IDENTIFYING MAELSTROM COMPLEXES

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Various classes of transposable elements occupy vast genomic territories and pose significant ganger to the genomic integrity. In mammalian species, including humans, the negative effect of transposons is countered by sophisticated cellular machinery that involves DNA methylation and a recently discovered small RNA-based mechanism known as the piRNA pathway. This novel defensive mechanism involves effector proteins of the PIWI subclade of Argonautes, associated small RNAs and a cadre of auxiliary proteins. One of such proteins aiding in piRNA production and function is Maelstrom (MAEL), an evolutionarily conserved protein with a non-canonical HMG box nucleic acid binding domain and a putative RNAseH fold domain of unknown biochemical function. Our prior genetic and cell biological studies have demonstrated the essential role of mouse MAEL for the robust activity of the piRNA pathway. To further elucidate the mechanism of piRNA biogenesis and function in the mouse male germ cell lineage, we propose to systematically characterize MAEL-associated proteome and RNAome. To this end, we will perform affinity purification of MAEL-containing complexes, identify MAEL-interacting proteins (MIPs) by mass spectrometry, and characterize their interactions with MAEL by means of yeast two hybrid, immunofluorescent analysis, and in vitro binding approaches. We will also identify small and large RNA composition of MAEL-containing complexes, characterize their subcellular distribution by RNA fluorescent in situ hybridization in wild type and Mael mutant germ cells, and determine the effect of the Mael mutation on their expression. The combination of these approaches will provide essential new clues about piRNA biogenesis and functioning in the mouse male germline.