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.

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.

HuR protein attenuates miRNA-mediated repression by promoting miRISC dissociation from the target RNA.

The microRNA (miRNA)-mediated repression of protein synthesis in mammalian cells is a reversible process. Target mRNAs with regulatory AU-rich elements (AREs) in their 3'-untranslated regions (3'-UTR) can be relieved of miRNA repression under cellular stress in a process involving the embryonic lethal and altered vision family ARE-binding protein HuR. The HuR-mediated derepression occurred even when AREs were positioned at a considerable distance from the miRNA sites raising questions about the mechanism of HuR action. Here, we show that the relief of miRNA-mediated repression involving HuR can be recapitulated in different in vitro systems in the absence of stress, indicating that HuR alone is sufficient to relieve the miRNA repression upon binding to RNA ARE. Using in vitro assays with purified miRISC and recombinant HuR and its mutants, we show that HuR, likely by its property to oligomerize along RNA, leads to the dissociation of miRISC from target RNA even when miRISC and HuR binding sites are positioned at a distance. Further, we demonstrate that HuR association with AREs can also inhibit miRNA-mediated deadenylation of mRNA in the Krebs-2 ascites extract, in a manner likewise depending on the potential of HuR to oligomerize.

dsRNA expression in the mouse elicits RNAi in oocytes and low adenosine deamination in somatic cells.

Double-stranded RNA (dsRNA) can enter different pathways in mammalian cells, including sequence-specific RNA interference (RNAi), sequence-independent interferon (IFN) response and editing by adenosine deaminases. To study the routing of dsRNA to these pathways in vivo, we used transgenic mice ubiquitously expressing from a strong promoter, an mRNA with a long hairpin in its 3'-UTR. The expressed dsRNA neither caused any developmental defects nor activated the IFN response, which was inducible only at high expression levels in cultured cells. The dsRNA was poorly processed into siRNAs in somatic cells, whereas, robust RNAi effects were found in oocytes, suggesting that somatic cells lack some factor(s) facilitating siRNA biogenesis. Expressed dsRNA did not cause transcriptional silencing in trans. Analysis of RNA editing revealed that a small fraction of long dsRNA is edited. RNA editing neither prevented the cytoplasmic localization nor processing into siRNAs. Thus, a long dsRNA structure is well tolerated in mammalian cells and is mainly causing a robust RNAi response in oocytes.

miRNA repression involves GW182-mediated recruitment of CCR4-NOT through conserved W-containing motifs.

miRNA-mediated repression in animals is dependent on the GW182 protein family. GW182 proteins are recruited to the miRNA repression complex through direct interaction with Argonaute proteins, and they function downstream to repress target mRNA. Here we demonstrate that in human and Drosophila melanogaster cells, the critical repressive features of both the N-terminal and C-terminal effector domains of GW182 proteins are Gly/Ser/Thr-Trp (G/S/TW) or Trp-Gly/Ser/Thr (WG/S/T) motifs. These motifs, which are dispersed across both domains and act in an additive manner, function by recruiting components of the CCR4-NOT deadenylation complex. A heterologous yeast polypeptide with engineered WG/S/T motifs acquired the ability to repress tethered mRNA and to interact with the CCR4-NOT complex. These results identify previously unknown effector motifs functioning as important mediators of miRNA-induced silencing in both species, and they reveal that recruitment of the CCR4-NOT complex by tryptophan-containing motifs acts downstream of GW182 to repress mRNAs, including inhibiting translation independently of deadenylation.

Properties of the Regulatory RNA-Binding Protein HuR and its Role in Controlling miRNA Repression.

Gene expression in eukaryotes is subject to extensive regulation at posttranscriptional levels. One of the most important sites of control involves mRNA 3' untranslated regions (3'utrs), which are recognized by RNA-binding proteins (RBPs) and microRNAs (miRNAs). These factors greatly influence translational efficiency and stability of target mRNAs and often also determine their cellular localization. HuR, a ubiquitously expressed member of the ElaV family of RBPs, has been implicated in regulation of stability and translation of over one hundred mRNAs in mammalian cells. Recent data indicate that some of the effects of HuR can be explained by its interplay with miRNAs. Binding of HuR may suppress the inhibitory effect of mirNAs interacting with the 3'UTR and redirect the repressed mRNA to polysomes for active translation. However, HuR can also synergize with miRNAs. The finding that HuR is able to disengage miRNAs from the repressed mrNa, or render them inactive, provides evidence that miRNa regulation is much more dynamic then originally anticipated. In this chapter we review properties of HuR and describe examples of the cross-talk between the protein and miRNAs, with emphasis on response of the regulation to cellular stress.

The liver-specific microRNA miR-122: biology and therapeutic potential.

MicroRNAs (miRNAs) are small noncoding RNAs that regulate the expression of a large fraction of genes in animals, plants, and protozoa. miRNA-mediated gene repression occurs posttranscriptionally, generally by base-pairing to the 3'-untranslated regions of target mRNAs, which inhibits protein synthesis and destabilizes the mRNA. In this chapter, we discuss the biological functions of miR-122, a highly abundant, liver-specific miRNA. We will review how studies of miR-122 helped to establish important new paradigms of miRNA-mediated regulation, as well as identifying miR-122 as a factor implicated in important human diseases, including cancer and hepatitis C. We discuss antisense strategies targeting miR-122 as a potential therapeutic approach to treat hepatitis C and possibly other diseases.

Twenty-second annual Pezcoller Symposium: RNA biology and cancer.

The 22nd annual Pezcoller Symposium titled "RNA Biology and Cancer" was held in Trento, Italy, on June 10-12, 2010. The program of the symposium was developed by Drs. Rene Bernards, Witold Filipowicz, and David Livingston; they cochaired the meeting in cooperation with Dr. Enrico Mihich. The topics discussed included the opportunities offered by small RNA as tools for cancer drug development, the role of noncoding RNAs, the biochemistry of small RNAs, the function of micro RNA in cancer, and RNAs as diagnostics and therapeutics in cancer.

KSRP promotes the maturation of a group of miRNA precursors.

microRNAs (miRNAs) are small noncodingRNAs that down-regulate gene expression by reducing stability and/or translation of target mRNAs. In animals, miRNAs arise from sequential processing of hairpin primary transcripts by two RNAse III domain-containing enzymes, namely Drosha and Dicer, to generate a mature form of about 22 nucleotides. In this chapter we discuss our latest findings indicating that KSRP is an integral component of both Drosha and Dicer complexes. KSRP binds to the terminal loop sequence of a subset of miRNA precursors promoting their maturation. Our data indicate that the terminal loop is a pivotal structure where activators of miRNA processing as well as repressors of miRNA processing act in a coordinated way to convert cellular signals into changes in miRNA expression processing. This uncovers a new level of complexity of miRNA mechanisms for gene expression regulation.

Properties of the regulatory RNA-binding protein HuR and its role in controlling miRNA repression.

Gene expression in eukaryotes is subject to extensive regulation at posttranscriptional levels. One of the most important sites of control involves mRNA 3' untranslated regions (3'UTRs), which are recognized by RNA-binding proteins (RBPs) and microRNAs (miRNAs). These factors greatly influence translational efficiency and stability of target mRNAs and often also determine their cellular localization. HuR, a ubiquitously expressed member of the ELAV family of RBPs, has been implicated in regulation of stability and translation of over one hundred mRNAs in mammalian cells. Recent data indicate that some of the effects of HuR can be explained by its interplay with miRNAs. Binding of HuR may suppress the inhibitory effect of miRNAs interacting with the 3'UTR and redirect the repressed mRNA to polysomes for active translation. However, HuR can also synergize with miRNAs. The finding that HuR is able to disengage miRNAs from the repressed mRNA, or render them inactive, provides evidence that miRNA regulation is much more dynamic then originally anticipated. In this chapter we review properties of HuR and describe examples of the cross-talk between the protein and miRNAs, with emphasis on response of the regulation to cellular stress.

Mammalian miRNA RISC recruits CAF1 and PABP to affect PABP-dependent deadenylation.

MicroRNAs (miRNAs) inhibit mRNA expression in general by base pairing to the 3'UTR of target mRNAs and consequently inhibiting translation and/or initiating poly(A) tail deadenylation and mRNA destabilization. Here we examine the mechanism and kinetics of miRNA-mediated deadenylation in mouse Krebs-2 ascites extract. We demonstrate that miRNA-mediated mRNA deadenylation occurs subsequent to initial translational inhibition, indicating a two-step mechanism of miRNA action, which serves to consolidate repression. We show that a let-7 miRNA-loaded RNA-induced silencing complex (miRISC) interacts with the poly(A)-binding protein (PABP) and the CAF1 and CCR4 deadenylases. In addition, we demonstrate that miRNA-mediated deadenylation is dependent upon CAF1 activity and PABP, which serves as a bona fide miRNA coactivator. Importantly, we present evidence that GW182, a core component of the miRISC, directly interacts with PABP via its C-terminal region and that this interaction is required for miRNA-mediated deadenylation.

The RNA-binding protein KSRP promotes the biogenesis of a subset of microRNAs.

Consistent with the role of microRNAs (miRNAs) in down-regulating gene expression by reducing the translation and/or stability of target messenger RNAs, the levels of specific miRNAs are important for correct embryonic development and have been linked to several forms of cancer. However, the regulatory mechanisms by which primary miRNAs (pri-miRNAs) are processed first to precursor miRNAs (pre-miRNAs) and then to mature miRNAs by the multiprotein Drosha and Dicer complexes, respectively, remain largely unknown. The KH-type splicing regulatory protein (KSRP, also known as KHSRP) interacts with single-strand AU-rich-element-containing mRNAs and is a key mediator of mRNA decay. Here we show in mammalian cells that KSRP also serves as a component of both Drosha and Dicer complexes and regulates the biogenesis of a subset of miRNAs. KSRP binds with high affinity to the terminal loop of the target miRNA precursors and promotes their maturation. This mechanism is required for specific changes in target mRNA expression that affect specific biological programs, including proliferation, apoptosis and differentiation. These findings reveal an unexpected mechanism that links KSRP to the machinery regulating maturation of a cohort of miRNAs that, in addition to its role in promoting mRNA decay, independently serves to integrate specific regulatory programs of protein expression.

Decreased levels of microRNA miR-122 in individuals with hepatitis C responding poorly to interferon therapy.

Several microRNAs (miRNAs), including liver-specific miR-122, have been implicated in the control of hepatitis C virus (HCV) RNA replication and its response to interferon (IFN) in human hepatoma cells. Our analysis of liver biopsies from subjects with chronic hepatitis C (CHC) undergoing IFN therapy revealed no correlation of miR-122 expression with viral load and markedly decreased pretreatment miR-122 levels in subjects who had no virological response during later IFN therapy; other investigated miRNAs showed only limited changes. These data have implications for the prospect of targeting miRNAs for CHC therapy.

Interferon signaling and treatment outcome in chronic hepatitis C.

Hepatitis C virus (HCV) infection is a major cause of chronic liver disease worldwide. The current standard therapy for chronic hepatitis C (CHC) consists of a combination of pegylated IFN alpha (pegIFNalpha) and ribavirin. It achieves a sustained viral clearance in only 50-60% of patients. To learn more about molecular mechanisms underlying treatment failure, we investigated IFN-induced signaling in paired liver biopsies collected from CHC patients before and after administration of pegIFNalpha. In patients with a rapid virological response to treatment, pegIFNalpha induced a strong up-regulation of IFN-stimulated genes (ISGs). As shown previously, nonresponders had high expression levels of ISGs before therapy. Analysis of posttreatment biopsies of these patients revealed that pegIFNalpha did not induce expression of ISGs above the pretreatment levels. In accordance with ISG expression data, phosphorylation, DNA binding, and nuclear localization of STAT1 indicated that the IFN signaling pathway in nonresponsive patients is preactivated and refractory to further stimulation. Some features characteristic of nonresponders were more accentuated in patients infected with HCV genotypes 1 and 4 compared with genotypes 2 and 3, providing a possible explanation for the poor response of the former group to therapy. Taken together with previous findings, our data support the concept that activation of the endogenous IFN system in CHC not only is ineffective in clearing the infection but also may impede the response to therapy, most likely by inducing a refractory state of the IFN signaling pathway.

MicroRNAs control de novo DNA methylation through regulation of transcriptional repressors in mouse embryonic stem cells.

Loss of microRNA (miRNA) pathway components negatively affects differentiation of embryonic stem (ES) cells, but the underlying molecular mechanisms remain poorly defined. Here we characterize changes in mouse ES cells lacking Dicer (Dicer1). Transcriptome analysis of Dicer-/- cells indicates that the ES-specific miR-290 cluster has an important regulatory function in undifferentiated ES cells. Consistently, many of the defects in Dicer-deficient cells can be reversed by transfection with miR-290 family miRNAs. We demonstrate that Oct4 (also known as Pou5f1) silencing in differentiating Dicer-/- ES cells is accompanied by accumulation of repressive histone marks but not by DNA methylation, which prevents the stable repression of Oct4. The methylation defect correlates with downregulation of de novo DNA methyltransferases (Dnmts). The downregulation is mediated by Rbl2 and possibly other transcriptional repressors, potential direct targets of miR-290 cluster miRNAs. The defective DNA methylation can be rescued by ectopic expression of de novo Dnmts or by transfection of the miR-290 cluster miRNAs, indicating that de novo DNA methylation in ES cells is controlled by miRNAs.

MicroRNA inhibition of translation initiation in vitro by targeting the cap-binding complex eIF4F.

MicroRNAs (miRNAs) play an important role in gene regulatory networks in animals. Yet, the mechanistic details of their function in translation inhibition or messenger RNA (mRNA) destabilization remain controversial. To directly examine the earliest events in this process, we have developed an in vitro translation system using mouse Krebs-2 ascites cell-free extract that exhibits an authentic miRNA response. We show here that translation initiation, specifically the 5' cap recognition process, is repressed by endogenous let-7 miRNAs within the first 15 minutes of mRNA exposure to the extract when no destabilization of the transcript is observed. Our results indicate that inhibition of translation initiation is the earliest molecular event effected by miRNAs. Other mechanisms, such as mRNA degradation, may subsequently consolidate mRNA silencing.

Argonautes and company: sailing against the wind.

The 3'-untranslated region (UTR) of mRNA is crucial for posttranscriptional regulation. In this issue, Vasudevan and Steitz (2007) report a new function for AGO2 and FXR1--two proteins that have been linked to the regulation of microRNAs and translation repression. AGO2 and FXR1 bind to the AU-rich element in the 3'-UTR of TNFalpha mRNA, unexpectedly activating its translation in a cell growth-dependent manner.

Relief of microRNA-mediated translational repression in human cells subjected to stress.

In metazoans, most microRNAs imperfectly base-pair with the 3' untranslated region (3'UTR) of target mRNAs and prevent protein accumulation by either repressing translation or inducing mRNA degradation. Examples of specific mRNAs undergoing microRNA-mediated repression are numerous, but whether the repression is a reversible process remains largely unknown. Here we show that cationic amino acid transporter 1 (CAT-1) mRNA and reporters bearing its 3'UTR can be relieved from the microRNA miR-122-induced inhibition in human hepatocarcinoma cells subjected to different stress conditions. The derepression of CAT-1 mRNA is accompanied by its release from cytoplasmic processing bodies and its recruitment to polysomes. The derepression requires binding of HuR, an AU-rich-element binding protein, to the 3'UTR of CAT-1 mRNA. We propose that proteins interacting with the 3'UTR will generally act as modifiers altering the potential of miRNAs to repress gene expression.

ADP-ribose-1"-monophosphatase: a conserved coronavirus enzyme that is dispensable for viral replication in tissue culture.

Replication of the approximately 30-kb plus-strand RNA genome of coronaviruses and synthesis of an extensive set of subgenome-length RNAs are mediated by the replicase-transcriptase, a membrane-bound protein complex containing several cellular proteins and up to 16 viral nonstructural proteins (nsps) with multiple enzymatic activities, including protease, polymerase, helicase, methyltransferase, and RNase activities. To get further insight into the replicase gene-encoded functions, we characterized the coronavirus X domain, which is part of nsp3 and has been predicted to be an ADP-ribose-1"-monophosphate (Appr-1"-p) processing enzyme. Bacterially expressed forms of human coronavirus 229E (HCoV-229E) and severe acute respiratory syndrome-coronavirus X domains were shown to dephosphorylate Appr-1"-p, a side product of cellular tRNA splicing, to ADP-ribose in a highly specific manner. The enzyme had no detectable activity on several other nucleoside phosphates. Guided by the crystal structure of AF1521, an X domain homolog from Archaeoglobus fulgidus, potential active-site residues of the HCoV-229E X domain were targeted by site-directed mutagenesis. The data suggest that the HCoV-229E replicase polyprotein residues, Asn 1302, Asn 1305, His 1310, Gly 1312, and Gly 1313, are part of the enzyme's active site. Characterization of an Appr-1"-pase-deficient HCoV-229E mutant revealed no significant effects on viral RNA synthesis and virus titer, and no reversion to the wild-type sequence was observed when the mutant virus was passaged in cell culture. The apparent dispensability of the conserved X domain activity in vitro indicates that coronavirus replicase polyproteins have evolved to include nonessential functions. The biological significance of the novel enzymatic activity in vivo remains to be investigated.

ATP modulates siRNA interactions with an endogenous human Dicer complex.

Short interfering RNA (siRNA) binding by Dicer is important for RNA interference in Drosophila, but human Dicer (hDcr) has been reported to lack siRNA binding activity. We used native gel electrophoresis to characterize the siRNA-binding activity of endogenous hDcr-containing complexes in extracts from human cells. We identified a complex (D) that contains hDcr, as demonstrated by antibody supershift. Complex D appears to contain double-stranded siRNAs, and requires structural features of authentic siRNAs. Glycerol gradient sedimentation indicates that Complex D is ~250 kDa, slightly larger than hDcr alone. In addition, we found that purified recombinant hDcr (rhDcr) alone has siRNA binding activity. Complex D migrates more slowly than the rhDcr/siRNA complex in a native gel, suggesting that it contains at least one additional factor. hDcr directly contacts siRNAs within Complex D, as indicated by crosslinking. The endogenous complex is significantly enhanced by ATP, unlike the siRNA-binding activity of purified rhDcr, suggesting the existence of additional factors that can enforce the ATP dependence of endogenous hDcr/siRNA interactions. Complex D could impinge upon the RISC assembly pathway in humans, similar to an analogous complex in Drosophila.