Quantitative functions of Argonaute proteins in mammalian development

MicroRNA (miRNA) is a class of tiny (∼19- to 24-nucleotide [nt]), noncoding RNA species that play important roles in the regulation of mammalian gene expression at post-transcriptional levels (Bartel 2009). To execute their diverse physiological functions, miRNAs are loaded into the miRNA-induced silencing complex (miRISC) and, in turn, guide the miRISC to their cognate mRNA targets by base-pairing between the miRNAs and their mRNA targets, usually located within the 3′ untranslated region (3′ UTR) (Bartel 2009). Argonaute proteins are the essential components of the miRISC that directly recruit miRNAs and function as the interface between miRNAs and their mRNA targets (Hock and Meister 2008; Czech and Hannon 2011). Mammals have four Argonautes (Ago1–4) that are involved in the miRNA pathway (Hock and Meister 2008; Czech and Hannon 2011). Among them, Ago2 is unique, with the slicer activity that mediates the cleavage of perfectly matched targets for miRNAs and siRNAs (Liu et al. 2004; Meister et al. 2004; Yekta et al. 2004; Hock and Meister 2008; Czech and Hannon 2011). When individual Argonautes are ablated constitutively in mice, only the loss of Ago2 causes embryonic lethality, whereas single losses of Ago1, Ago3, or Ago4 are dispensable for animal development (Liu et al. 2004; Morita et al. 2007). In addition, the biogenesis of endogenous siRNA (endo-siRNA) in the oocytes and miR-451, a blood-specific miRNA, has been shown to require the slicer activity of Ago2 (Tam et al. 2008; Watanabe et al. 2008; Cheloufi et al. 2010; Cifuentes et al. 2010). Collectively, these observations lead to a widely recognized view that Ago2 is a specialized Argonaute, probably due to its slicer activity. However, exhaustive bioinformatic and experimental analyses have failed to identify a large number of perfectly matched miRNA:mRNA regulatory sequences. It remains unclear why Ago2 is universally important for miRNA functions in diverse organs and tissues of mammals. In addition, the functional significance of Ago1, Ago3, and Ago4 for miRNA activity is poorly understood, probably due to the lack of developmental phenotypes in knockout (KO) mouse models. Interestingly, novel regulatory mechanisms begin to emerge in which Ago1 and their associated miRISCs are asymmetrically segregated to dictate cell fate decision during stem cell division (Neumuller et al. 2008; Schwamborn et al. 2009). Finally, recent studies suggest that miRNA-mediated regulation is highly quantitative and dependent on the relative concentration of miRNA and their target mRNAs (Mukherji et al. 2011; Tay et al. 2011). Therefore, it is critical to determine the function of individual Argonautes and understand the quantitative nature of their contribution to the miRNA pathway during mammalian development. In this study, we investigate the functions of individual Argonautes by quantitatively cloning and sequencing miRNAs that associate with endogenous Argonautes and examining the developmental defects in Ago1 and Ago2 single- or double-KO mouse models in the skin. Surprisingly, the slicer activity of Ago2 is dispensable for both miRNA biogenesis and function in the skin. Instead, miRNAs are randomly sorted to miRISCs that contain individual Argonautes in proportion to the abundance of each protein. Genome-wide shotgun proteomics and absolute quantification of Argonautes demonstrates that Ago2 is the most abundant Argonaute and so associates with the largest pool of miRNAs in both mouse epidermal cells and human melanoma cells. Finally, genome-wide protein quantification reveals a quantitative landscape for the key components of the miRNA pathway. The demonstration of the quantitative distribution of miRNAs to individual Argonautes in mammals has important implications for our understanding of the function of Argonautes in diverse biological events, and these findings raise a possibility that miRNA activity can be quantitatively manipulated by controlling individual Argonautes.