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


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.

Download full-text


Available from: William E Salomon, May 31, 2015
  • Source
    • "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. "
    [Show abstract] [Hide abstract]
    ABSTRACT: MicroRNAs are small RNAs that regulate protein levels. It is commonly assumed that the expression level of a microRNA is directly correlated with its repressive activity - that is, highly expressed microRNAs will repress their target mRNAs more. Here we investigate the quantitative relationship between endogenous microRNA expression and repression for 32 mature microRNAs in Drosophila melanogaster S2 cells. In general, we find that more abundant microRNAs repress their targets to a greater degree. However, the relationship between expression and repression is nonlinear, such that a 10-fold greater microRNA concentration produces only a 10% increase in target repression. The expression/repression relationship is the same for both dominant guide microRNAs and minor mature products (so-called passenger strands/microRNA* sequences). However, we find examples of microRNAs whose cellular concentrations differ by several orders of magnitude, yet induce similar repression of target mRNAs. Likewise, microRNAs with similar expression can have very different repressive abilities. We show that the association of microRNAs with Argonaute proteins does not explain this variation in repression. The observed relationship is consistent with the limiting step in target repression being the association of the microRNA/RISC complex with the target site. These findings argue that modest changes in cellular microRNA concentration will have minor effects on repression of targets.
    PLoS ONE 08/2014; 9(8):e104286. DOI:10.1371/journal.pone.0104286 · 3.23 Impact Factor
  • Source
    • "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. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Background MiRNAs are essential mediators of many biological processes. The aim of this study was to investigate the dynamics of miRNA-mRNA regulatory networks during exercise and the subsequent recovery period. Results Here we monitored the transcriptome changes using microarray analysis of the whole blood of eight highly trained athletes before and after 30 min of moderate exercise followed by 30 min and 60 min of recovery period. We combined expression profiling and bioinformatics and analysed metabolic pathways enriched with differentially expressed mRNAs and mRNAs which are known to be validated targets of differentially expressed miRNAs. Finally we revealed four dynamically regulated networks comprising differentially expressed miRNAs and their known target mRNAs with anti-correlated expression profiles over time. The data suggest that hsa-miR-21-5p regulated TGFBR3, PDGFD and PPM1L mRNAs. Hsa-miR-24-2-5p was likely to be responsible for MYC and KCNJ2 genes and hsa-miR-27a-5p for ST3GAL6. The targets of hsa-miR-181a-5p included ROPN1L and SLC37A3. All these mRNAs are involved in processes highly relevant to exercise response, including immune function, apoptosis, membrane traffic of proteins and transcription regulation. Conclusions We have identified metabolic pathways involved in response to exercise and revealed four miRNA-mRNA networks dynamically regulated following exercise. This work is the first study to monitor miRNAs and mRNAs in parallel into the recovery period. The results provide a novel insight into the regulatory role of miRNAs in stress adaptation.
    BMC Physiology 06/2013; 13(1). DOI:10.1186/1472-6793-13-9
  • Source
    • "In contrast, the Ago1 binding to miR-U2 sequences could induce a shift to the endonucleolytic cleavages pathway which cleaves at positions 70–80 and 145 respectively. Multiple and adjacent small RNA-binding sites for Ago1 are known to facilitate cooperative interactions that stabilize Argonaute binding [54]. Since Ago1 has no enzymatic activity, an RNase must be recruited for miR-U2 maturation, similarly to miR-like ncRNAs produced from snoRNAs and tRNAs. "
    [Show abstract] [Hide abstract]
    ABSTRACT: RNU2 exists in two functional forms (RNU2-1 and RNU2-2) distinguishable by the presence of a unique 4-bases motif. Detailed investigation of datasets obtained from deep sequencing of five human lung primary tumors revealed that both forms express at a high rate a 19-22nt fragment (miR-U2-1 and -2) from its 3' region and contains the 4-bases motif. Deep sequencing of independent pools of serum samples from healthy donors and lung cancer patients revealed that miR-U2-1 and -2 are pervasively processed in lung tissue by means of endonucleolytic cleavages and stably exported to the blood. Then, microarrays hybridization experiments of matched normal/tumor samples revealed a significant over-expression of miR-U2-1 in 14 of 18 lung primary tumors. Subsequently, qRT-PCR of miR-U2-1 using serum from 62 lung cancer patients and 96 various controls demonstrated that its expression levels identify lung cancer patients with 79% sensitivity and 80% specificity. miR-U2-1 expression correlated with the presence or absence of lung cancer in patients with chronic obstructive pulmonary disease (COPD), other diseases of the lung - not cancer, and in healthy controls. These data suggest that RNU2-1 is a new bi-functional ncRNA that produces a 19-22nt fragment which may be useful in detecting lung cancer non-invasively in high risk patients.
    PLoS ONE 03/2013; 8(3):e60134. DOI:10.1371/journal.pone.0060134 · 3.23 Impact Factor
Show more