RESUMO
MicroRNAs (miRNAs) regulate a wide variety of biological processes by silencing their target genes. Argonaute (AGO) proteins load miRNAs to form an RNA-induced silencing complex (RISC), which mediates translational repression and/or mRNA decay of the targets. A scaffold protein called GW182 directly binds AGO and the CCR4-NOT deadenylase complex, initiating the mRNA decay reaction. Although previous studies have demonstrated the critical role of GW182 in cultured cells as well as in cell-free systems, its biological significance in living organisms remains poorly explored, especially in Drosophila melanogaster. Here, we generated gw182-null flies using the CRISPR/Cas9 system and found that, unexpectedly, they can survive until an early second-instar larval stage. Moreover, in vivo miRNA reporters can be effectively repressed in gw182-null first-instar larvae. Nevertheless, gw182-null flies have defects in the expression of chitin-related genes and the formation of the larval trachea system, preventing them from completing larval development. Our results highlight the importance of both GW182-dependent and -independent silencing mechanisms in vivo.
RESUMO
The path of ribosomes on mRNAs can be impeded by various obstacles. One such example is halting of ribosome movement by microRNAs, but the exact mechanism and physiological role remain unclear. Here, we find that ribosome stalling caused by the Argonaute-microRNA-SGS3 complex regulates production of secondary small interfering RNAs (siRNAs) in plants. We show that the double-stranded RNA-binding protein SGS3 interacts directly with the 3' end of the microRNA in an Argonaute protein, resulting in ribosome stalling. Importantly, microRNA-mediated ribosome stalling correlates positively with efficient production of secondary siRNAs from target mRNAs. Our results illustrate a role of paused ribosomes in regulation of small RNA function that may have broad biological implications across the plant kingdom.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Proteínas Argonautas/metabolismo , MicroRNAs/metabolismo , RNA de Plantas/metabolismo , RNA Interferente Pequeno/metabolismo , Ribossomos/metabolismo , Arabidopsis/metabolismo , Sequência de Bases , Linhagem Celular , Elementos Facilitadores Genéticos/genética , MicroRNAs/genética , Modelos Biológicos , Ligação Proteica , RNA de Cadeia Dupla/metabolismo , RNA de Plantas/genética , Complexo de Inativação Induzido por RNA/metabolismoRESUMO
The Argonaute subfamily of proteins (AGO) loads microRNAs (miRNAs) to form the effector complex that mediates target gene silencing. Empty AGO, but not miRNA-loaded AGO, is selectively degraded across species. We have reported that the degradation of empty AGO is part of a quality control pathway that eliminates dysfunctional AGO. However, how empty AGO is degraded remains unclear. Here we show that the empty state of Drosophila Ago1 is degraded by autophagy. Comprehensive LC-MS/MS analyses, together with manipulation of the Ago1 ubiquitination level, revealed that VCP, which mediates selective autophagy, recognizes empty Ago1 via the Ufd1-Npl4 heterodimer. Depletion of VCP-Ufd1-Npl4 machinery impairs degradation of empty Ago1 and miRNA-mediated target gene silencing. Our findings reveal a direct link between empty AGO degradation and selective autophagy that ensures efficient miRNA function.
Assuntos
Proteínas Argonautas/metabolismo , Morte Celular Autofágica , Proteínas de Drosophila/metabolismo , Proteólise , Proteína com Valosina/metabolismo , Animais , Proteínas Argonautas/genética , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster , Proteína com Valosina/genéticaRESUMO
MicroRNAs (miRNAs) are loaded into the Argonaute subfamily of proteins (AGO) to form an effector complex that silences target genes. Empty but not miRNA-loaded AGO is selectively degraded across species. However, the mechanism and biological significance of selective AGO degradation remain unclear. We discovered a RING-type E3 ubiquitin ligase we named Iruka (Iru), which selectively ubiquitinates the empty form of Drosophila Ago1 to trigger its degradation. Iru preferentially binds empty Ago1 and ubiquitinates Lys514 in the L2 linker, which is predicted to be inaccessible in the miRNA-loaded state. Depletion of Iru results in global impairment of miRNA-mediated silencing of target genes and in the accumulation of aberrant Ago1 that is dysfunctional for canonical protein-protein interactions and miRNA loading. Our findings reveal a sophisticated mechanism for the selective degradation of empty AGO that underlies a quality control process to ensure AGO function.