RESUMO
The evolutionarily conserved nuclear Piwi protein of Drosophila melanogaster is a representative of the Argonaute small RNA binding protein family. Guided by small piRNAs, Piwi functions in transposon silencing in somatic and germ cells of the gonad. We found that in ovarian somatic and germ cells, as well as in the established ovarian somatic cell line, Piwi is concentrated predominantly in the nucleolus--the main nuclear compartment, participating not only in rRNA synthesis, but also in various cell stress responses. We demonstrated the colocalization of Piwi with nucleolar marker proteins--fibrillarin and Nopp140. A mutation preventing Piwi transport to the nucleus and disturbing transposon silencing (piwi(Nt)) leads to 6-8-fold upregulation of rRNA genes expression, as evaluated by the level of transcripts of transposon insertions in 28S rRNA genes. RNase treatment of live cultured ovarian somatic cells depletes Piwi from the nucleolus. The same effect is observed upon inhibiting RNA polymerase I which transcribes rRNA, but not RNA polymerase II. In contrast, upon heat shock Piwi is concentrated in the nucleolus and is depleted from the nucleoplasm. These results implicate Piwi in RNA polymerase activity modulation and stress response in the nucleolus. We discuss possible noncanonical Piwi functions along with its canonical role in transposon silencing by piRNAs.
Assuntos
Proteínas Argonautas/metabolismo , Nucléolo Celular/genética , RNA Polimerases Dirigidas por DNA/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , RNA Ribossômico 28S/genética , Animais , Proteínas Argonautas/genética , Nucléolo Celular/metabolismo , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Elementos de DNA Transponíveis/genética , Proteínas de Drosophila/genética , Feminino , Inativação Gênica , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Ovário/citologia , Ovário/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNARESUMO
Complexes of Piwi proteins and Piwi-interacting RNAs (piRNAs) carry out the repression of transposable elements in animal gonads. The Piwi protein clade is represented in D. melanogaster by three members: Piwi, Aub and Ago3. Piwi protein functions in the nuclei of somatic and germinal ovarian cells, whereas Aub and Ago3 are cytoplasmic proteins of germinal cells. Aub and Ago3 interact with each other in the perinuclear nuage organelle to perform piRNA amplification via the ping-pong mechanism. Previously, derepression of several transposable elements as a result of mutations in the piRNA silencing system was shown. Here we quantify the increase in expression level of an enlarged number of retrotransposons due to the mutations in the piwi gene, nuage components coding aub, mael and spn-E genes and the RNA helicase armi gene mutation that impairs Piwi nuclear localization, but not the ping-pong cycle. We reveal that piwi, armi, aub, spn-E and mael genes participate together in the repression of several transposons (HMS-Beagle, Gate and HeT-A), whereas silencing of land G elements requires the same genes except piwi. We suggest that Armi has other functions besides the localizing of Piwi protein in the nuclei. Our data suggest also a role of cytoplasmic Aub, Spn-E and Mael nuage proteins in Piwi-mediated repression of Gate and HMS-Beagle transposons in the germline nuclei. As a whole, our results corroborate the idea that genome stabilization in the germline is realized by different silencing strategies specific for different transposable elements. At the same time, our data suggest the existence of yet unknown mechanisms of interplay between nuclear and cytoplasmic components of the piRNA machinery in the germline.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Fatores de Iniciação de Peptídeos/metabolismo , RNA Interferente Pequeno/genética , Retroelementos/genética , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Animais , Proteínas Argonautas , Proteínas de Drosophila/genética , Feminino , Inativação Gênica , Ovário/metabolismo , Fatores de Iniciação de Peptídeos/genética , RNA Helicases/genética , RNA Helicases/metabolismo , RNA Interferente Pequeno/metabolismo , Complexo de Inativação Induzido por RNA/genética , Complexo de Inativação Induzido por RNA/metabolismoRESUMO
Two main types of short RNAs, 21 to 25 nucleotides long, are involved in the negative regulation of gene expression in eukaryotes: microRNAs and small interfering RNAs (siRNAs) of the RNA interference system. MicroRNAs predominantly suppress the translation of mRNA targets, while siRNAs not only prevent mRNA translation and/or lead to mRNA degradation, but are also involved in the regulation of gene expression at the transcriptional level. In germ cells translational regulation of gene expression plays a significant role and its mechanism has been extensively studied in oogenesis of Drosophila@. The role of heterochromatization and chromatin compaction, which can repress the expression of mobile elements and other repeated elements of the genome, was studied to a lesser extent. Activation and transposition of mobile elements accompanied by mutations and chromosome rearrangements are especially dangerous in germline cells. It has been proposed that a specialized class of short RNAs, repeat associated siRNAs (rasiRNAs), can be involved in repression of the expression of mobile elements in Drosophila germ cells. Here we describe the findings on subcellular ribonucleoprotein structures characteristic of germ cells: perinuclear and polar granules containing proteins of the RNA interference and microRNA maturation system. Also, we present our own results revealing the role of genes of the RNA interference system in mobile element silencing in Drosophila.