RESUMEN
The C-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II) is a regulatory hub for transcription and RNA processing. Here, we identify PHD-finger protein 3 (PHF3) as a regulator of transcription and mRNA stability that docks onto Pol II CTD through its SPOC domain. We characterize SPOC as a CTD reader domain that preferentially binds two phosphorylated Serine-2 marks in adjacent CTD repeats. PHF3 drives liquid-liquid phase separation of phosphorylated Pol II, colocalizes with Pol II clusters and tracks with Pol II across the length of genes. PHF3 knock-out or SPOC deletion in human cells results in increased Pol II stalling, reduced elongation rate and an increase in mRNA stability, with marked derepression of neuronal genes. Key neuronal genes are aberrantly expressed in Phf3 knock-out mouse embryonic stem cells, resulting in impaired neuronal differentiation. Our data suggest that PHF3 acts as a prominent effector of neuronal gene regulation by bridging transcription with mRNA decay.
Asunto(s)
Neuronas/metabolismo , ARN Polimerasa II/química , ARN Polimerasa II/metabolismo , ARN , Factores de Transcripción/metabolismo , Animales , Línea Celular , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Ratones Noqueados , Neuronas/química , Fosforilación , Dominios Proteicos , ARN/química , ARN/genética , ARN/metabolismo , ARN Polimerasa II/genética , Procesamiento Postranscripcional del ARN , Estabilidad del ARN , Factores de Transcripción/genética , Transcripción GenéticaRESUMEN
Poly(ADP-ribose) glycohydrolase (PARG) regulates cellular poly(ADP-ribose) (PAR) levels by rapidly cleaving glycosidic bonds between ADP-ribose units. PARG interacts with proliferating cell nuclear antigen (PCNA) and is strongly recruited to DNA damage sites in a PAR- and PCNA-dependent fashion. Here we identified PARG acetylation site K409 that is essential for its interaction with PCNA, its localization within replication foci and its recruitment to DNA damage sites. We found K409 to be part of a non-canonical PIP-box within the PARG disordered regulatory region. The previously identified putative N-terminal PIP-box does not bind PCNA directly but contributes to PARG localization within replication foci. X-ray structure and MD simulations reveal that the PARG non-canonical PIP-box binds PCNA in a manner similar to other canonical PIP-boxes and may represent a new type of PIP-box. While the binding of previously described PIP-boxes is based on hydrophobic interactions, PARG PIP-box binds PCNA via both stabilizing hydrophobic and fine-tuning electrostatic interactions. Our data explain the mechanism of PARG-PCNA interaction through a new PARG PIP-box that exhibits non-canonical sequence properties but a canonical mode of PCNA binding.
Asunto(s)
Glicósido Hidrolasas/metabolismo , Antígeno Nuclear de Célula en Proliferación/metabolismo , Acetilación , Calorimetría/métodos , Cromatina/metabolismo , Cristalografía por Rayos X , Daño del ADN , Transferencia Resonante de Energía de Fluorescencia , Glicósido Hidrolasas/química , Glicósido Hidrolasas/genética , Células HeLa , Humanos , Inmunoprecipitación , Rayos Láser , Lisina/genética , Lisina/metabolismo , Simulación de Dinámica Molecular , Antígeno Nuclear de Célula en Proliferación/química , Conformación Proteica , Fase S/genética , Electricidad EstáticaRESUMEN
Sirtuin 2 (SIRT2) is an NAD-dependent deacetylase known to regulate microtubule dynamics and cell cycle progression. SIRT2 has also been implicated in the pathology of cancer, neurodegenerative diseases and progeria. Here, we show that SIRT2 depletion or overexpression causes nuclear envelope reassembly defects. We link this phenotype to the recently identified regulator of nuclear envelope reassembly ANKLE2. ANKLE2 acetylation at K302 and phosphorylation at S662 are dynamically regulated throughout the cell cycle by SIRT2 and are essential for normal nuclear envelope reassembly. The function of SIRT2 therefore extends beyond the regulation of microtubules to include the regulation of nuclear envelope dynamics.
