Akira Fukao

MicroRNAs Trigger Dissociation of eIF4AI and eIF4AII from Target mRNAs in Humans

In animals, key functions of microRNA-induced silencing complex (miRISC) are translational repression and deadenylation followed by mRNA decay. While miRISC represses translation initiation, it is poorly understood how miRISC exerts this function. Here we assessed the effect of miRISC on synergistic recruitment of translation initiation factors to target mRNAs by using direct biochemical assays. We show that miRISC promotes eIF4AI and eIF4AII release from target mRNAs prior to dissociation of eIF4E and eIF4G in a deadenylation-independent manner. Strikingly, miRISC-induced release of eIF4AI and eIF4AII from target mRNAs and miRISC-induced inhibition of cap-dependent translation can both be counteracted by the RNA-binding protein HuD via a direct interaction of HuD with eIF4A. Furthermore, the pharmacological eIF4A inhibitor silvestrol, which locks eIF4A on mRNAs, conferred resistance to miRNA-mediated translational repression. In summary, we propose that both eIF4AI and eIF4AII are functionally important targets in miRISC-mediated translation control.

Translational inhibition by deadenylation-independent mechanisms is central to microRNA-mediated silencing in zebrafish

MicroRNA (miRNA) is a class of small noncoding RNA approximately 22 nt in length. Animal miRNA silences complementary mRNAs via translational inhibition, deadenylation, and mRNA degradation. However, the underlying molecular mechanisms remain unclear. A key question is whether these three outputs are independently induced by miRNA through distinct mechanisms or sequentially induced within a single molecular pathway. Here, we successfully dissected these intricate outputs of miRNA-mediated repression using zebrafish embryos as a model system. Our results indicate that translational inhibition and deadenylation are independent outputs mediated by distinct domains of TNRC6A, which is an effector protein in the miRNA pathway. Translational inhibition by TNRC6A is divided into two mechanisms: PAM2 motif-mediated interference of poly(A)-binding protein (PABP), and inhibition of 5′ cap- and poly(A) tail-independent step(s) by a previously undescribed P-GL motif. Consistent with these observations, we show that, in zebrafish embryos, miRNA inhibits translation of the target mRNA in a deadenylation- and PABP-independent manner at early time points. These results indicate that miRNA exerts multiple posttranscriptional outputs via physically and functionally independent mechanisms and that direct translational inhibition is central to miRNA-mediated repression.

Microbial fluorescence sensing for human neurotensin receptor type 1 using Gα-engineered yeast cells

Neurotensin receptor type-1 (NTSR1) is a member of the G-protein-coupled receptor (GPCR) family. The natural ligand of NTSR1 is neurotensin (NT), a neuromodulator of the central nervous system. Because NT is also involved in many oncogenic actions, the signaling mediator NTSR1 is a significant molecular target in medicinal and therapeutic fields. In the current study, we constructed a fluorescence-based microbial yeast biosensor that can monitor the activation of human NTSR1 signaling responding to its agonist. To increase the sensitivity of the biosensor, a yeast strain with the green fluorescent protein (GFP) reporter gene was genetically engineered to enhance binding with human NTSR1 expressed on the membrane. Following previous reports, the 5 carboxy-terminal amino acid residues of the guanine nucleotide binding protein α-subunit (Gα) in yeast Gpa1p were substituted with the equivalent human Gα(q) sequences (Gpa1/Gα(q) transplant). After optimizing the assay conditions, the Gα-engineered yeast demonstrated significantly improved sensing for NTSR1 signaling. Because detection using a GFP fluorescence reporter considerably simplifies the measurement procedure, this microbial fluorescence sensor holds promise for use in the diagnosis of NTSR1-associated diseases and the development of agonists.

Functional and direct interaction between the RNA binding protein HuD and active Akt1

The RNA binding protein HuD plays essential roles in neuronal development and plasticity. We have previously shown that HuD stimulates translation. Key for this enhancer function is the linker region and the poly(A) binding domain of HuD that are also critical for its function in neurite outgrowth. Here, we further explored the underlying molecular interactions and found that HuD but not the ubiquitously expressed HuR interacts directly with active Akt1. We identify that the linker region of HuD is required for this interaction. We also show by using chimeric mutants of HuD and HuR, which contain the reciprocal linker between RNA-binding domain 2 (RBD2) and RBD3, respectively, and by overexpressing a dominant negative mutant of Akt1 that the HuD–Akt1 interaction is functionally important, as it is required for the induction of neurite outgrowth in PC12 cells. These results suggest the model whereby RNA-bound HuD functions as an adapter to recruit Akt1 to trigger neurite outgrowth. These data might also help to explain how HuD enhances translation of mRNAs that encode proteins involved in neuronal development.

The coupled and uncoupled mechanisms by which trans-acting factors regulate mRNA stability and translation.

In mammals, spatiotemporal control of protein synthesis plays a key role in the post-transcriptional regulation of gene expression during cell proliferation, development and differentiation and RNA-binding proteins (RBPs) and microRNAs (miRNAs) are required for this phenomenon. RBPs and miRNAs control the levels of mRNA protein products by regulating mRNA stability and translation. Recent studies have shown that RBPs and miRNAs simultaneously regulate mRNA stability and translation, and that the differential functions of RBPs and miRNAs are dependent on their interaction partners. Here, we summarize the coupled- and uncoupled mechanisms by which trans-acting factors regulate mRNA stability and translation.

The CCR4-NOT deadenylase complex controls Atg7-dependent cell death and heart function

Destabilization of Atg7 mRNA by the CCR4-NOT complex prevents p53-dependent cell death in the heart. Protecting the heart by destabilizing mRNA The removal of polyadenylate tails from mRNAs by the CCR4-NOT complex marks these mRNAs for degradation. Yamaguchi et al. (see also the Focus by Das) found that this activity of this complex was required to prevent cell death in the heart. Mice deficient in a component of this complex suffered from cardiac dysfunction and died of heart failure due to cardiomyocyte death. The CCR4-NOT complex deadenylated Atg7 mRNA, which encodes a protein required for autophagy, a process by which cellular constituents and organelles are digested. The increase in Atg7 in the mutant mice resulted in activation of cell death–associated genes by the transcription factor p53. Drugs that increase autophagy have been explored for the treatment of various diseases, but the authors note that their results raise the possibility of cardiovascular side effects for such drugs. Shortening and removal of the polyadenylate [poly(A)] tail of mRNA, a process called deadenylation, is a key step in mRNA decay that is mediated through the CCR4-NOT (carbon catabolite repression 4–negative on TATA-less) complex. In our investigation of the regulation of mRNA deadenylation in the heart, we found that this complex was required to prevent cell death. Conditional deletion of the CCR4-NOT complex components Cnot1 or Cnot3 resulted in the formation of autophagic vacuoles and cardiomyocyte death, leading to lethal heart failure accompanied by long QT intervals. Cnot3 bound to and shortened the poly(A) tail of the mRNA encoding the key autophagy regulator Atg7. In Cnot3-depleted hearts, Atg7 expression was posttranscriptionally increased. Genetic ablation of Atg7, but not Atg5, increased survival and partially restored cardiac function of Cnot1 or Cnot3 knockout mice. We further showed that in Cnot3-depleted hearts, Atg7 interacted with p53 and modulated p53 activity to induce the expression of genes encoding cell death–promoting factors in cardiomyocytes, indicating that defects in deadenylation in the heart aberrantly activated Atg7 and p53 to promote cell death. Thus, mRNA deadenylation mediated by the CCR4-NOT complex is crucial to prevent Atg7-induced cell death and heart failure, suggesting a role for mRNA deadenylation in targeting autophagy genes to maintain normal cardiac homeostasis.

The molecular mechanism of translational control via the communication between the microRNA pathway and RNA-binding proteins.

MicroRNAs (miRNAs) are evolutionarily conserved small noncoding RNAs found in most plants and animals. The miRNA pathway regulates posttranscriptional gene expression through the deadenylation and translation repression of target mRNAs. Recent studies revealed that the early step of translation initiation is the target of “pure” translation repression by the miRNA pathway. Moreover, particularly in animals, the miRNA pathway is required for neuronal development, differentiation, and plasticity. In addition, some functions of miRNAs are regulated by RNA-binding proteins (RBPs) in neuronal cells. This review summarizes new insights about the molecular mechanisms of pure translation repression by miRNA pathway and the communication between the miRNA pathway and RBPs in neuronal local translation.

Spatial regulation of the KH domain RNA-binding protein Rnc1 mediated by a Crm1-independent nuclear export system in Schizosaccharomyces pombe: Nuclear export system of Rnc1

RNA‐binding proteins (RBPs) play important roles in the posttranscriptional regulation of gene expression, including mRNA stability, transport and translation. Fission yeast rnc1+ encodes a K Homology (KH)‐type RBP, which binds and stabilizes the Pmp1 MAPK phosphatase mRNA thereby suppressing the Cl− hypersensitivity of calcineurin deletion and MAPK signaling mutants. Here, we analyzed the spatial regulation of Rnc1 and discovered a putative nuclear export signal (NES)Rnc1, which dictates the cytoplasmic localization of Rnc1 in a Crm1‐independent manner. Notably, mutations in the NESRnc1 altered nucleocytoplasmic distribution of Rnc1 and abolished its function to suppress calcineurin deletion, although the Rnc1 NES mutant maintains the ability to bind Pmp1 mRNA. Intriguingly, the Rnc1 NES mutant destabilized Pmp1 mRNA, suggesting the functional importance of the Rnc1 cytoplasmic localization. Mutation in Rae1, but not Mex67 deletion or overproduction, induced Rnc1 accumulation in the nucleus, suggesting that Rnc1 is exported from the nucleus to the cytoplasm via the mRNA export pathway involving Rae1. Importantly, mutations in the Rnc1 KH‐domains abolished the mRNA‐binding ability and induced nuclear localization, suggesting that Rnc1 may be exported from the nucleus together with its target mRNAs. Collectively, the functional Rae1‐dependent mRNA export system may influence the cytoplasmic localization and function of Rnc1.

Translation of Hepatitis A Virus IRES Is Upregulated by a Hepatic Cell-Specific Factor

Many viruses strongly prefer to infect certain cell types, a phenomenon known as “tropism.” Understanding tropism’s molecular basis is important for the design of vaccines and antiviral therapy. A common mechanism involves viral protein interactions with cell-specific surface receptors, but intracellular mechanisms involving translation have also been described. In this report, we focus on Hepatitis A Virus (HAV) tissue tropism from the standpoint of the translational machinery. HAV genomic RNA, like other positive stranded RNA viruses, is devoid of a cap structure and its translation is driven by highly structured RNA sequences termed internal ribosome entry site (IRES) in the 5′ untranslated region (UTR). Unlike most viral IRESs, HAV IRES-mediated translation requires eIF4E and the 3′ end of HAV RNA is polyadenylated. However, the molecular mechanism of HAV IRES-mediated translation initiation remains poorly understood. We analyzed HAV-IRES-mediated translation in a cell-free system derived from either non-hepatic cells (HeLa) or hepatoma cells (Huh-7) that enables investigation of the contribution of the cap and the poly(A) tail. This revealed that HAV IRES-mediated translation activity in hepatoma cell extracts is higher as compared to extracts derived from a non-hepatic line. Our data suggest that HAV IRES-mediated translation is upregulated by a hepatic cell-specific activator in a poly(A) tail-independent manner.

The essential role for the RNA triphosphatase Cet1p in nuclear import of the mRNA capping enzyme Cet1p-Ceg1p complex of Saccharomyces cerevisiae.

mRNA capping is the first cotranscriptional modification of mRNA in the nucleus. In Saccharomyces cerevisiae, the first two steps of mRNA capping are catalyzed by the RNA triphosphatase Cet1p and the RNA guanylyltransferase Ceg1p. Cet1p and Ceg1p interact to form a mRNA capping enzyme complex and the guanylyltransferase activity of Ceg1p is stimulated by binding with Cet1p. The Cet1p-Ceg1p complex needs to be transported into the nucleus, where mRNA capping occurs. However, the molecular mechanism of nuclear transport of the Cet1p-Ceg1p complex is not known. Here, we show that Cet1p is responsible and that the Cet1p-Ceg1p interaction is essential for the nuclear localization of the Cet1p-Ceg1p complex. The results indicate that the Cet1p-Ceg1p interaction is important not only for the activation of Ceg1p, but also for nuclear import of the complex.