RESUMEN
PIWI proteins and their associated PIWI-interacting RNAs (piRNAs) protect genome integrity by silencing transposons in animal germlines. The molecular mechanisms and components responsible for piRNA biogenesis remain elusive. PIWI proteins contain conserved symmetrical dimethylarginines (sDMAs) that are specifically targeted by TUDOR domain-containing proteins. Here we report that the sDMAs of PIWI proteins play crucial roles in PIWI localization and piRNA biogenesis in Bombyx mori-derived BmN4 cells, which harbor fully functional piRNA biogenesis machinery. Moreover, RNAi screenings for Bombyx genes encoding TUDOR domain-containing proteins identified BmPAPI, a Bombyx homolog of Drosophila PAPI, as a factor modulating the length of mature piRNAs. BmPAPI specifically recognized sDMAs and interacted with PIWI proteins at the surface of the mitochondrial outer membrane. BmPAPI depletion resulted in 3'-terminal extensions of mature piRNAs without affecting the piRNA quantity. These results reveal the BmPAPI-involved piRNA precursor processing mechanism on mitochondrial outer membrane scaffolds.
Asunto(s)
Arginina/análogos & derivados , Bombyx/metabolismo , Proteínas Portadoras/metabolismo , Proteínas de Drosophila/metabolismo , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Ovario/metabolismo , ARN Interferente Pequeño/metabolismo , Animales , Arginina/metabolismo , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Northern Blotting , Western Blotting , Bombyx/genética , Proteínas Portadoras/genética , Cartilla de ADN/química , Cartilla de ADN/genética , Cartilla de ADN/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Femenino , Técnica del Anticuerpo Fluorescente , Células Germinativas , Inmunoprecipitación , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Mitocondrias/genética , Proteínas Mitocondriales/genética , Ovario/citología , Estructura Terciaria de Proteína , ARN Mensajero/genética , ARN Interferente Pequeño/química , ARN Interferente Pequeño/genética , Complejo Silenciador Inducido por ARN/genética , Complejo Silenciador Inducido por ARN/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Resonancia por Plasmón de SuperficieRESUMEN
Piwi proteins and their bound Piwi-interacting RNAs (piRNAs) are predominantly expressed in the germline and play crucial roles in germline development by silencing transposons and other targets. Bombyx mori BmN4 cells are culturable germ cells that equip the piRNA pathway. Because of the scarcity of piRNA-expressing culturable cells, BmN4 cells are being utilized for the analyses of piRNA biogenesis. We here report that the piRNA biogenesis in BmN4 cells is regulated by cell density. As cell density increased, the abundance of Piwi proteins and piRNA biogenesis factors was commonly upregulated, resulting in an increased number of perinuclear nuage-like granules where Piwi proteins localize. Along with these phenomena, the abundance of mature piRNAs also globally increased, whereas levels of long piRNA precursor and transposons decreased, suggesting that increasing cell density promotes piRNA biogenesis pathway and that the resultant accumulation of mature piRNAs is functionally significant for transposon silencing. Our study reveals a previously uncharacterized link between cell density and piRNA biogenesis, designates cell density as a critical variable in piRNA studies using BmN4 cell system, and suggests the alteration of cell density as a useful tool to monitor piRNA biogenesis and function.
Asunto(s)
Bombyx/genética , Células Germinativas/metabolismo , ARN Interferente Pequeño/genética , Animales , Recuento de Células , Línea Celular , Células Cultivadas , Biología Computacional , Gránulos Citoplasmáticos/metabolismo , Elementos Transponibles de ADN , Proteínas de Insectos/genética , Proteínas de Insectos/metabolismoRESUMEN
Arginine methylation is an important posttranslational protein modification that modulates protein function for a wide range of biological processes. PIWI proteins, a subclade of the Argonaute family proteins, contain evolutionarily conserved symmetrical dimethylarginines (sDMAs). It has become increasingly apparent that the sDMAs of PIWI proteins serve as binding elements for TUDOR domain-containing proteins and that sDMA-dependent protein interactions play crucial roles in the biogenesis and function of PIWI-interacting RNAs (piRNAs). We describe a method for detecting PIWI sDMAs and purifying PIWI/piRNA complexes using anti-sDMA antibodies.