Regulation of the multisubunit CCR4-NOT deadenylase in the initiation of mRNA degradation

Abstract

The conserved CCR4-NOT complex initiates the decay of mRNAs by catalyzing the shortening of their poly(A) tails in a process known as deadenylation. Recent studies have provided mechanistic insights into the action and regulation of this molecular machine. The two catalytic enzymatic subunits of the complex hydrolyze polyadenosine RNA. Notably, the non-catalytic subunits substantially enhance the complex's affinity and sequence selectivity for polyadenosine by directly contacting the RNA. An additional regulatory mechanism is the active recruitment of the CCR4-NOT to transcripts targeted for decay by RNA-binding proteins that recognize motifs or sequences residing predominantly in untranslated regions. This targeting and strict control of the mRNA deadenylation process emerges as a crucial nexus during post-transcriptional regulation of gene expression.

Figure 1.. The architecture of the mammalian CCR4-NOT complex.

A schematic view of the domain organization of NOT1 and bound subunits, together with the structures and structural models of the characterized modules and subassemblies. The experimental structures shown here are the N-terminal part of S. cerevisiae NOT1 (PDB 4B8B) [17], the human CAF40 module (PDB 4CRU) [21], the C. thermophilum NOT1 MIF4G-C (PDB 6H3Z) [23] and the human NOT module (PDB 4C0D) [24]. The model of the human nuclease module was prepared by superposing the NOT1-MIF4G-CAF1 dimer (PDB 4GMJ) [18] as well as the CCR4 LRR and the CCR4 EEP domains, respectively (both PDB 7AX1) [19] onto the S. cerevisiae nuclease module (PDB 4B8C) [17]. No experimental structural information was reported for the NOT10–11 module. AlphaFold2 structure prediction suggests that NOT10 (UniProt ID Q9H9A5) and NOT11 (UniProt ID Q9UKZ1) are composed primarily of helical repeats. NOT10 and NOT11 do not exist in S. cerevisiae and other ascomycetes; all other subunits are conserved throughout eukaryotes.

Figure 2.. Transcript-specific recruitment of CCR4-NOT by RNA-binding proteins.

A) A gallery of co-structures of CCR4-NOT subunits or subassemblies with interaction partners. CAF40 is a hotspot of interactions and possesses tandem tryptophan (W) binding pockets on its convex side that are used by GW182 proteins and TTP (PDB 4CRV) [21,42], and a peptide-binding pocket on the convex side which recognizes Bag-of-marbles (Bam; PDB 5ONA) [40], metazoan NOT4 (PDB 6HOM) [35], Roquin (PDB 5LSW) [34] and RNF219 [41]. Tristetraprolin (TTP) binds the small helical domain N-terminal of the central MIF4G (PDB 4J8S) [26]. Tob folds into a globular domain associated with CAF1 (PDB 2D5R) [16]. The NOT module is contacted and recruited by Drosophila Nanos (PDB 5FU7) [32], Hs Nanos (PDB 4CQO) [33], and yeast NOT4 [36]. B) A schematic depiction of recruitment of the CCR4-NOT complex by RNA-associated proteins. Recruiting factors interact with target transcripts indirectly (GW182, mediated by miRNA-AGO complex; Tob/BTG, mediated by PABP) or directly (most likely all other displayed proteins) and then contact CCR4-NOT on one or multiple surfaces. RNA-binding domains are typically globular folds, but interactions with CCR4-NOT are mediated mainly by short peptides; a notable exception is Tob which binds CAF1 using a folded domain.

Figure 3.. Mechanisms of CCR4-NOT complex activation.

A) Competition of transcripts for CCR4-NOT recruitment. Some binding sites on the CCR4-NOT surface are not unique but are shared by several RNA-binding proteins. Consequently, those proteins might, in principle, be able to compete for recruitment of CCR4-NOT to their bound transcripts. One example is the peptide-binding site on the concave side of CAF40 (A) which is used by evolutionarily unrelated peptides of proteins Bag-of-marbles (Bam) [40], Roquin [34], and metazoan NOT4 [35]. The tandem W-binding sites on the convex side of CAF40 (B) can accommodate Trp residues from GW182 proteins [21,22] as well as TTP [42]. B) Additive effects might enhance CCR4-NOT recruitment efficiency. Many transcripts contain several or multiple sequence motifs and miRNA-target sites and thus could be bound simultaneously by multiple CCR4-NOT recruiters. These proteins could, in turn, recruit one CCR4-NOT complex by multiple interactions, as shown here by the example of a hypothetical transcript containing one miRNA-binding site and a stem-loop structure that Roquin binds, leading to a tighter interaction and more efficient recruitment than in the situation when only one recruitment factor would be present. Alternatively, several RNA-associated proteins bound on one mRNA could recruit several CCR4-NOT complexes. C) Substrate RNA binding may enhance CCR4-NOT activity. Several RNA-binding interfaces have been identified or proposed on different parts of CCR4-NOT, i.e., (1) the nuclease module [19], (2) CAF40 [48], (3) the NOT module [25], and (4) the NOT10–11 module [29]. Binding of the substrate transcript might enhance the overall affinity of CCR4-NOT, improving deadenylation efficiency and processivity, stabilizing a favorable conformation of the complex, position the 3′-end of the substrate close to the active sites, and terminate the exonuclease activity at the end of the poly(A) tail. D) Model of general and specific mRNA deadenylation. Newly synthesized mRNAs possess long poly(A) tails coated by copies of PABP. In general, bulk mRNA decay, PAN2-PAN3, is recruited to the transcript by directly binding PABP, and the 3’-end is positioned optimally to reach the active site [4]. At this stage, CCR4-NOT can already be recruited by BTG/Tob (not shown) bound to PABP but cannot yet outcompete PAN2-PAN3. Once the penultimate PABP dissociates, PAN2-PAN3 loses affinity for the transcript. At the same time, CCR4-NOT can remove the terminal PABP, associate with the naked substrate, and distributively shorten the remaining tail. This pathway can be circumvented by RNA-binding proteins recruiting CCR4-NOT to specific transcripts to elicit fast and efficient displacement of PABP and subsequent processive deadenylation. In both cases, deadenylation is followed by rapid 5′-decapping and exonucleolytic decay of the mRNA body.