microRNA-induced translational control of antiviral immunity by the cap-binding protein 4EHP.

Type I interferons (IFNs) are critical cytokines in the host defense against invading pathogens. Sustained production of IFNs, however, is detrimental to the host, as it provokes autoimmune diseases. Thus, the expression of IFNs is tightly controlled. We report that the mRNA 5' cap-binding protein 4EHP plays a key role in regulating type I IFN concomitant with controlling virus replication, both in vitro and in vivo. Mechanistically, 4EHP suppresses IFN-ß production by effecting the miR-34a-induced translational silencing of Ifnb1 mRNA. miR-34a is upregulated by both RNA virus infection and IFN-ß induction, prompting a negative feedback regulatory mechanism that represses IFN-ß expression via 4EHP. These findings demonstrate the direct involvement of 4EHP in virus-induced host response, underscoring a critical translational silencing mechanism mediated by 4EHP and miR-34a to impede sustained IFN production. This study highlights an intrinsic regulatory function for miRNA and the translation machinery in maintaining host homeostasis.

Cap-binding protein 4EHP effects translation silencing by microRNAs.

MicroRNAs (miRNAs) play critical roles in a broad variety of biological processes by inhibiting translation initiation and by destabilizing target mRNAs. The CCR4-NOT complex effects miRNA-mediated silencing, at least in part through interactions with 4E-T (eIF4E transporter) protein, but the precise mechanism is unknown. Here we show that the cap-binding eIF4E-homologous protein 4EHP is an integral component of the miRNA-mediated silencing machinery. We demonstrate that the cap-binding activity of 4EHP contributes to the translational silencing by miRNAs through the CCR4-NOT complex. Our results show that 4EHP competes with eIF4E for binding to 4E-T, and this interaction increases the affinity of 4EHP for the cap. We propose a model wherein the 4E-T/4EHP interaction engenders a closed-loop mRNA conformation that blocks translational initiation of miRNA targets.

Alternative splicing of CNOT7 diversifies CCR4-NOT functions.

The CCR4-associated factor CAF1, also called CNOT7, is a catalytic subunit of the CCR4-NOT complex, which has been implicated in all aspects of the mRNA life cycle, from mRNA synthesis in the nucleus to degradation in the cytoplasm. In human cells, alternative splicing of the CNOT7 gene yields a second CNOT7 transcript leading to the formation of a shorter protein, CNOT7 variant 2 (CNOT7v2). Biochemical characterization indicates that CNOT7v2 interacts with CCR4-NOT subunits, although it does not bind to BTG proteins. We report that CNOT7v2 displays a distinct expression profile in human tissues, as well as a nuclear sub-cellular localization compared to CNOT7v1. Despite a conserved DEDD nuclease domain, CNOT7v2 is unable to degrade a poly(A) tail in vitro and preferentially associates with the protein arginine methyltransferase PRMT1 to regulate its activity. Using both in vitro and in cellulo systems, we have also demonstrated that CNOT7v2 regulates the inclusion of CD44 variable exons. Altogether, our findings suggest a preferential involvement of CNOT7v2 in nuclear processes, such as arginine methylation and alternative splicing, rather than mRNA turnover. These observations illustrate how the integration of a splicing variant inside CCR4-NOT can diversify its cell- and tissue-specific functions.

A continuum of mRNP complexes in embryonic microRNA-mediated silencing.

MicroRNAs (miRNAs) impinge on the translation and stability of their target mRNAs, and play key roles in development, homeostasis and disease. The gene regulation mechanisms they instigate are largely mediated through the CCR4­NOT deadenylase complex, but the molecular events that occur on target mRNAs are poorly resolved. We observed a broad convergence of interactions of germ granule and P body mRNP components on AIN-1/GW182 and NTL-1/CNOT1 in Caenorhabditis elegans embryos. We show that the miRISC progressively matures on the target mRNA from a scanning form into an effector mRNP particle by sequentially recruiting the CCR4­NOT complex, decapping and decay, or germ granule proteins. Finally, we implicate intrinsically disordered proteins, key components in mRNP architectures, in the embryonic function of lsy-6 miRNA. Our findings define dynamic steps of effector mRNP assembly in miRNA-mediated silencing, and identify a functional continuum between germ granules and P bodies in the C. elegans embryo.

hCAF1/CNOT7 regulates interferon signalling by targeting STAT1.

Stringent regulation of the interferon (IFN) signalling pathway is essential for maintaining the immune response to pathogens and tumours. The transcription factor STAT1 is a crucial mediator of this response. Here, we show that hCAF1/CNOT7 regulates class I and II IFN pathways at different crucial steps. In resting cells, hCAF1 can control STAT1 trafficking by interacting with the latent form of STAT1 in the cytoplasm. IFN treatment induces STAT1 release, suggesting that hCAF1 may shield cytoplasmic STAT1 from undesirable stimulation. Consistently, hCAF1 silencing enhances STAT1 basal promoter occupancy associated with increased expression of a subset of STAT1-regulated genes. Consequently, hCAF1 knockdown cells exhibit an increased protection against viral infection and reduced viral replication. Furthermore, hCAF1 participates in the extinction of the IFN signal, through its deadenylase activity, by speeding up the degradation of some STAT1-regulated mRNAs. Since abnormal and unbalanced JAK/STAT activation is associated with immune disorders and cancer, hCAF1 could play a major role in innate immunity and oncogenesis, contributing to tumour escape.

The ribosome-associated protein RACK1 represses Kir4.1 translation in astrocytes and influences neuronal activity.

The regulation of translation in astrocytes, the main glial cells in the brain, remains poorly characterized. We developed a high-throughput proteomics screen for polysome-associated proteins in astrocytes and focused on ribosomal protein receptor of activated protein C kinase 1 (RACK1), a critical factor in translational regulation. In astrocyte somata and perisynaptic astrocytic processes (PAPs), RACK1 preferentially binds to a number of mRNAs, including Kcnj10, encoding the inward-rectifying potassium (K+) channel Kir4.1. By developing an astrocyte-specific, conditional RACK1 knockout mouse model, we show that RACK1 represses production of Kir4.1 in hippocampal astrocytes and PAPs. Upregulation of Kir4.1 in the absence of RACK1 increases astrocytic Kir4.1-mediated K+ currents and volume. It also modifies neuronal activity attenuating burst frequency and duration. Reporter-based assays reveal that RACK1 controls Kcnj10 translation through the transcript's 5' untranslated region. Hence, translational regulation by RACK1 in astrocytes represses Kir4.1 expression and influences neuronal activity.

The SARS-CoV-2 protein NSP2 impairs the silencing capacity of the human 4EHP-GIGYF2 complex.

There is an urgent need for a molecular understanding of how SARS-CoV-2 influences the machineries of the host cell. Herein, we focused our attention on the capacity of the SARS-CoV-2 protein NSP2 to bind the human 4EHP-GIGYF2 complex, a key factor involved in microRNA-mediated silencing of gene expression. Using in vitro interaction assays, our data demonstrate that NSP2 physically associates with both 4EHP and a central segment in GIGYF2 in the cytoplasm. We also provide functional evidence showing that NSP2 impairs the function of GIGYF2 in mediating translation repression using reporter-based assays. Collectively, these data reveal the potential impact of NSP2 on the post-transcriptional silencing of gene expression in human cells, pointing out 4EHP-GIGYF2 targeting as a possible strategy of SARS-CoV-2 to take over the silencing machinery and to suppress host defenses.

m6A mRNA Destiny: Chained to the rhYTHm by the YTH-Containing Proteins.

The control of gene expression is a multi-layered process occurring at the level of DNA, RNA, and proteins. With the emergence of highly sensitive techniques, new aspects of RNA regulation have been uncovered leading to the emerging field of epitranscriptomics dealing with RNA modifications. Among those post-transcriptional modifications, N6-methyladenosine (m6A) is the most prevalent in messenger RNAs (mRNAs). This mark can either prevent or stimulate the formation of RNA-protein complexes, thereby influencing mRNA-related mechanisms and cellular processes. This review focuses on proteins containing a YTH domain (for YT521-B Homology), a small building block, that selectively detects the m6A nucleotide embedded within a consensus motif. Thereby, it contributes to the recruitment of various effectors involved in the control of mRNA fates through adjacent regions present in the different YTH-containing proteins.

Translational control of ERK signaling through miRNA/4EHP-directed silencing.

MicroRNAs (miRNAs) exert a broad influence over gene expression by directing effector activities that impinge on translation and stability of mRNAs. We recently discovered that the cap-binding protein 4EHP is a key component of the mammalian miRNA-Induced Silencing Complex (miRISC), which mediates gene silencing. However, little is known about the mRNA repertoire that is controlled by the 4EHP/miRNA mechanism or its biological importance. Here, using ribosome profiling, we identify a subset of mRNAs that are translationally controlled by 4EHP. We show that the Dusp6 mRNA, which encodes an ERK1/2 phosphatase, is translationally repressed by 4EHP and a specific miRNA, miR-145. This promotes ERK1/2 phosphorylation, resulting in augmented cell growth and reduced apoptosis. Our findings thus empirically define the integral role of translational repression in miRNA-induced gene silencing and reveal a critical function for this process in the control of the ERK signaling cascade in mammalian cells.

The E3 ubiquitin ligase and RNA-binding protein ZNF598 orchestrates ribosome quality control of premature polyadenylated mRNAs.

Cryptic polyadenylation within coding sequences (CDS) triggers ribosome-associated quality control (RQC), followed by degradation of the aberrant mRNA and polypeptide, ribosome disassembly and recycling. Although ribosomal subunit dissociation and nascent peptide degradation are well-understood, the molecular sensors of aberrant mRNAs and their mechanism of action remain unknown. We studied the Zinc Finger Protein 598 (ZNF598) using PAR-CLIP and revealed that it cross-links to tRNAs, mRNAs and rRNAs, thereby placing the protein on translating ribosomes. Cross-linked reads originating from AAA-decoding tRNALys(UUU) were 10-fold enriched over its cellular abundance, and poly-lysine encoded by poly(AAA) induced RQC in a ZNF598-dependent manner. Encounter with translated polyA segments by ZNF598 triggered ubiquitination of several ribosomal proteins, requiring the E2 ubiquitin ligase UBE2D3 to initiate RQC. Considering that human CDS are devoid of >4 consecutive AAA codons, sensing of prematurely placed polyA tails by a specialized RNA-binding protein is a novel nucleic-acid-based surveillance mechanism of RQC.

Metformin ameliorates core deficits in a mouse model of fragile X syndrome.

Fragile X syndrome (FXS) is the leading monogenic cause of autism spectrum disorders (ASD). Trinucleotide repeat expansions in FMR1 abolish FMRP expression, leading to hyperactivation of ERK and mTOR signaling upstream of mRNA translation. Here we show that metformin, the most widely used drug for type 2 diabetes, rescues core phenotypes in Fmr1-/y mice and selectively normalizes ERK signaling, eIF4E phosphorylation and the expression of MMP-9. Thus, metformin is a potential FXS therapeutic.

Novel roles of the CCR4-NOT complex.

The CCR4-NOT complex is a multi-subunit protein complex evolutionarily conserved across eukaryotes which regulates several aspects of gene expression. A fascinating model is emerging in which this complex acts as a regulation platform, controlling gene products 'from birth to death' through the coordination of different cellular machineries involved in diverse cellular functions. Recently the CCR4-NOT functions have been extended to the control of the innate immune response through the regulation of interferon signaling. Thus, a more comprehensive picture of how CCR4-NOT allows the rapid adaptation of cells to external stress, from transcription to mRNA and protein decay, is presented and discussed here. Overall, CCR4-NOT permits the efficient and rapid adaptation of cellular gene expression in response to changes in environmental conditions and stimuli.

beta-Nerve growth factor participates in an auto/paracrine pathway of regulation of the meiotic differentiation of rat spermatocytes.

NGF appears to be involved in spermatogenesis. However, mice lacking NGF or TrkA genes do not survive more than a few days whereas p75(NTR) knockout mice are viable and fertile. Therefore, we addressed the effect of betaNGF on spermatogenesis by using the systems of rat germ cell culture we established previously. betaNGF did not modify the number of Sertoli cells, pachytene spermatocytes, secondary spermatocytes nor the half-life of round spermatids, but increased the number of secondary meiotic metaphases and decreased the number of round spermatids formed in vitro. These effects of betaNGF were reversible and maximal at about 4 x 10(-11) M. Conversely, K252a, a Trk-specific kinase inhibitor, enhanced the number of round spermatids above that of control cultures. The presence of betaNGF and its receptors TrkA and p75(NTR) was investigated in testis sections, in Sertoli cell and germ cell fractions, and in germ cell and Sertoli cell co-cultures. betaNGF was detected only in germ cells from pachytene spermatocytes of stages VII up to spermatids of stages IX-X. TrkA and p75(NTR) were detected in Sertoli cells and in these germ cells. Taken together, these results indicate that betaNGF should participate in an auto/paracrine pathway of regulation of the second meiotic division of rat spermatocytes in vivo.