RESUMEN
MicroRNAs (miRNAs) play an important role in gene regulation. In Arabidopsis, mature miRNAs are processed from primary miRNA transcripts by the Dicing complex that contains Dicer-like 1 (DCL1), SERRATE (SE), and Hyponastic Leaves 1 (HYL1). The Dicing complex can form nuclear dicing bodies (D-bodies) through SE phase separation. Here, we report that Cyclophilin71 (CYP71), a peptidyl-prolyl isomerase (PPIase), positively regulates miRNA processing. We show that CYP71 directly interacts with SE and enhances its phase separation, thereby promoting the formation of D-body and increasing the activity of the Dicing complex. We further show that the PPIase activity is important for the function of CYP71 in miRNA production. Our findings reveal orchestration of miRNA processing by a cyclophilin protein and suggest the involvement of peptidyl-prolyl cis-trans isomerization, a structural mechanism, in SE phase separation and miRNA processing.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , MicroARNs , Arabidopsis/genética , Ciclofilinas/genética , MicroARNs/genética , Hojas de la Planta , Proteínas de Unión al ARN , Proteínas de Arabidopsis/genéticaRESUMEN
MicroRNA (miRNA) production entails the step-wise processing of primary miRNAs (pri-miRNAs) into precursor miRNAs (pre-miRNAs) and miRNA/* duplexes by Dicing complexes containing DCL1, HYL1 and SE, which are localized in nuclear dicing bodies (D-bodies)1,2. Here, we show that D-bodies are phase-separated condensates. SE forms droplets and drives DCL1, HYL1 and pri/pre-miRNAs into the droplets in vitro, and mutation of SE abrogates the formation of D-bodies in vivo, which indicates that D-bodies arise through SE-mediated phase separation. Disruption of SE phase separation greatly reduces its activity in promoting miRNA processing both in vitro and in vivo. We further show that pre-miRNAs are processed into miRNA/* duplexes in the droplets and, after processing, miRNA/* duplexes are bound by HYL1 and released from the droplets. Our findings provide evidence that efficient miRNA processing depends on the SE-phase-separation-mediated formation of D-bodies and suggest a paradigm that the products made in phase-separated condensates can be shipped out for subsequent processes.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Regulación de la Expresión Génica de las Plantas , MicroARNs/metabolismo , Procesamiento Postranscripcional del ARN , Proteínas de Unión al ARN/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Núcleo Celular , MicroARNs/genética , Transición de Fase , Proteínas de Unión al ARN/genéticaRESUMEN
Chemical modifications on histones constitute a key mechanism for gene regulation in chromatin context. Recently, histone lysine ß-hydroxybutyrylation (Kbhb) was identified as a new form of histone acylation that connects starvation-responsive metabolism to epigenetic regulation. Sirtuins are a family of NAD+-dependent deacetylases. Through systematic profiling studies, we show that human SIRT3 displays class-selective histone de-ß-hydroxybutyrylase activities with preference for H3 K4, K9, K18, K23, K27, and H4K16, but not for H4 K5, K8, K12, which distinguishes it from the Zn-dependent HDACs. Structural studies revealed a hydrogen bond-lined hydrophobic pocket favored for the S-form Kbhb recognition and catalysis. ß-backbone but not side chain-mediated interactions around Kbhb dominate sequence motif recognition, explaining the broad site-specificity of SIRT3. The observed class-selectivity of SIRT3 is due to an entropically unfavorable barrier associated with the glycine-flanking motif that the histone Kbhb resides in. Collectively, we reveal the molecular basis for class-selective histone de-ß-hydroxybutyrylation by SIRT3, shedding lights on the function of sirtuins in Kbhb biology through hierarchical deacylation.