Our research on AGO4 began with the observation that two Argonautes in particular are highly expressed in mouse spermatocytes. These are AGO3 and AGO4. We generated mouse mutants for Ago4 and discovered that two key processes are affected during gametogenesis. Firstly, we find that meiotic sex chromosome silencing is reduced in the absence of AGO4, coincident with a loss of sex body integrity and the expression of genes from the Y chromosome that are toxic to meiotic cells. The sex body is a unique feature of pachytene cells and harbors the mostly unsynapsed X and Y chromosomes, presumably sequestering them away from the synapsis checkpoint machinery that would otherwise trigger apoptotic cell death upon encountering asynaptic chromosomal regions. Co-incident with this is the transcriptional silencing of these asynapsed regions, in a process known as meiotic sex chromosome inactivation (MSCI). Silencing also occurs on the autosomes if synapsis is disrupted, a process known as meiotic silencing of unpaired chromatin (MSUC), as observed in mouse mutants that exhibit different degrees of synapsis failure. Indeed, in such mutants, we find that AGO4 localizes to these sites of asynapsis during prophase I, as well as to the sex body of normal spermatocytes. Thus, AGO4 localizes to sites of meiotic silencing and brings with it small RNAs, the identity of which has yet to be determined. This localization of AGO4 is unique since most Argoautes reside within the cytoplasm of mammalian cells, suggesting unique roles of AGO4 in regulating chromatin-related events during silencing (see figure A below).
Our second observation was the unexpected early entry into meiosis in Ago4 mutant males. Ago4 mutant males enter meiotic prophase I approximately 4-6 days earlier during postnatal life, suggesting that appropriate timing of meiotic initiation is dependent on small RNAs bound to AGO4. Coincident with this, we see premature expression of genes required for retinoic acid metabolism. Since retinoic acid is the major initiator of meiosis in mammals, these observations suggest that AGO4-bound small RNAs influence the timing of retinoic acid production in the mouse testis (see figure B below).
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The image below shows the localization of a specific microRNA (mir24) in the sex body of prophase 1 nuclei of wildtype and Ago4 homozygous mutant males:
The discovery in the late 1980s that genome-encoded double-stranded RNA (dsRNA) could serve as a precursor for small RNA-mediated translational repression led to the identification of RNA interference (RNAi) as a major mechanism for control of gene expression in many organisms, and the awarding of the Nobel Prize to Andrew Fire and Craig Mello in 2006. Since then, many classes of small RNA have been identified, including small-interfering RNA (siRNA), micro-RNA (miRNA), and the germline specific piwi-interacting RNA (piRNA). One of the basic principles that governs the functioning of any small RNA species is that their activity is determined by, and dependent on, their binding to an Argonaute protein, of which more than 40 have been identified across eukaryotes. Two sub-clades of Argonautes exist in mammals: the PIWI subfamily that specifically bind piRNAs, and the AGO subfamily that bind both miRNA and siRNA classes. While the four mammalian AGO family members are thought to share redundant functions in the microRNA pathway, only AGO2 possesses the catalytic “slicer” function that underlies the process of RNAi. Whether AGO1, AGO3, or AGO4 possess specialized functions in mammals remains unclear, but given the high expression of AGO3 and AGO4 in the mammalian germ line, we are interested in the role that these Argonautes play in gametogenesis. For more information on RNA interference, see this site.
Modzelewski A.J., Holmes R.J., Hilz S., Grimson A., Cohen P.E. AGO4 regulates entry into meiosis and influences silencing of sex chromosomes in the male mouse germline. Developmental Cell. 23: 251-264 (2012) DOWNLOAD