RESUMEN
INO80/SWR1 family chromatin remodelers are complexes composed of >15 subunits and molecular masses exceeding 1 MDa. Their important role in transcription and genome maintenance is exchanging the histone variants H2A and H2A.Z. We report the architecture of S. cerevisiae INO80 using an integrative approach of electron microscopy, crosslinking and mass spectrometry. INO80 has an embryo-shaped head-neck-body-foot architecture and shows dynamic open and closed conformations. We can assign an Rvb1/Rvb2 heterododecamer to the head in close contact with the Ino80 Snf2 domain, Ies2, and the Arp5 module at the neck. The high-affinity nucleosome-binding Nhp10 module localizes to the body, whereas the module that contains actin, Arp4, and Arp8 maps to the foot. Structural and biochemical analyses indicate that the nucleosome is bound at the concave surface near the neck, flanked by the Rvb1/2 and Arp8 modules. Our analysis establishes a structural and functional framework for this family of large remodelers.
Asunto(s)
Nucleosomas/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Ensamble y Desensamble de Cromatina , Espectrometría de Masas , Modelos Moleculares , Nucleosomas/metabolismo , Estructura Terciaria de Proteína , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/ultraestructura , Factores de Transcripción/química , Factores de Transcripción/metabolismoRESUMEN
Control of messenger RNA (mRNA) decay rate is intimately connected to translation elongation, but the spatial coordination of these events is poorly understood. The Ccr4-Not complex initiates mRNA decay through deadenylation and activation of decapping. We used a combination of cryo-electron microscopy, ribosome profiling, and mRNA stability assays to examine the recruitment of Ccr4-Not to the ribosome via specific interaction of the Not5 subunit with the ribosomal E-site in Saccharomyces cerevisiae This interaction occurred when the ribosome lacked accommodated A-site transfer RNA, indicative of low codon optimality. Loss of the interaction resulted in the inability of the mRNA degradation machinery to sense codon optimality. Our findings elucidate a physical link between the Ccr4-Not complex and the ribosome and provide mechanistic insight into the coupling of decoding efficiency with mRNA stability.
Asunto(s)
Codón , Extensión de la Cadena Peptídica de Translación , Estabilidad del ARN , Proteínas Represoras/metabolismo , Ribonucleasas/metabolismo , Ribosomas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Microscopía por Crioelectrón , Complejos Multiproteicos/química , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Factores de Iniciación de Péptidos/metabolismo , Conformación Proteica en Hélice alfa , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas Represoras/química , Proteínas Represoras/genética , Ribonucleasas/química , Ribonucleasas/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Factores de Transcripción/química , Factores de Transcripción/genética , Ubiquitina-Proteína Ligasas/química , Ubiquitina-Proteína Ligasas/genética , Ubiquitinación , Factor 5A Eucariótico de Iniciación de TraducciónRESUMEN
Protein inhibitors of activated STATs (PIAS) represent a small family of nuclear proteins that modulate the activity of many transcription factors and act as E3 ligases for covalent modification of proteins with the small ubiquitin-like modifier (SUMO). In particular, PIASy has been shown to inhibit the activation of gene expression by the IFN-responsive transcription factor STAT1 and the Wnt-responsive transcription factor LEF1. To assess the function of PIASy in vivo, we generated and analyzed mice carrying a targeted mutation of the Piasy gene. We find that homozygous mutant mice have no obvious morphological defects and have a normal distribution of lymphocyte populations. Molecular analysis of signaling in response to IFN-gamma and Wnt agonists revealed a modest reduction in the activation of endogenous and transfected target genes. Two-dimensional analysis of total proteins and SUMO-modified proteins in transformed pre-B cells showed no significant differences between wild-type mice and homozygous mutant mice. Taken together, our data indicate that PIASy has a modest effect on cytokine and Wnt signaling, suggesting a redundancy with other members of the family of PIAS proteins.