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

Csilla Keskeny,Tobias Raisch,Oliver Weichenrieder,Elisa Izaurralde,Annamaria Sgromo,Dipankar Bhandari,Cátia Igreja

doi:10.1101/gad.320952.118

Csilla Keskeny, Tobias Raisch + Show 5 more

Open Access

https://doi.org/10.1101/gad.320952.118

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Abstract

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.

Highlights

The multisubunit CCR4–NOT mRNA deadenylase complex plays important roles in the posttranscriptional regulation of gene expression
The CCR4–NOT complex (Fig. 1A) is a multisubunit complex (Chen et al 2001; Lau et al 2009; Temme et al 2010) that assembles on the NOT1 scaffold protein, which consists of several α-helical domains that serve to dock the other subunits of the complex (Bawankar et al 2013)
Hs NOT4-C, showed a stable interaction with the CCR4–NOT complex, as indicated by the detection of endogenous NOT1, NOT2, and NOT3 subunits in the pull-down fraction (Fig. 1C, lane 8)

Summary

Introduction

The multisubunit CCR4–NOT mRNA deadenylase complex plays important roles in the posttranscriptional regulation of gene expression. 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. The catalytic module and the NOT module are connected by the CAF40-binding domain of NOT1 (labeled “CN9BD”) and a connector domain (labeled “MIF4G-C”) of unknown function (Chen et al 2014; Mathys et al 2014; Raisch et al 2018) Both the NOT module and the CAF40 subunit of the CCR4–NOT complex have been reported as important peptide-docking sites for the recruitment of the complex by mRNA-associated proteins. NOT4 has been implicated in cotranslational mRNA quality control and translational repression in the context of stalled ribosomes, such as in the “No-Go”

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