NOT10 and C2orf29/NOT11 form a conserved module of the CCR4-NOT complex that docks onto the NOT1 N-terminal domain

Abstract

The CCR4-NOT complex plays a crucial role in post-transcriptional mRNA regulation in eukaryotes. This complex catalyzes the removal of mRNA poly(A) tails, thereby repressing translation and committing an mRNA to degradation. The conserved core of the complex is assembled by the interaction of at least two modules: the NOT module, which minimally consists of NOT1, NOT2 and NOT3, and a catalytic module comprising two deadenylases, CCR4 and POP2/CAF1. Additional complex subunits include CAF40 and two newly identified human subunits, NOT10 and C2orf29. The role of the NOT10 and C2orf29 subunits and how they are integrated into the complex are unknown. Here, we show that the Drosophila melanogaster NOT10 and C2orf29 orthologs form a complex that interacts with the N-terminal domain of NOT1 through C2orf29. These interactions are conserved in human cells, indicating that NOT10 and C2orf29 define a conserved module of the CCR4-NOT complex. We further investigated the assembly of the D. melanogaster CCR4-NOT complex, and demonstrate that the conserved armadillo repeat domain of CAF40 interacts with a region of NOT1, comprising a domain of unknown function, DUF3819. Using tethering assays, we show that each subunit of the CCR4-NOT complex causes translational repression of an unadenylated mRNA reporter and deadenylation and degradation of a polyadenylated reporter. Therefore, the recruitment of a single subunit of the complex to an mRNA target induces the assembly of the complete CCR4-NOT complex, resulting in a similar regulatory outcome.

The Ccr4-Not deadenylase complex constitutes the major poly(A) removal activity inC. elegans

“…CNOT10) of the human Ccr4-Not complex subunits are evolutionarily conserved in C. elegans. Overall, the nematode Ccr4-Not complex is more similar to flies and humans than to yeast (Bawankar et al, 2013;Mauxion et al, 2012). In parallel to humans and flies (Albert et al, 2000;Temme et al, 2010), the two yeast paralogs, Not3p and Not5p, are represented by only one gene in C. elegans, i.e.…”

“…With the exception of yeast Caf130p, all are evolutionarily conserved and orthologs have been described in flies and mammals (Denis and Chen, 2003;Temme et al, 2004). Furthermore, the complex is extended in Trypanosomes by NOT10, and in flies and mammals by NOT10 and NOT11 (Bawankar et al, 2013;Färber et al, 2013;Mauxion et al, 2012;Temme et al, 2010). The enzymatic balance between CCR4 and CAF1 changed during evolution.…”

mi RISC and the CCR 4– NOT complex silence mRNA targets independently of 43S ribosomal scanning

“…This model is based on the following observations: First, the interaction of GW182 proteins with the CCR4–NOT complex is not only required for degradation but also required for translational repression of miRNA reporters (Braun et al , 2011; Chekulaeva et al , 2011; Fabian et al , 2011; Huntzinger et al , 2013; Zekri et al , 2013; Chen et al , 2014; Mathys et al , 2014). Second, like miRISC, the CCR4–NOT complex represses translation in the absence of deadenylation (Cooke et al , 2010; Braun et al , 2011; Chekulaeva et al , 2011; Bawankar et al , 2013; Zekri et al , 2013). Translational repression by the CCR4–NOT complex can, at least in part, be explained by a direct interaction between the NOT1 subunit and the DEAD‐box ATPase DDX6 (also known as RCK).…”

The deadenylase components Not2p, Not3p, and Not5p promote mRNA decapping

“…Previous studies of NOT2, NOT3, and NOT5 have shown that these proteins have closely related activities (Bai et al 1999;Bawankar et al 2013). Moreover, Not2p may be a core member of the Ccr4p/Not1p-5p deadenylase complex that recruits Not3p and/or Not5p into the complex (Bhaskar et al 2013).…”