Fabienne Mauxion

Accumulation of aberrant and unfolded proteins induce cellular stresses and contributes in human to diseases and aging. Several cellular pathways combat the formation of these deleterious molecules. Recently, processes limiting the production of unwanted polypeptides through the co-translational degradation of their messenger RNAs (mRNAs) have been identified but remain poorly understood. In Saccharomyces cerevisiae , Caf130 was proposed to regulate the levels of mRNAs encoding some ribosomal proteins, principally Rpl4, when they do not fold properly. We report the functional and structural characterization this process. Biochemical purifications, interaction assays, and in vivo analyses combined with Alphafold modelling define the organization and function of the Caf130 module of the CCR4-NOT complex and associated factors that mediate this process. Our data reveal the critical role of different forms of the Nascent protein Associated Complex (NAC) to dock the CCR4-NOT complex onto translating ribosomes. The CCR4-NOT complex directly recognizes some nascent unfolded proteins emerging from the ribosome tunnel triggering deadenylation of the mRNAs encoding them. Our data further reveal unsuspected evolutionary and functional similarities between yeast and mammalian proteins involved in the quality control of nascent proteins through mRNA degradation. Altogether, our data indicate the existence of a conserved machinery contributing to protein quality control by triggering the decay of mRNAs encoding deleterious polypeptides in eukaryotes.

The CCR4–NOT complex is a multi-subunit assembly found in all eukaryotic cells. Yet, its composition varies across organisms, with a universally conserved core enriched by lineage-specific subunits. Further, heterogeneity results from the occurrence of paralogous proteins, substoichiometric subunits, transient partners, and protein isoforms. Altogether, multiple CCR4–NOT complexes exist, and some even coexist within a single cell. The CCR4–NOT complex is an essential actor of gene expression through its roles in messenger RNA (mRNA) deadenylation, decay, and translation. Over time, support for the originally proposed role of the CCR4–NOT complex in transcription has been waning. Consistent with a role in post-transcriptional regulation, ribosomes appear to be major partners of the CCR4–NOT complex to coordinate translation and mRNA decay. Further, the CCR4–NOT complex is at the center of a network involving RNA-binding proteins and ubiquitin ligases, as well as factors of currently unknown function. Structural and functional analyses indicate that the CCR4–NOT complex integrates different levels of information present in mRNAs to control their stability and translation, thereby contributing to diverse functions including intricate processes such as human brain or pancreas development. It is thus not surprising that genetic alteration of this essential cellular machine, or impairment of its activity by pathogens, contributes to human diseases.

In eukaryotes, a non-templated poly-adenosine (poly(A)) tail is added co-transcriptionally to almost every messenger RNA (mRNA). The length of this poly(A) tail changes during the lifetime of mRNAs and has been shown in many circumstances to be an important factor controlling transcript fates. Yet, the measure of the length of this homogenous nucleotide sequence is technically challenging, making it difficult to assess its dynamic variation. In this chapter, we describe an RNA-ligation-based RACE-PAT (Rapid Amplification of cDNA End—Poly(A) Tail) assay to monitor the poly(A) tail length of mRNAs. In the first step, an RNA oligonucleotide is ligated to mRNA 3′ ends providing an anchoring site to prime cDNA synthesis, avoiding the bias introduced by oligo(dT)-derived primers. Afterward, reverse transcription is performed with an anchor primer with a unique 5′ extension. The choice of the oligonucleotide 3′ end at this step allows further flexibility to amplify modified tails, for example, by uridylation. Next, short DNA fragments encompassing the poly(A) tails are amplified by Polymerase Chain Reaction (PCR) using as forward primer, a transcript-specific primer hybridizing close to the transcript polyadenylation signal, and as reverse primer, an oligonucleotide corresponding to the 5′ extension of the primer used for cDNA synthesis, ensuring that only cDNAs are amplified. The resulting DNA fragments are then visualized after size fractionation by electrophoresis. This method does not provide exact nucleotide count and composition but has the advantage of allowing the processing of many samples in parallel at a low cost. Key words Poly(A) tail Polyadenylation Deadenylation RT-PCR RNA ligation Uridylation

Antiproliferative BTG/Tob proteins interact directly with the CAF1 deadenylase subunit of the CCR4-NOT complex. This binding requires the presence of two conserved motifs, boxA and boxB, characteristic of the BTG/Tob APRO domain. Consistently, these proteins were shown to stimulate mRNA deadenylation and decay in several instances. Two members of the family, BTG1 and BTG2, were reported further to associate with the protein arginine methyltransferase PRMT1 through a motif, boxC, conserved only in this subset of proteins. We recently demonstrated that BTG1 and BTG2 also contact the first RRM domain of the cytoplasmic poly(A) binding protein PABPC1. To decipher the mode of interaction of BTG1 and BTG2 with partners, we performed nuclear magnetic resonance experiments as well as mutational and biochemical analyses. Our data demonstrate that, in the context of an APRO domain, the boxC motif is necessary and sufficient to allow interaction with PABPC1 but, unexpectedly, that it is not required for BTG2 association with PRMT1. We show further that the presence of a boxC motif in an APRO domain endows it with the ability to stimulate deadenylation in cellulo and in vitro. Overall, our results identify the molecular interface allowing BTG1 and BTG2 to activate deadenylation, a process recently shown to be necessary for maintaining T-cell quiescence. GRAPHICAL ABSTRACT

While BTG2 plays an important role in cellular differentiation and cancer, its precise molecular function remains unclear. BTG2 interacts with CAF1 deadenylase through its APRO domain, a defining feature of BTG/Tob factors. Our previous experiments revealed that expression of BTG2 promoted mRNA poly(A) tail shortening through an undefined mechanism. Here we report that the APRO domain of BTG2 interacts directly with the first RRM domain of the poly(A)-binding protein PABPC1. Moreover, PABPC1 RRM and BTG2 APRO domains are sufficient to stimulate CAF1 deadenylase activity in vitro in the absence of other CCR4–NOT complex subunits. Our results unravel thus the mechanism by which BTG2 stimulates mRNA deadenylation, demonstrating its direct role in poly(A) tail length control. Importantly, we also show that the interaction of BTG2 with the first RRM domain of PABPC1 is required for BTG2 to control cell proliferation.

The CCR4-NOT complex was originally identified and its composition and organization characterized in the yeast Saccharomyces cerevisiae. It was first suggested to participate in transcription regulation, but since then it has become clear that it plays a key role in mRNA decay in all eukaryotes, thereby contributing importantly to gene expression regulation. Hence, the mammalian CCR4-NOT complex was recently shown to participate in miRNA-mediated mRNA repression. A better characterization of the composition and organization of this complex in higher eukaryotes is thus warranted. Purifications of the CCR4-NOT complex, performed by others and us, suggest that the protein of unknown function C2ORF29 is associated with this assembly. We demonstrate here that C2ORF29 is indeed a bona fide subunit of the human CCR4-NOT complex and propose to rename it CNOT11. In addition, we show that CNOT11 interacts with the first amino acids of CNOT1 and with CNOT10 and is required for the association of CNOT10 with the CCR4-NOT complex. Thus, the human CCR4-NOT complex possesses in addition to the CCR4-CAF1 deadenylase module and to the NOT module, a module composed of CNOT10 and CNOT11 that interacts with the N-terminal part of CNOT1. Phylogenetic analyses indicate that the CNOT10/CNOT11 module is conserved in all eukaryotes except fungi.

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