RESUMO
2'-O-Methylation (Nm) represents one of the most common RNA modifications. Nm affects RNA structure and function with crucial roles in various RNA-mediated processes ranging from RNA silencing, translation, self versus non-self recognition to viral defense mechanisms. Here, we identify two Nm methyltransferases (Nm-MTases) in Drosophila melanogaster (CG7009 and CG5220) as functional orthologs of yeast TRM7 and human FTSJ1. Genetic knockout studies together with MALDI-TOF mass spectrometry and RiboMethSeq mapping revealed that CG7009 is responsible for methylating the wobble position in tRNAPhe, tRNATrp and tRNALeu, while CG5220 methylates position C32 in the same tRNAs and also targets additional tRNAs. CG7009 or CG5220 mutant animals were viable and fertile but exhibited various phenotypes such as lifespan reduction, small RNA pathways dysfunction and increased sensitivity to RNA virus infections. Our results provide the first detailed characterization of two TRM7 family members in Drosophila and uncover a molecular link between enzymes catalyzing Nm at specific tRNAs and small RNA-induced gene silencing pathways.
Assuntos
Drosophila melanogaster/genética , Inativação Gênica , RNA de Transferência/genética , tRNA Metiltransferases/genética , Animais , Regulação da Expressão Gênica/genética , Humanos , Metilação , Metiltransferases/genética , Proteínas Nucleares/genética , Interferência de RNA , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Small RNAs target their complementary chromatin regions for gene silencing through nascent long non-coding RNAs (lncRNAs). In the ciliated protozoan Tetrahymena, the interaction between Piwi-associated small RNAs (scnRNAs) and the nascent lncRNA transcripts from the somatic genome has been proposed to induce target-directed small RNA degradation (TDSD), and scnRNAs not targeted for TDSD later target the germline-limited sequences for programmed DNA elimination. In this study, we show that the SUMO E3 ligase Ema2 is required for the accumulation of lncRNAs from the somatic genome and thus for TDSD and completing DNA elimination to make viable sexual progeny. Ema2 interacts with the SUMO E2 conjugating enzyme Ubc9 and enhances SUMOylation of the transcription regulator Spt6. We further show that Ema2 promotes the association of Spt6 and RNA polymerase II with chromatin. These results suggest that Ema2-directed SUMOylation actively promotes lncRNA transcription, which is a prerequisite for communication between the genome and small RNAs.
Assuntos
RNA Longo não Codificante , Ubiquitina-Proteína Ligases , Ubiquitina-Proteína Ligases/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo , RNA Interferente Pequeno/metabolismo , DNA de Protozoário/genética , Cromatina , SumoilaçãoRESUMO
Piwi-bound small RNAs induce programmed DNA elimination in the ciliated protozoan Tetrahymena. Using the phenomenon called codeletion, this process can be reprogrammed to induce ectopic DNA elimination at basically any given genomic location. Here, we describe the usage of codeletion for genetic studies in Tetrahymena and for investigations of the molecular mechanism of Piwi-directed programmed DNA elimination.