Article

Argonaute protein identity and pairing geometry determine cooperativity in mammalian RNA silencing

University of Massachusetts Medical School, Worcester, MA 01605, USA.
RNA (Impact Factor: 4.94). 08/2011; 17(10):1858-69. DOI: 10.1261/rna.2778911
Source: PubMed

ABSTRACT

Small RNAs loaded into Argonaute proteins direct silencing of complementary target mRNAs. It has been proposed that multiple, imperfectly complementary small interfering RNAs or microRNAs, when bound to the 3' untranslated region of a target mRNA, function cooperatively to silence target expression. We report that, in cultured human HeLa cells and mouse embryonic fibroblasts, Argonaute1 (Ago1), Ago3, and Ago4 act cooperatively to silence both perfectly and partially complementary target RNAs bearing multiple small RNA-binding sites. Our data suggest that for Ago1, Ago3, and Ago4, multiple, adjacent small RNA-binding sites facilitate cooperative interactions that stabilize Argonaute binding. In contrast, small RNAs bound to Ago2 and pairing perfectly to an mRNA target act independently to silence expression. Noncooperative silencing by Ago2 does not require the endoribonuclease activity of the protein: A mutant Ago2 that cannot cleave its mRNA target also silences noncooperatively. We propose that Ago2 binds its targets by a mechanism fundamentally distinct from that used by the three other mammalian Argonaute proteins.

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Available from: William E Salomon, May 31, 2015
    • "These sites are not randomly distributed but often occur in pairs or clusters, which can cooperatively repress mutual targets. The ideal distance for a cooperative miRNA effect was determined to have 13–35 nt between the recognized sites that are complementary to the seed sequences (Grimson et al, 2007; Saetrom et al, 2007; Broderick et al, 2011). Ablation of miRNA biogenesis promotes Th1 differentiation but impairs Treg cell development (Muljo et al, 2005; Cobb et al, 2006; Chong et al, 2008; Steiner et al, 2011). "
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    ABSTRACT: Peripheral induction of regulatory T (Treg) cells provides essential protection from inappropriate immune responses. CD4(+) T cells that lack endogenous miRNAs are impaired to differentiate into Treg cells, but the relevant miRNAs are unknown. We performed an overexpression screen with T-cell-expressed miRNAs in naive mouse CD4(+) T cells undergoing Treg differentiation. Among 130 candidates, the screen identified 29 miRNAs with a negative and 10 miRNAs with a positive effect. Testing reciprocal Th17 differentiation revealed specific functions for miR-100, miR-99a and miR-10b, since all of these promoted the Treg and inhibited the Th17 program without impacting on viability, proliferation and activation. miR-99a cooperated with miR-150 to repress the expression of the Th17-promoting factor mTOR. The comparably low expression of miR-99a was strongly increased by the Treg cell inducer "retinoic acid", and the abundantly expressed miR-150 could only repress Mtor in the presence of miR-99a. Our data suggest that induction of Treg cell differentiation is regulated by a miRNA network, which involves cooperation of constitutively expressed as well as inducible miRNAs. © 2015 The Authors.
    No preview · Article · Feb 2015 · The EMBO Journal
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    • "For example, repression is affected by varying degrees of complementarity between the microRNA and its target, but to different extents with different microRNAs. Multiple copies of microRNA target sites in the UTRs of reporter constructs have been shown to enable greater levels of repression, and cooperativity has been demonstrated for closely positioned microRNA target sites [47]. We have attempted to remove the complexity and variability between different microRNAs and target contexts by examining a constrained case where targets have a single perfectly complementary site in a homogeneous cell population. "
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    • "It has been demonstrated that the same mRNAs can be targeted by more than one miRNA which provides for more efficient and specific regulation [20,21]. We found 49 mRNAs which are known to be validated targets for 2 or even 3 differentially expressed miRNAs. "
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