to Ago proteins, miRNA-mediated silencing requires proteins of the GW182 family3. Tethering of GW182 proteins to mRNA reporters induces translational repression and mRNA decay in the absence of miRNAs and Ago, suggesting that GW182 proteins have silencing functions downstream of Ago4. A direct interaction between Ago and GW182, Users may view, print, copy, download and text and data- mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use

Ago-TNRC6 triggers microRNA-mediated decay by promoting two deadenylation steps.

MicroRNAs (miRNAs) silence the expression of their mRNA targets mainly by promoting mRNA decay. The mechanism, kinetics and participating enzymes for miRNA-mediated decay in mammalian cells remain largely unclear. Combining the approaches of transcriptional pulsing, RNA tethering, overexpression of dominant-negative mutants, and siRNA-mediated gene knockdown, we show that let-7 miRNA-induced silencing complexes (miRISCs), which contain the proteins Argonaute (Ago) and TNRC6 (also known as GW182), trigger very rapid mRNA decay by inducing accelerated biphasic deadenylation mediated by Pan2-Pan3 and Ccr4-Caf1 deadenylase complexes followed by Dcp1-Dcp2 complex-directed decapping in mammalian cells. When tethered to mRNAs, all four human Ago proteins and TNRC6C are each able to recapitulate the two deadenylation steps. Two conserved human Ago2 phenylalanines (Phe470 and Phe505) are critical for recruiting TNRC6 to promote deadenylation. These findings indicate that promotion of biphasic deadenylation to trigger mRNA decay is an intrinsic property of miRISCs