Micrographs of empty cryo-EM grids that are used in training of crYOLO

Micrographs of empty cryo-EM grids that are used in training of crYOLO. The micrographs do not contain any protein, only ice and contamination. Datasets were recorded with different cameras (Falcon 3 / K2) and grid types

A conserved CAF40-binding motif in metazoan NOT4 mediates association with the CCR4-NOT complex

The multisubunit CCR4-NOT mRNA deadenylase complex plays important roles in the posttranscriptional regulation of gene expression. The NOT4 E3 ubiquitin ligase is a stable component of the CCR4-NOT complex in yeast but does not copurify with the human or Drosophila melanogaster complex. Here we show that the C-terminal regions of human and D. melanogaster NOT4 contain a conserved sequence motif that directly binds the CAF40 subunit of the CCR4-NOT complex (CAF40-binding motif [CBM]). In addition, nonconserved sequences flanking the CBM also contact other subunits of the complex. Crystal structures of the CBM-CAF40 complex reveal a mutually exclusive binding surface for NOT4 and Roquin or Bag of marbles mRNA regulatory proteins. Furthermore, CAF40 depletion or structure-guided mutagenesis to disrupt the NOT4-CAF40 interaction impairs the ability of NOT4 to elicit decay of tethered reporter mRNAs in cells. Together with additional sequence analyses, our results reveal the molecular basis for the association of metazoan NOT4 with the CCR4-NOT complex and show that it deviates substantially from yeast. They mark the NOT4 ubiquitin ligase as an ancient but nonconstitutive cofactor of the CCR4-NOT deadenylase with potential recruitment and/or effector functions

Drosophila Bag-of-marbles directly interacts with the CAF40 subunit of the CCR4-NOT complex to elicit repression of mRNA targets

Drosophila melanogaster Bag-of-marbles (Bam) promotes germline stem cell (GSC) differentiation by repressing the expression of mRNAs encoding stem cell maintenance factors. Bam interacts with Benign gonial cell neoplasm (Bgcn) and the CCR4 deadenylase, a catalytic subunit of the CCR4-NOT complex. Bam has been proposed to bind CCR4 and displace it from the CCR4-NOT complex. Here, we investigated the interaction of Bam with the CCR4-NOT complex by using purified recombinant proteins. Unexpectedly, we found that Bam does not interact with CCR4 directly but instead binds to the CAF40 subunit of the complex in a manner mediated by a conserved N-terminal CAF40-binding motif (CBM). The crystal structure of the Bam CBM bound to CAF40 reveals that the CBM peptide adopts an α-helical conformation after binding to the concave surface of the crescent-shaped CAF40 protein. We further show that Bam-mediated mRNA decay and translational repression depend entirely on Bam's interaction with CAF40. Thus, Bam regulates the expression of its mRNA targets by recruiting the CCR4-NOT complex through interaction with CAF40

Structure and assembly of the NOT10:11 module of the CCR4-NOT complex

Abstract NOT1, NOT10, and NOT11 form a conserved module in the CCR4-NOT complex, critical for post-transcriptional regulation in eukaryotes, but how this module contributes to the functions of the CCR4-NOT remains poorly understood. Here, we present cryo-EM structures of human and chicken NOT1:NOT10:NOT11 ternary complexes to sub-3 Å resolution, revealing an evolutionarily conserved, flexible structure. Through biochemical dissection studies, which include the Drosophila orthologs, we show that the module assembly is hierarchical, with NOT11 binding to NOT10, which then organizes it for binding to NOT1. A short proline-rich motif in NOT11 stabilizes the entire module assembly

HELZ directly interacts with CCR4-NOT and causes decay of bound mRNAs

Eukaryotic superfamily (SF) 1 helicases have been implicated in various aspects of RNA metabolism, including transcription, processing, translation, and degradation. Nevertheless, until now, most human SF1 helicases remain poorly understood. Here, we have functionally and biochemically characterized the role of a putative SF1 helicase termed "helicase with zinc-finger," or HELZ. We discovered that HELZ associates with various mRNA decay factors, including components of the carbon catabolite repressor 4-negative on TATA box (CCR4-NOT) deadenylase complex in human and Drosophila melanogaster cells. The interaction between HELZ and the CCR4-NOT complex is direct and mediated by extended low-complexity regions in the C-terminal part of the protein. We further reveal that HELZ requires the deadenylase complex to mediate translational repression and decapping-dependent mRNA decay. Finally, transcriptome-wide analysis of Helz-null cells suggests that HELZ has a role in the regulation of the expression of genes associated with the development of the nervous system

Structure and assembly of the NOT module of the human CCR4–NOT complex

The CCR4-NOT deadenylase complex is a master regulator of translation and mRNA stability. Its NOT module orchestrates recruitment of the catalytic subunits to target mRNAs. We report the crystal structure of the human NOT module formed by the CNOT1, CNOT2 and CNOT3 C-terminal (-C) regions. CNOT1-C provides a rigid scaffold consisting of two perpendicular stacks of HEAT-like repeats. CNOT2-C and CNOT3-C heterodimerize through their SH3-like NOT-box domains. The heterodimer is stabilized and tightly anchored to the surface of CNOT1 through an unexpected intertwined arrangement of peptide regions lacking defined secondary structure. These assembly peptides mold onto their respective binding surfaces and form extensive interfaces. Mutagenesis of individual interfaces and perturbation of endogenous protein ratios cause defects in complex assembly and mRNA decay. Our studies provide a structural framework for understanding the recruitment of the CCR4-NOT complex to mRNA targets