RESUMO
Colonization of genomes by a new selfish genetic element is detrimental to the host species and must lead to an efficient, repressive response. In vertebrates as well as in Drosophila, piRNAs repress transposons in the germ line, whereas endogenous siRNAs take on this role in somatic cells. We show that their biogenesis depends on a new isoform of the Drosophila TRBP homologue loquacious, which arises by alternative polyadenylation and is distinct from the one that functions during the biogenesis of miRNAs. For endo-siRNAs and piRNAs, it is unclear how an efficient response can be initiated de novo. Our experiments establish that the endo-siRNA pathway will target artificially introduced sequences without the need for a pre-existing template in the genome. This response is also triggered in transiently transfected cells, thus genomic integration is not essential. Deep sequencing showed that corresponding endo-siRNAs are generated throughout the sequence, but preferentially from transcribed regions. One strand of the dsRNA precursor can come from spliced mRNA, whereas the opposite strand derives from independent transcripts in antisense orientation.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , RNA Interferente Pequeno/genética , Proteínas de Ligação a RNA/metabolismo , Animais , Drosophila melanogaster/metabolismo , Proteínas de Fluorescência Verde/genética , Isoformas de Proteínas/metabolismo , Interferência de RNA , Splicing de RNA , RNA de Cadeia Dupla/genética , RNA de Cadeia Dupla/metabolismo , RNA Mensageiro/genética , RNA Interferente Pequeno/metabolismo , Transfecção , TransgenesRESUMO
In Drosophila, three types of endogenous small RNAs-microRNAs (miRNAs), PIWI-interacting RNAs (piRNAs), and endogenous small-interfering RNAs (endo-siRNAs or esiRNAs)-function as triggers in RNA silencing. Although piRNAs are produced independently of Dicer, miRNA and esiRNA biogenesis pathways require Dicer1 and Dicer2, respectively. Recent studies have shown that among the four isoforms of Loquacious (Loqs), Loqs-PB and Loqs-PD are involved in miRNA and esiRNA processing pathways, respectively. However, how these Loqs isoforms function in their respective small RNA biogenesis pathways remains elusive. Here, we show that Loqs-PD associates specifically with Dicer2 through its C-terminal domain. The Dicer2-Loqs-PD complex contains R2D2, another known Dicer2 partner, and excises both exogenous siRNAs and esiRNAs from their corresponding precursors in vitro. However, Loqs-PD, but not R2D2, enhanced Dicer2 activity. The Dicer2-Loqs-PD complex processes esiRNA precursor hairpins with long stems, which results in the production of AGO2-associated small RNAs. Interestingly, however, small RNAs derived from terminal hairpins of esiRNA precursors are loaded onto AGO1; thus, they are classified as a new subset of miRNAs. These results suggest that the precursor RNA structure determines the biogenesis mechanism of esiRNAs and miRNAs, thereby implicating hairpin structures with long stems as intermediates in the evolution of Drosophila miRNA.
Assuntos
Drosophila melanogaster/metabolismo , MicroRNAs/biossíntese , RNA Interferente Pequeno/biossíntese , Animais , Vias Biossintéticas , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , MicroRNAs/química , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Helicases/química , RNA Helicases/metabolismo , RNA Interferente Pequeno/química , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Ribonuclease III/química , Ribonuclease III/metabolismoRESUMO
In addition to miRNAs and siRNAs, a third small RNA silencing system has been uncovered that prevents the spreading of selfish genetic elements. Production of the Piwi-associated RNAs (piRNAs), which mediate the silencing activity in this pathway, is initiated at a few master control regions within the genome. The nature of the primary piRNA-generating transcript is still unknown, but RNA interference (RNAi)-like cleavage events are likely defining the 5'-ends of mature piRNAs. We summarize the recent literature on piRNA biogenesis and function with an emphasis on work in Drosophila, where genetics and biochemistry have met very successfully.