20 research outputs found
miRNA-Dependent Translational Repression in the Drosophila Ovary
Background: The Drosophila ovary is a tissue rich in post-transcriptional regulation of gene expression. Many of the regulatory factors are proteins identified via genetic screens. The more recent discovery of microRNAs, which in other animals and tissues appear to regulate translation of a large fraction of all mRNAs, raised the possibility that they too might act during oogenesis. However, there has been no direct demonstration of microRNA-dependent translational repression in the ovary. Methodology/Principal Findings: Here, quantitative analyses of transcript and protein levels of transgenes with or without synthetic miR-312 binding sites show that the binding sites do confer translational repression. This effect is dependent on the ability of the cells to produce microRNAs. By comparison with microRNA-dependent translational repression in other cell types, the regulated mRNAs and the protein factors that mediate repression were expected to be enriched in sponge bodies, subcellular structures with extensive similarities to the P bodies found in other cells. However, no such enrichment was observed. Conclusions/Significance: Our results reveal the variety of post-transcriptional regulatory mechanisms that operate in the Drosophila ovary, and have implications for the mechanisms of miRNA-dependent translational control used in the ovary.This work was supported in part by NIH grant GM54409 and in part by Research Grant No. 1-FY08-445. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Cellular and Molecular Biolog
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