The human CNOT1-CNOT10-CNOT11 complex forms a structural platform for protein-protein interactions.

The evolutionary conserved CCR4-NOT complex functions in the cytoplasm as the main mRNA deadenylase in both constitutive mRNA turnover and regulated mRNA decay pathways. The versatility of this complex is underpinned by its modular multi-subunit organization, with distinct structural modules actuating different functions. The structure and function of all modules are known, except for that of the N-terminal module. Using different structural approaches, we obtained high-resolution data revealing the architecture of the human N-terminal module composed of CNOT1, CNOT10, and CNOT11. The structure shows how two helical domains of CNOT1 sandwich CNOT10 and CNOT11, leaving the most conserved domain of CNOT11 protruding into solvent as an antenna. We discovered that GGNBP2, a protein identified as a tumor suppressor and spermatogenic factor, is a conserved interacting partner of the CNOT11 antenna domain. Structural and biochemical analyses thus pinpoint the N-terminal CNOT1-CNOT10-CNOT11 module as a conserved protein-protein interaction platform.

A conserved motif in human BTG1 and BTG2 proteins mediates interaction with the poly(A) binding protein PABPC1 to stimulate mRNA deadenylation.

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.

BTG2 bridges PABPC1 RNA-binding domains and CAF1 deadenylase to control cell proliferation.

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.

Intracellular ribonucleases involved in transcript processing and decay: precision tools for RNA.

In order to adapt to changing environmental conditions and regulate intracellular events such as division, cells are constantly producing new RNAs while discarding old or defective transcripts. These functions require the coordination of numerous ribonucleases that precisely cleave and trim newly made transcripts to produce functional molecules, and rapidly destroy unnecessary cellular RNAs. In recent years our knowledge of the nature, functions and structures of these enzymes in bacteria, archaea and eukaryotes has dramatically expanded. We present here a synthetic overview of the recent development in this dynamic area which has seen the identification of many new endoribonucleases and exoribonucleases. Moreover, the increasing pace at which the structures of these enzymes, or of their catalytic domains, have been solved has provided atomic level detail into their mechanisms of action. Based on sequence conservation and structural data, these proteins have been grouped into families, some of which contain only ribonuclease members, others including a variety of nucleolytic enzymes that act upon DNA and/or RNA. At the other extreme some ribonucleases belong to families of proteins involved in a wide variety of enzymatic reactions. Functional characterization of these fascinating enzymes has provided evidence for the extreme diversity of their biological functions that include, for example, removal of poly(A) tails (deadenylation) or poly(U) tails from eukaryotic RNAs, processing of tRNA and mRNA 3' ends, maturation of rRNAs and destruction of unnecessary mRNAs. This article is part of a Special Issue entitled: RNA Decay mechanisms.

C2ORF29/CNOT11 and CNOT10 form a new module of the CCR4-NOT complex.

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.

BTG/TOB factors impact deadenylases.

BTG/TOB factors are a family of antiproliferative proteins whose expression is altered in numerous cancers. They have been implicated in cell differentiation, development and apoptosis. Although proposed to affect transcriptional regulation, these factors interact with CAF1, a subunit of the main eukaryotic deadenylase, and with poly(A)-binding-proteins, strongly suggesting a role in post-transcriptional regulation of gene expression. The recent determination of the structures of BTG2, TOB1 N-terminal domain (TOB1N138) and TOB1N138-CAF1 complexes support a role for BTG/TOB proteins in mRNA deadenylation, a function corroborated by recently published functional characterizations. We highlight molecular mechanisms by which BTG/TOB proteins influence deadenylation and discuss the need for a better understanding of BTG/TOB physiological functions.

The BTG2 protein is a general activator of mRNA deadenylation.

BTG2 is a prototype member of the BTG/Tob family of antiproliferative proteins, originally identified as a primary response gene induced by growth factors and tumour promoters. Its expression has been linked to diverse cellular processes such as cell-cycle progression, differentiation or apoptosis. BTG2 has also been shown to interact with the Pop2/Caf1 deadenylase. Here, we demonstrate that BTG2 is a general activator of mRNA decay, thereby contributing to gene expression control. Detailed characterizations of BTG2 show that it enhances deadenylation of all transcripts tested. Our results demonstrate that Caf1 nuclease activity is required for efficient deadenylation in mammalian cells and that the deadenylase activities of both Caf1 and its Ccr4 partner are required for Btg2-induced poly(A) degradation. General activation of deadenylation may represent a new mode of global regulation of gene expression, which could be important to allow rapid resetting of protein production during development or after specific stresses. This may constitute a common function for BTG/Tob family members.

Conservation of the deadenylase activity of proteins of the Caf1 family in human.

The yeast Pop2 protein, belonging to the eukaryotic Caf1 family, is required for mRNA deadenylation in vivo. It also catalyzes poly(A) degradation in vitro, even though this property has been questioned. Caf1 proteins are related to RNase D, a feature supported by the recently published structure of Pop2. Yeast Pop2 contains, however, a divergent active site while its human homologs harbor consensus catalytic residues. Given these differences, we tested whether its deadenylase activity is conserved in the human homologs Caf1 and Pop2. Our data demonstrate that both human factors degrade poly(A) tails indicating their involvement in mRNA metabolism.

X-ray structure and activity of the yeast Pop2 protein: a nuclease subunit of the mRNA deadenylase complex.

In Saccharomyces cerevisiae, a large complex, known as the Ccr4-Not complex, containing two nucleases, is responsible for mRNA deadenylation. One of these nucleases is called Pop2 and has been identified by similarity with PARN, a human poly(A) nuclease. Here, we present the crystal structure of the nuclease domain of Pop2 at 2.3 A resolution. The domain has the fold of the DnaQ family and represents the first structure of an RNase from the DEDD superfamily. Despite the presence of two non-canonical residues in the active site, the domain displays RNase activity on a broad range of RNA substrates. Site-directed mutagenesis of active-site residues demonstrates the intrinsic ability of the Pop2 RNase D domain to digest RNA. This first structure of a nuclease involved in the 3'-5' deadenylation of mRNA in yeast provides information for the understanding of the mechanism by which the Ccr4-Not complex achieves its functions.