Structural and biochemical insights to the role of the CCR4-NOT complex and DDX6 ATPase in microRNA repression.

MicroRNAs (miRNAs) control gene expression by regulating mRNA translation and stability. The CCR4-NOT complex is a key effector of miRNA function acting downstream of GW182/TNRC6 proteins. We show that miRNA-mediated repression requires the central region of CNOT1, the scaffold protein of CCR4-NOT. A CNOT1 domain interacts with CNOT9, which in turn interacts with the silencing domain of TNRC6 in a tryptophan motif-dependent manner. These interactions are direct, as shown by the structure of a CNOT9-CNOT1 complex with bound tryptophan. Another domain of CNOT1 with an MIF4G fold recruits the DEAD-box ATPase DDX6, a known translational inhibitor. Structural and biochemical approaches revealed that CNOT1 modulates the conformation of DDX6 and stimulates ATPase activity. Structure-based mutations showed that the CNOT1 MIF4G-DDX6 interaction is important for miRNA-mediated repression. These findings provide insights into the repressive steps downstream of the GW182/TNRC6 proteins and the role of the CCR4-NOT complex in posttranscriptional regulation in general.

Structural analysis of the yeast Dhh1-Pat1 complex reveals how Dhh1 engages Pat1, Edc3 and RNA in mutually exclusive interactions.

Translational repression and deadenylation of eukaryotic mRNAs result either in the sequestration of the transcripts in a nontranslatable pool or in their degradation. Removal of the 5' cap structure is a crucial step that commits deadenylated mRNAs to 5'-to-3' degradation. Pat1, Edc3 and the DEAD-box protein Dhh1 are evolutionary conserved factors known to participate in both translational repression and decapping, but their interplay is currently unclear. We report the 2.8 Å resolution structure of yeast Dhh1 bound to the N-terminal domain of Pat1. The structure shows how Pat1 wraps around the C-terminal RecA domain of Dhh1, docking onto the Phe-Asp-Phe (FDF) binding site. The FDF-binding site of Dhh1 also recognizes Edc3, revealing why the binding of Pat1 and Edc3 on Dhh1 are mutually exclusive events. Using co-immunoprecipitation assays and structure-based mutants, we demonstrate that the mode of Dhh1-Pat1 recognition is conserved in humans. Pat1 and Edc3 also interfere and compete with the RNA-binding properties of Dhh1. Mapping the RNA-binding sites on Dhh1 with a crosslinking-mass spectrometry approach shows a large RNA-binding surface around the C-terminal RecA domain, including the FDF-binding pocket. The results suggest a model for how Dhh1-containing messenger ribonucleoprotein particles might be remodeled upon Pat1 and Edc3 binding.

Genome-wide assessment of AU-rich elements by the AREScore algorithm.

In mammalian cells, AU-rich elements (AREs) are well known regulatory sequences located in the 3' untranslated region (UTR) of many short-lived mRNAs. AREs cause mRNAs to be degraded rapidly and thereby suppress gene expression at the posttranscriptional level. Based on the number of AUUUA pentamers, their proximity, and surrounding AU-rich regions, we generated an algorithm termed AREScore that identifies AREs and provides a numerical assessment of their strength. By analyzing the AREScore distribution in the transcriptomes of 14 metazoan species, we provide evidence that AREs were selected for in several vertebrates and Drosophila melanogaster. We then measured mRNA expression levels genome-wide to address the importance of AREs in SL2 cells derived from D. melanogaster hemocytes. Tis11, a zinc finger RNA-binding protein homologous to mammalian tristetraprolin, was found to target ARE-containing reporter mRNAs for rapid degradation in SL2 cells. Drosophila mRNAs whose expression is elevated upon knock down of Tis11 were found to have higher AREScores. Moreover high AREScores correlate with reduced mRNA expression levels on a genome-wide scale. The precise measurement of degradation rates for 26 Drosophila mRNAs revealed that the AREScore is a very good predictor of short-lived mRNAs. Taken together, this study introduces AREScore as a simple tool to identify ARE-containing mRNAs and provides compelling evidence that AREs are widespread regulatory elements in Drosophila.

Posttranscriptional destabilization of the liver-specific long noncoding RNA HULC by the IGF2 mRNA-binding protein 1 (IGF2BP1).

UNLABELLED: Selected long noncoding RNAs (lncRNAs) have been shown to play important roles in carcinogenesis. Although the cellular functions of these transcripts can be diverse, many lncRNAs regulate gene expression. In contrast, factors that control the expression of lncRNAs remain largely unknown. Here we investigated the impact of RNA binding proteins on the expression of the liver cancer-associated lncRNA HULC (highly up-regulated in liver cancer). First, we validated the strong up-regulation of HULC in human hepatocellular carcinoma. To elucidate posttranscriptional regulatory mechanisms governing HULC expression, we applied an RNA affinity purification approach to identify specific protein interaction partners and potential regulators. This method identified the family of IGF2BPs (IGF2 mRNA-binding proteins) as specific binding partners of HULC. Depletion of IGF2BP1, also known as IMP1, but not of IGF2BP2 or IGF2BP3, led to an increased HULC half-life and higher steady-state expression levels, indicating a posttranscriptional regulatory mechanism. Importantly, HULC represents the first IGF2BP substrate that is destabilized. To elucidate the mechanism by which IGF2BP1 destabilizes HULC, the CNOT1 protein was identified as a novel interaction partner of IGF2BP1. CNOT1 is the scaffold of the human CCR4-NOT deadenylase complex, a major component of the cytoplasmic RNA decay machinery. Indeed, depletion of CNOT1 increased HULC half-life and expression. Thus, IGF2BP1 acts as an adaptor protein that recruits the CCR4-NOT complex and thereby initiates the degradation of the lncRNA HULC. CONCLUSION: Our findings provide important insights into the regulation of lncRNA expression and identify a novel function for IGF2BP1 in RNA metabolism.

Role of Rck-Pat1b binding in assembly of processing-bodies.

The DEAD box RNA helicase Rck and the scaffold protein Pat1b participate in controlling gene expression at the post-transcriptional level by suppressing mRNA translation and promoting mRNA decapping. In addition, both proteins are required for the assembly of processing (P)-bodies, cytoplasmic foci that contain stalled mRNAs and numerous components of the mRNA decay machinery. The C-terminal RecA-like domain of Rck interacts with the N-terminal acidic domain of Pat1b. Here, we identified point mutations in human Rck and Pat1b that prevent the two proteins from binding to each other. By analyzing interaction-deficient mutants in combination with knockdown and rescue strategies in human HeLa cells, we found that Pat1b assembles P-bodies and suppresses expression of tethered mRNAs in the absence of Rck binding. In contrast, Rck requires the Pat1b-binding site in order to promote P-body assembly and associate with the decapping enzyme Dcp2 as well as Ago2 and TNRC6A, two core components of the RNA-induced silencing complex. Our data indicate that P-body assembly occurs in a step-wise manner, where Rck participates in the initial suppression of mRNA translation, whereas Pat1b in a second step triggers P-body assembly and promotes mRNA decapping.

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.

On track with P-bodies.

P-bodies (processing bodies) are cytoplasmic foci visible by light microscopy in somatic cells of vertebrate and invertebrate origin as well as in yeast, plants and trypanosomes. At the molecular level, P-bodies are dynamic aggregates of specific mRNAs and proteins that serve a dual function: first, they harbour mRNAs that are translationally silenced, and such mRNA can exit again from P-bodies to re-engage in translation. Secondly, P-bodies recruit mRNAs that are targeted for deadenylation and degradation by the decapping/Xrn1 pathway. Whereas certain proteins are core constituents of P-bodies, others involved in recognizing short-lived mRNAs can only be trapped in P-bodies when mRNA decay is attenuated. This reflects the very transient interactions by which many proteins associate with P-bodies. In the present review, we summarize recent findings on the function, assembly and motility of P-bodies. An updated list of proteins and RNAs that localize to P-bodies will help in keeping track of this fast-growing field.

Dual RNA Processing Roles of Pat1b via Cytoplasmic Lsm1-7 and Nuclear Lsm2-8 Complexes.

Pat1 RNA-binding proteins, enriched in processing bodies (P bodies), are key players in cytoplasmic 5' to 3' mRNA decay, activating decapping of mRNA in complex with the Lsm1-7 heptamer. Using co-immunoprecipitation and immunofluorescence approaches coupled with RNAi, we provide evidence for a nuclear complex of Pat1b with the Lsm2-8 heptamer, which binds to the spliceosomal U6 small nuclear RNA (snRNA). Furthermore, we establish the set of interactions connecting Pat1b/Lsm2-8/U6 snRNA/SART3 and additional U4/U6.U5 tri-small nuclear ribonucleoprotein particle (tri-snRNP) components in Cajal bodies, the site of snRNP biogenesis. RNA sequencing following Pat1b depletion revealed the preferential upregulation of mRNAs normally found in P bodies and enriched in 3' UTR AU-rich elements. Changes in >180 alternative splicing events were also observed, characterized by skipping of regulated exons with weak donor sites. Our data demonstrate the dual role of a decapping enhancer in pre-mRNA processing as well as in mRNA decay via distinct nuclear and cytoplasmic Lsm complexes.

Structure of a Human 4E-T/DDX6/CNOT1 Complex Reveals the Different Interplay of DDX6-Binding Proteins with the CCR4-NOT Complex.

The DEAD-box protein DDX6 is a central component of translational repression mechanisms in maternal mRNA storage in oocytes and microRNA-mediated silencing in somatic cells. DDX6 interacts with the CCR4-NOT complex and functions in concert with several post-transcriptional regulators, including Edc3, Pat1, and 4E-T. We show that the conserved CUP-homology domain (CHD) of human 4E-T interacts directly with DDX6 in both the presence and absence of the central MIF4G domain of CNOT1. The 2.1-Å resolution structure of the corresponding ternary complex reveals how 4E-T CHD wraps around the RecA2 domain of DDX6 and contacts CNOT1. Although 4E-T CHD lacks recognizable sequence similarity with Edc3 or Pat1, it shares the same DDX6-binding surface. In contrast to 4E-T, however, the Edc3 and Pat1 FDF motifs dissociate from DDX6 upon CNOT1 MIF4G binding in vitro. The results underscore the presence of a complex network of simultaneous and/or mutually exclusive interactions in DDX6-mediated repression.

The conformational plasticity of eukaryotic RNA-dependent ATPases.

RNA helicases are present in all domains of life and participate in almost all aspects of RNA metabolism, from transcription and processing to translation and decay. The diversity of pathways and substrates that they act on is reflected in the diversity of their individual functions, structures, and mechanisms. However, RNA helicases also share hallmark properties. At the functional level, they promote rearrangements of RNAs and RNP particles by coupling nucleic acid binding and release with ATP hydrolysis. At the molecular level, they contain two domains homologous to the bacterial RecA recombination protein. This conserved catalytic core is flanked by additional domains, which typically regulate the ATPase activity in cis. Binding to effector proteins targets or regulates the ATPase activity in trans. Structural and biochemical studies have converged on the plasticity of RNA helicases as a fundamental property that is used to control their timely activation in the cell. In this review, we focus on the conformational regulation of conserved eukaryotic RNA helicases.

Human Pat1b connects deadenylation with mRNA decapping and controls the assembly of processing bodies.

In eukaryotic cells, degradation of many mRNAs is initiated by removal of the poly(A) tail followed by decapping and 5'-3' exonucleolytic decay. Although the order of these events is well established, we are still lacking a mechanistic understanding of how deadenylation and decapping are linked. In this report we identify human Pat1b as a protein that is tightly associated with the Ccr4-Caf1-Not deadenylation complex as well as with the Dcp1-Dcp2 decapping complex. In addition, the RNA helicase Rck and Lsm1 proteins interact with human Pat1b. These interactions are mediated via at least three independent domains within Pat1b, suggesting that Pat1b serves as a scaffold protein. By tethering Pat1b to a reporter mRNA, we further provide evidence that Pat1b is also functionally linked to both deadenylation and decapping. Finally, we report that Pat1b strongly induces the formation of processing (P) bodies, cytoplasmic foci that contain most enzymes of the RNA decay machinery. An amino-terminal region within Pat1b serves as an aggregation-prone domain that nucleates P bodies, whereas an acidic domain controls the size of P bodies. Taken together, these findings provide evidence that human Pat1b is a central component of the RNA decay machinery by physically connecting deadenylation with decapping.