The RNA-binding protein HuD/ELAVL4 is essential for neuronal development and synaptic plasticity by governing various post-transcriptional processes of target mRNAs, including stability, translation, and localization. We previously showed that the linker region and poly(A)-binding domain of HuD play a pivotal role in promoting translation and inducing neurite outgrowth. In addition, we found that HuD interacts exclusively with the active form of Akt1, through the linker region. Although this interaction is essential for neurite outgrowth, HuD is not a substrate for Akt1, raising questions about the dynamics between HuD-mediated translational stimulation and its association with active Akt1. Here, we demonstrate that active Akt1 interacts with the cap-binding complex via HuD. We identify key amino acids in linker region of HuD responsible for Akt1 interaction, leading to the generation of two point-mutated HuD variants: one that is incapable of binding to Akt1 and another that can interact with Akt1 regardless of its phosphorylation status. In vitro translation assays using these mutants reveal that HuD-mediated translation stimulation is independent of its binding to Akt1. In addition, it is evident that the interaction between HuD and active Akt1 is essential for HuD-induced neurite outgrowth, whereas a HuD mutant capable of binding to any form of Akt1 leads to aberrant neurite development. Collectively, our results revisit the understanding of the HuD-Akt1 interaction in translation and suggest that this interaction contributes to HuD-mediated neurite outgrowth via a unique molecular mechanism distinct from translation regulation.

Translation initiation is the rate-limiting step of protein synthesis and is the main target of translation regulation. RNA-binding proteins (RBPs) are key mediators of the spatiotemporal control of translation and are critical for cell proliferation, development, and differentiation. We have previously shown that HuD, one of the neuronal RBPs, enhances cap-dependent translation through the direct interaction with eukaryotic initiation factor 4A (eIF4A) and poly(A) tail using a HeLa-derived in vitro translation system. We have also found that translation stimulation of HuD is essential for HuD-induced neurite outgrowth in PC12 cells. However, it remains unclear how HuD is involved in the regulation of translation initiation. Here, we report that HuD binds to eukaryotic initiation factor 3 (eIF3) via the eIF3b subunit, which belongs to the functional core of mammalian eIF3. eIF3 plays an essential role in recruiting the 40S ribosomal subunit onto mRNA in translation initiation. We hypothesize that the interaction between HuD and eIF3 stabilizes the translation initiation complex and increases translation efficiency. We also showed that the linker region of HuD is required for the interaction with eIF3b. Moreover, we found that eIF3b-binding region of HuD is conserved in all Hu proteins (HuB, HuC, HuD, and HuR). These data might also help to explain how Hu proteins stimulate translation in a cap- and poly(A)-dependent way.

CCR4-NOT complex-mediated mRNA deadenylation serves critical functions in multiple biological processes, yet how this activity is regulated is not fully understood. Here, we show that osmotic stress induces MAPKAPK-2 (MK2)-mediated phosphorylation of CNOT2. Programmed cell death is greatly enhanced by osmotic stress in CNOT2-depleted cells, indicating that CNOT2 is responsible for stress resistance of cells. Although wild-type (WT) and non-phosphorylatable CNOT2 mutants reverse this sensitivity, a phosphomimetic form of CNOT2, in which serine at the phosphorylation site is replaced with glutamate, does not have this function. We also show that mRNAs have elongated poly(A) tails in CNOT2-depleted cells and that introduction of CNOT2 WT or a non-phosphorylatable mutant, but not phosphomimetic CNOT2, renders their poly(A) tail lengths comparable to those in control HeLa cells. Consistent with this, the CCR4-NOT complex containing phosphomimetic CNOT2 exhibits less deadenylase activity than that containing CNOT2 WT. These data suggest that CCR4-NOT complex deadenylase activity is regulated by post-translational modification, yielding dynamic control of mRNA deadenylation.

Protein synthesis is tightly regulated at each step of translation. In particular, the formation of the basic cap-binding complex, eukaryotic initiation factor 4F (eIF4F) complex, on the 5' cap structure of mRNA is positioned as the rate-limiting step, and various cis-elements on mRNA contribute to fine-tune spatiotemporal protein expression. The cis-element on mRNAs is recognized and bound to the trans-acting factors, which enable the regulation of the translation rate or mRNA stability. In this review, we focus on the molecular mechanism of how the assembly of the eIF4F complex is regulated on the cap structure of mRNAs. We also summarize the fine-tuned regulation of translation initiation by various trans-acting factors through cis-elements on mRNAs.

Protein kinase C (PKC) signaling is a highly conserved signaling module that plays a central role in a myriad of physiological processes, ranging from cell proliferation to cell death, via various signaling pathways, including MAPK signaling. Stress granules (SGs) are non-membranous cytoplasmic foci that aggregate in cells exposed to environmental stresses. Here, we explored the role of SGs in PKC/MAPK signaling activation in fission yeast. High-heat stress (HHS) induced Pmk1 MAPK activation and Pck2 translocation from the cell tips into poly(A)-binding protein (Pabp)-positive SGs. Pck2 dispersal from the cell tips required Pck2 kinase activity, and constitutively active Pck2 exhibited increased translocation to SGs. Importantly, Pmk1 deletion impaired Pck2 recruitment to SGs, indicating that MAPK activation stimulates Pck2 SG translocation. Consistently, HHS-induced SGs delayed Pck2 relocalization at the cell tips, thereby blocking subsequent Pmk1 reactivation after recovery from HHS. HHS partitioned Pck2 into the Pabp-positive SG-containing fraction, which resulted in reduced Pck2 abundance and kinase activity in the soluble fraction. Taken together, these results indicate that MAPK-dependent Pck2 SG recruitment serves as a feedback mechanism to intercept PKC/MAPK activation induced by HHS, which might underlie PKC-related diseases.

[This corrects the article DOI: 10.3389/fgene.2019.00332.].

Eukaryotic gene expression can be spatiotemporally tuned at the post-transcriptional level by cis-regulatory elements in mRNA sequences. An important example is the AU-rich element (ARE), which induces mRNA destabilization in a variety of biological contexts in mammals and can also mediate translational control. Regulation is mediated by trans-acting factors that recognize the ARE, such as Tristetraprolin (TTP) and BRF1/ZFP36L1. Although both proteins can destabilize their target mRNAs through the recruitment of the CCR4-NOT deadenylation complex, TTP also directly regulates translation. Whether ZFP36L1 can directly repress translation remains unknown. Here, we used an in vitro translation system derived from mammalian cell lines to address this key mechanistic issue in ARE regulation by ZFP36L1. Functional assays with mutant proteins reveal that ZFP36L1 can repress translation via AU-Rich elements independent of deadenylation. ZFP36L1-mediated translation repression requires interaction between ZFP36L1 and CNOT1, suggesting that it might use a repression mechanism similar to either TPP or miRISC. However, several lines of evidence suggest that the similarity ends there. Unlike, TTP, it does not efficiently interact with either 4E-HP or GIGYF2, suggesting it does not repress translation by recruiting these proteins to the mRNA cap. Moreover, ZFP36L1 could not repress ECMV-IRES driven translation and was resistant to pharmacological eIF4A inhibitor silvestrol, suggesting fundamental differences with miRISC repression via eIF4A. Collectively, our results reveal that ZFP36L1 represses translation directly and suggest that it does so via a novel mechanism distinct from other translational regulators that interact with the CCR4-NOT deadenylase complex.

Codon bias has been implicated as one of the major factors contributing to mRNA stability in several model organisms. However, the molecular mechanisms of codon bias on mRNA stability remain unclear in humans. Here, we show that human cells possess a mechanism to modulate RNA stability through a unique codon bias. Bioinformatics analysis showed that codons could be clustered into two distinct groups-codons with G or C at the third base position (GC3) and codons with either A or T at the third base position (AT3): the former stabilizing while the latter destabilizing mRNA. Quantification of codon bias showed that increased GC3-content entails proportionately higher GC-content. Through bioinformatics, ribosome profiling, and in vitro analysis, we show that decoupling the effects of codon bias reveals two modes of mRNA regulation, one GC3- and one GC-content dependent. Employing an immunoprecipitation-based strategy, we identify ILF2 and ILF3 as RNA-binding proteins that differentially regulate global mRNA abundances based on codon bias. Our results demonstrate that codon bias is a two-pronged system that governs mRNA abundance.

Although KRAS and TP53 mutations are major drivers of pancreatic ductal adenocarcinoma (PDAC), the incurable nature of this cancer still remains largely elusive. ARF6 and its effector AMAP1 are often overexpressed in different cancers and regulate the intracellular dynamics of integrins and E-cadherin, thus promoting tumor invasion and metastasis when ARF6 is activated. Here we show that the ARF6-AMAP1 pathway is a major target by which KRAS and TP53 cooperatively promote malignancy. KRAS was identified to promote eIF4A-dependent ARF6 mRNA translation, which contains a quadruplex structure at its 5'-untranslated region, by inducing TEAD3 and ETV4 to suppress PDCD4; and also eIF4E-dependent AMAP1 mRNA translation, which contains a 5'-terminal oligopyrimidine-like sequence, via up-regulating mTORC1. TP53 facilitated ARF6 activation by platelet-derived growth factor (PDGF), via its known function to promote the expression of PDGF receptor β (PDGFRβ) and enzymes of the mevalonate pathway (MVP). The ARF6-AMAP1 pathway was moreover essential for PDGF-driven recycling of PD-L1, in which KRAS, TP53, eIF4A/4E-dependent translation, mTOR, and MVP were all integral. We moreover demonstrated that the mouse PDAC model KPC cells, bearing KRAS/TP53 mutations, express ARF6 and AMAP1 at high levels and that the ARF6-based pathway is closely associated with immune evasion of KPC cells. Expression of ARF6 pathway components statistically correlated with poor patient outcomes. Thus, the cooperation among eIF4A/4E-dependent mRNA translation and MVP has emerged as a link by which pancreatic driver mutations may promote tumor cell motility, PD-L1 dynamics, and immune evasion, via empowering the ARF6-based pathway and its activation by external ligands.

RNA-binding proteins (RBPs) are key regulators of posttranscriptional gene expression and control many important biological processes including cell proliferation, development, and differentiation. RBPs bind specific motifs in their target mRNAs and regulate mRNA fate at many steps. The AU-rich element (ARE) is one of the major cis-regulatory elements in the 3' untranslated region (UTR) of labile mRNAs. Many of these encode factors requiring very tight regulation, such as inflammatory cytokines and growth factors. Disruption in the control of these factors' expression can cause autoimmune diseases, developmental disorders, or cancers. Therefore, these mRNAs are strictly regulated by various RBPs, particularly ARE-binding proteins (ARE-BPs). To regulate mRNA metabolism, ARE-BPs bind target mRNAs and affect some factors on mRNAs directly, or recruit effectors, such as mRNA decay machinery and protein kinases to target mRNAs. Importantly, some ARE-BPs have stabilizing roles, whereas others are destabilizing, and ARE-BPs appear to compete with each other when binding to target mRNAs. The function of specific ARE-BPs is modulated by posttranslational modifications (PTMs) including methylation and phosphorylation, thereby providing a means for cellular signaling pathways to regulate stability of specific target mRNAs. In this review, we summarize recent studies which have revealed detailed molecular mechanisms of ARE-BP-mediated regulation of gene expression and also report on the importance of ARE-BP function in specific physiological contexts and how this relates to disease. We also propose an mRNP regulatory network based on competition between stabilizing ARE-BPs and destabilizing ARE-BPs.

© 2018 Sadahiro, Fukao, Kosaka, Funakami, Takizawa, Takeuchi, Duncan and Fujiwara. 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.

Staphylococcal superantigen like 5 (SSL5) is an exotoxin produced by S. aureus and has a strong inhibitory effect on MMP-9 enzymatic activity. However, the mechanism of inhibition remains unclear. We sought to identify the responsible regions of SSL5 for the interaction with MMP-9 by comparing a series of domain swap and deletion mutants of SSL5. Binding analyses revealed that SSL5 had two regions for binding to MMP-9 catalytic domain, β1-3 region (25SKELKNVTGY RYSKGGKHYL IFDKNRKFTR VQIFGK60) in N-terminal half and α4β9 region (138KELDFKLRQY LIQNFDLYKK FPKDSKIKVI MKD170) in C-terminal half. The collagen binding domain and zinc-chelating histidine residues of MMP-9 were not essential for the specific binding to SSL5. The domain swap mutants of SSL5 that conserved β1-3 but not α4β9 region inhibited the gelatinolysis by MMP-9, and the mutant of SSL7 that substituted β1-3 region to that of SSL5 acquired the binding and inhibitory activity. Furthermore, the polypeptide that harbored β1-3 region of SSL5 inhibited gelatinolysis by MMP-9. Taken together, SSL5 inhibits the MMP9 activity through binding to the catalytic domain, and the β1-3 region is responsible for the inhibition of proteolytic activity of MMP-9.

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.

Yeast capping enzymes differ greatly from those of mammalian, both structurally and mechanistically. Yeast-type capping enzyme repressors are therefore candidate antifungal drugs. The 5'-guanine-N7 cap structure of mRNAs are an essential feature of all eukaryotic organisms examined to date and is the first co-transcriptional modification of cellular pre-messenger RNA. Inhibitors of the RNA 5'-triphosphatase in yeast are likely to show fungicidal effects against pathogenic yeast such as Candida. We discovered a new RNA 5'-triphosphatase inhibitor, designated as the kribellosides, by screening metabolites from actinomycetes. Kribellosides belong to the alkyl glyceryl ethers. These novel compounds inhibit the activity of Cet1p (RNA 5'-triphosphatase) from Saccharomyces cerevisiae in vitro with IC(50)s of 5-8 mu M and show antifungal activity with MICs ranging from 3.12 to 100 mu g ml(-1) against S. cerevisiae.

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.

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.

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.

Cellular translation is down-regulated by host antiviral responses. Picornaviridae and Flaviviridae including hepatitis C virus (HCV) evade this process using internal ribosomal entry sequences (IRESs). Although HCV IRES translation is a prerequisite for HCV replication, only few host factors critical for IRES activity are known and the global regulator network remains largely unknown. Since signal transduction is an import regulator of viral infections and the host antiviral response we combined a functional RNAi screen targeting the human signaling network with a HCV IRES-specific reporter mRNA assay. We demonstrate that the HCV host cell cofactors PI4K and MKNK1 are positive regulators of HCV IRES translation representing a novel pathway with a functional relevance for the HCV life cycle and IRES-mediated translation of viral RNA.

Regnase-1 and Roquin are RNA binding proteins essential for degradation of inflammation-related mRNAs and maintenance of immune homeostasis. However, their mechanistic relationship has yet to be clarified. Here, we show that, although Regnase-1 and Roquin regulate an overlapping set of mRNAs via a common stem-loop structure, they function in distinct subcellular locations: ribosome/endoplasmic reticulum and processing-body/stress granules, respectively. Moreover, Regnase-1 specifically cleaves and degrades translationally active mRNAs and requires the helicase activity of UPF1, similar to the decay mechanisms of nonsense mRNAs. In contrast, Roquin controls translationally inactive mRNAs, independent of UPF1. Defects in both Regnase-1 and Roquin lead to large increases in their target mRNAs, although Regnase-1 tends to control the early phase of inflammation when mRNAs are more actively translated. Our findings reveal that differential regulation of mRNAs by Regnase-1 and Roquin depends on their translation status and enables elaborate control of inflammation.

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.

Protein kinase B (PKB) also known as Akt is involved in many signal transduction pathways. As alterations of the PKB pathway are found in a number of human malignancies, PKB is considered an important drug target for cancer therapy. However, production of sufficient amounts of active PKB for biochemical and structural studies is very costly because of the necessity of using a higher organism expression system to obtain phosphorylated PKB. Here, we report efficient production of active PKB alpha using the BmNPV bacmid expression system with silkworm larvae. Following direct injection of bacmid DNA, recombinant PKB alpha protein was highly expressed in the fat bodies of larvae, and could be purified using a GST-tag and then cleaved. A final yield of approximately 1 mg PKB alpha/20 larvae was recorded. Kinase assays showed that the recombinant PKB alpha possessed high phosphorylation activity. We further confirmed phosphorylation on the activation loop by mass spectrometric analysis. Our results indicate that the silkworm expression system is of value for preparation of active-form PKB alpha with phosphorylation on the activation loop. This efficient production of the active protein will facilitate further biochemical and structural studies and stimulate subsequent drug development.

Neurotensin receptor type-I (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 NTSRI expressed on the membrane. Following previous reports, the 5 carboxy-terminal amino acid residues of the guanine nucleotide binding protein a-subunit (G alpha) in yeast Gpalp were substituted with the equivalent human G alpha, sequences (Gpal/G alpha(q) transplant). After optimizing the assay conditions, the G alpha-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. (C) 2013 Elsevier Inc. All rights reserved.

The transcriptional corepressors SMRT/NCoR, components of histone deacetylase complexes, interact with nuclear receptors and many other transcription factors. SMRT is a target for the ubiquitously expressed protein kinase CK2, which is known to phosphorylate a wide variety of substrates. Increasing evidence suggests that CK2 plays a regulatory role in many cellular events, particularly, in transcription. However, little is known about the precise mode of action involved. Here, we report the three-dimensional structure of a SMRT/HDAC1-associated repressor protein (SHARP) in complex with phosphorylated SMRT, as determined by solution NMR. Phosphorylation of the CK2 site on SMRT significantly increased affinity for SHARP. We also confirmed the significance of CK2 phosphorylation by reporter assay and propose a mechanism involving the process of phosphorylation acting as a molecular switch. Finally, we propose that the SPOC domain functions as a phosphorylation binding module.

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.

The staphylococcal superantigen-like protein (SSL) family is composed of 14 exoproteins sharing structural similarity with superantigens but no superantigenic activity. Target proteins of four SSLs have been identified to be involved in host immune responses. However, the counterparts of other SSLs have been functionally uncharacterized. In this study, we have identified porcine plasma prothrombin as SSL10-binding protein by affinity purification using SSL10-conjugated Sepharose. The resin recovered the prodomain of prothrombin (fragment 1 + 2) as well as factor Xa in pull-down analysis. The equilibrium dissociation constant between SSL10 and prothrombin was 1.36 x 10(-7) m in surface plasmon resonance analysis. On the other hand, the resin failed to recover -carboxyglutamic acid (Gla) domain-less coagulation factors and prothrombin from warfarin-treated mice, suggesting that the Gla domain of the coagulation factors is essential for the interaction. SSL10 prolonged plasma clotting induced by the addition of Ca2+ and factor Xa. SSL10 did not affect the protease activity of thrombin but inhibited the generation of thrombin activity in recalcified plasma. S. aureus produces coagulase that non-enzymatically activates prothrombin. SSL10 attenuated clotting induced by coagulase, but the inhibitory effect was weaker than that on physiological clotting, and SSL10 did not inhibit protease activity of staphylothrombin, the complex of prothrombin with coagulase. These results indicate that SSL10 inhibits blood coagulation by interfering with activation of coagulation cascade via binding to the Gla domain of coagulation factor but not by directly inhibiting thrombin activity. This is the first finding that the bacterial protein inhibits blood coagulation via targeting the Gla domain of coagulation factors.

Negative regulator of differentiation 1 (Nrd1) is known as a negative regulator of sexual differentiation in fission yeast. Recently, it has been revealed that Nrd1 also regulates cytokinesis, in which physical separation of the cell is achieved by a contractile ring comprising many proteins including actin and myosin. Cdc4, a myosin II light chain, is known to be required for cytokinesis. Nrd1 binds and stabilizes Cdc4 mRNA, and thereby suppressing the cytokinesis defects of the cdc4 mutants. Interestingly, Pmk1 MAPK phosphorylates Nrd1, resulting in markedly reduced RNA binding activity. Furthermore, Nrd1 localizes to stress granules in response to various stresses, and Pmk1 phosphorylation enhances the localization. Nrd1 consists of four RRM domains, although the mechanism by which Pmk1 regulates the RNA binding activity of Nrd1 is unknown. In an effort to delineate the relationship between Nrd1 structure and function, we prepared each RNA binding domain of Nrd1 and examined RNA binding to chemically synthesized oligo RNA using NMR. The structure of the second RRM domain of Nrd1 was determined and the RNA binding site on the second RRM domain was mapped by NMR. A plausible mechanism pertaining to the regulation of RNA binding activity by phosphorylation is also discussed. (c) 2013 Elsevier Inc. All rights reserved.

Background: Small interfering RNA (siRNA) has attracted attention in the field of nucleic acid medicine as a RNA interference (RNAi) application that leads to gene silencing due to specific messenger RNA (mRNA) destruction. However, since siRNA is unstable in blood and unable to cross the cell membrane, encapsulation of siRNA into a carrier is required. Results: In this study, we used a carrier that combined Z(HER2)-displaying bio-nanocapsule (derived from hepatitis B virus surface antigen) and liposomes in a complex in order to investigate the feasibility of effective and target-cell-specific RNAi applications. As a result, by observing RNAi only in HER2-expressing breast cancer cells, using our proposed methodology, we successfully demonstrated target-cell-specific delivery and effective function expression of siRNA. Conclusions: These findings show that, in the field of nucleic acid medicine, Z(HER2)-BNC/ LP can be a useful carrier for siRNA delivery, and could also become a useful tool for gene silencing and to accomplish protein knock-down.

We have previously shown that DPF2 (requiem/REQ) functions as a linker protein between the SWI/SNF complex and RelB/p52 NF-kappa B heterodimer and plays important roles in NF-kappa B transactivation via its noncanonical pathway. Using sensitive 293FT reporter cell clones that had integrated a SWI/SNF-dependent NF-kappa B reporter gene, we find in this study that the overexpression of DPF1, DPF2, DPF3a, DPF3b, and PHF10 significantly potentiates the transactivating activity of typical NF-kappa B dimers. Knockdown analysis using 293FT reporter cells that endogenously express these five proteins at low levels clearly showed that DPF3a and DPF3b, which are produced from the DPF3 gene by alternative splicing, are the most critical for the RelA/p50 NF-kappa B heterodimer transactivation induced by TNF-alpha stimulation. Our data further show that this transactivation requires the SWI/SNF complex. DPF3a and DPF3b are additionally shown to interact directly with RelA, p50, and several subunits of the SWI/SNF complex in vitro and to be co-immunoprecipitated with RelA/p50 and the SWI/SNF complex from the nuclear fractions of cells treated with TNF-alpha. In ChIP experiments, we further found that endogenous DPF3a/b and the SWI/SNF complex are continuously present on HIV-1 LTR, whereas the kinetics of RelA/p50 recruitment after TNF-alpha treatment correlate well with the viral transcriptional activation levels. Additionally, re-ChIP experiments showed DPF3a/b and the SWI/SNF complex associate with RelA on the endogenous IL-6 promoter after TNF-alpha treatment. In conclusion, our present data indicate that by linking RelA/p50 to the SWI/SNF complex, DPF3a/b induces the transactivation of NF-kappa B target gene promoters in relatively inactive chromatin contexts.

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.

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.

The translation factor eIF5 is an important partner of eIF2, directly modulating its function in several critical steps. First, eIF5 binds eIF2/GTP/Met-tRNA(i)(Met) ternary complex (TC), promoting its recruitment to 40S ribosomal subunits. Secondly, its GTPase activating function promotes eIF2 dissociation for ribosomal subunit joining. Finally, eIF5 GDP dissociation inhibition (GDI) activity can antagonize eIF2 reactivation by competing with the eIF2 guanine exchange factor (GEF), eIF2B. The C-terminal domain (CTD) of eIF5, a W2-type HEAT domain, mediates its interaction with eIF2. Here, we characterize a related human protein containing MA3- and W2-type HEAT domains, previously termed BZW2 and renamed here as eIF5-mimic protein 1 (5MP1). Human 5MP1 interacts with eIF2 and eIF3 and inhibits general and gene-specific translation in mammalian systems. We further test whether 5MP1 is a mimic or competitor of the GEF catalytic subunit eIF2B epsilon or eIF5, using yeast as a model. Our results suggest that 5MP1 interacts with yeast eIF2 and promotes TC formation, but inhibits TC binding to the ribosome. Moreover, 5MP1 is not a GEF but a weak GDI for yeast eIF2. We propose that 5MP1 is a partial mimic and competitor of eIF5, interfering with the key steps by which eIF5 regulates eIF2 function.

RNA-binding proteins (RBPs) play a vital role in the post-transcriptional regulation of gene expression during neuronal differentiation and synaptic plasticity. One such RBP family, the neuronal Hu protein family, serves as an early marker of neuronal differentiation and targets several mRNAs containing adenine/uridine-rich elements. Recently, we reported that one of the neuronal Hu proteins, HuD stimulates cap-dependent translation through interactions with eIF4A and poly (A) tail. Nevertheless, little is known with respect to how neuronal Hu proteins contribute to the local translation of target mRNAs in neuronal differentiation. Here, we found that neuronal Hu proteins, but not the ubiquitously expressed HuR protein, directly interact with the light chain of microtubule-associated proteins MAP1B (LC1). We also show that HuD simultaneously binds both RNA and LC1 in vitro and that it tightly associates with microtubules in cells in an LC1-dependent manner, raising the possibility that HuD recruits target mRNAs to microtubules. These results uncover the neuronal binding partners for neuron-specific Hu proteins and suggest the involvement of Hu proteins in microtubule-mediated regulation of mRNA expression within neuronal processes. (C) 2011 Elsevier Masson SAS. All rights reserved.

Metabolites, the end products of gene expression in living organisms, are tightly correlated with an organism's development and growth. Thus, metabolic profiling is a potentially important tool for understanding the events that have occurred in cells, tissues, and individual organisms. Here, we present a method for predicting the developmental stage of zebrafish embryos using novel metabolomic non-target fingerprints of "single-embryos". With this method, we observed the rate of development at different temperatures. Our results suggest that this method allows us to analyse the condition, or distinguish the genotype, of single-embryos before expression of their ultimate phenotype.

CeR-2 RNA is one of the newly identified Caenorhabditis elegans noncoding RNAs (ncRNAs). The characterization of CeR-2 by RNomic studies has failed to classify it into any known ncRNA family. In this study, we examined the spatiotemporal expression patterns of CeR-2 to gain insight into its function. CeR-2 is expressed in most cells from the early embryo to adult stages. The subcellular localization of this RNA is analogous to that of fibrillarin, a major protein of the nucleolus. It was observed that knockdown of C/D small nucleolar ribonucleoproteins (snoRNPs), but not of H/ACA snoRNPs, resulted in the aberrant nucleolar localization of CeR-2 RNA. A mutant worm with a reduced amount of cellular CeR-2 RNA showed changes in its pre-rRNA processing pattern compared with that of the wild-type strain N2. These results suggest that CeR-2 RNA is a C/D snoRNA involved in the processing of rRNAs.

The RNA-binding protein HuD promotes neuronal differentiation by an unknown mechanism. Here we identify an enhancer function of HuD in translation. Translation stimulation by HuD requires both a 3' poly(A) tail and a 5' m(7)G cap structure. We also show that HuD directly interacts with eIF4A. This interaction and the poly(A)-binding activity of HuD are critical for its translational enhancer function because HuD-eIF4A- and HuD-poly(A)-binding mutants fail to stimulate translation. We show that translation of HCV IRES mRNA, which is eIF4A independent, is not stimulated by HuD. We also find that the eIF4A and poly(A)-binding activities of HuD are not only important for stimulating translation but also are essential for HuD-induced neurite outgrowth in PC12 cells. This example of cap-dependent translational regulation might explain at least in part how HuD triggers the induction of neuronal differentiation.

Background: During zebrafish embryogenesis, microRNA (miRNA) miR-430 contributes to restrict Nanos1 and TDRD7 to primordial germ cells (PGCs) by inducing mRNA deadenylation, mRNA degradation, and translational repression of nanos1 and tdrd7 mRNAs in somatic cells. The nanos1 and tdrd7 3'UTRs include cis-acting elements that allow activity in PGCs even in the presence of miRNA-mediated repression. Methodology/Principal Findings: Using a GFP reporter mRNA that was fused with tdrd7 3'UTR, we show that a germline-specific RNA-binding protein DAZ-like (DAZL) can relieve the miR-430-mediated repression of tdrd7 mRNA by inducing poly(A) tail elongation (polyadenylation) in zebrafish. We also show that DAZL enhances protein synthesis via the 3'UTR of dazl mRNA, another germline mRNA targeted by miR-430. Conclusions/Significance: Our present study indicated that DAZL acts as an "anti-miRNA factor'' during vertebrate germ cell development. Our data also suggested that miRNA-mediated regulation can be modulated on specific target mRNAs through the poly(A) tail control.

Many Studies have demonstrated the functions of individual genes associated with embryogenesis and have determined the genome sequences of several organisms. Despite the availability of enormous amount of genetic information, dynamic changes that occur during embryogenesis have not yet been completely understood. In order to understand the dynamic processes involved in embryogenesis, we employed the metabolomic approach. The results of our study indicated that there is a close correlation between metabolomes and developmental stages. Our method enables the identification of embryonic stages using metabolomes as "fingerprints." In this manner, we could successfully predict embryonic development on the basis of metabolomic fingerprints. This is the first report describing a model for predicting vertebrate development by using metabolomics. (C) 2009 Elsevier Inc. All rights reserved.

The thymus is a vertebrate-specific organ where T lymphocytes are generated. Genetic programs that lead to thymus development are incompletely understood. We previously screened ethylnitrosourea-induced medaka mutants for recessive defects in thymus development. Here we report that one of those mutants is caused by a missense mutation in a gene encoding the previously uncharacterized protein WDR55 carrying the tryptophan-aspartate-repeat motif. We find that WDR55 is a novel nucleolar protein involved in the production of ribosomal RNA (rRNA). Defects in WDR55 cause aberrant accumulation of rRNA intermediates and cell cycle arrest. A mutation in WDR55 in zebrafish also leads to analogous defects in thymus development, whereas WDR55-null mice are lethal before implantation. These results indicate that WDR55 is a nuclear modulator of rRNA synthesis, cell cycle progression, and embryonic organogenesis including teleost thymus development.

The thymus is a vertebrate-specific organ where T lymphocytes are generated. Genetic programs that lead to thymus development are incompletely understood. We previously screened ethylnitrosourea-induced medaka mutants for recessive defects in thymus development. Here we report that one of those mutants is caused by a missense mutation in a gene encoding the previously uncharacterized protein WDR55 carrying the tryptophan- aspartate-repeat motif. We find that WDR55 is a novel nucleolar protein involved in the production of ribosomal RNA ( rRNA). Defects in WDR55 cause aberrant accumulation of rRNA intermediates and cell cycle arrest. A mutation in WDR55 in zebrafish also leads to analogous defects in thymus development, whereas WDR55-null mice are lethal before implantation. These results indicate that WDR55 is a nuclear modulator of rRNA synthesis, cell cycle progression, and embryonic organogenesis including teleost thymus development.

Selenium (Se) is an essential trace element. Se is found as selenocysteine (See) in Se-proteins. See is the 21(st) amino acid, because See has its tRNA, the codon UGA and those components in its translational machinery. See UGA codon shares with major stop codon UGA. We purified See synthesizing enzymes, such as seryl-tRNA synthetase (SerRS), See synthetase (SecS) and selenophosphate synthetase (SePS). I described the procedures to prepare See tRNA, SerRS, SecS, SePS and [Se-75] H2Se in detail. We clarified that SecS composed of two proteins, SecS alpha and SecS beta. See synthesizing and incorporating systems present in Monela, Animalia and Protoctista but not in Plantae and Fungi. We showed that protozoa had See tRNA on which See was synthesized from Ser-tRNA by bovine and protozoa SecS. Some worms, such as Caenorhabditis elegans and Fasiola gigantica, also had See tRNA on which See was synthesized by bovine liver SecS or C. elegans enzymes. We showed recognition sites of mammalian See tRNA by SecS. The identitiy units of See tRNA are 9 bp aminoacyl- and 6 bp D-stems. This recognition is not the base-specific manner but the length-specific manner. From comparison of the phylogeny trees of See synthesizing system and translation system, we concluded that the evolution of See synthesizing system is older than that of the translation system.

Fox-1 is a regulator of tissue-specific splicing, via binding to the element (U) GCAUG in mRNA precursors, in muscles and neuronal cells. Fox-1 can regulate splicing positively or negatively, most likely depending on where it binds relative to the regulated exon. In cases where the (U) GCAUG element lies in an intron upstream of the alternative exon, Fox-1 protein functions as a splicing repressor to induce exon skipping. Here we report the mechanism of exon skipping regulated by Fox-1, using the hF1 gamma gene as a model system. We found that Fox-1 induces exon 9 skipping by repressing splicing of the downstream intron 9 via binding to the GCAUG repressor elements located in the upstream intron 8. In vitro splicing analyses showed that Fox-1 prevents formation of the pre-spliceosomal early (E) complex on intron 9. In addition, we located a region of the Fox-1 protein that is required for inducing exon skipping. Taken together, our data show a novel mechanism of how RNA-binding proteins regulate alternative splicing.

Early in development, primordial germ cells (PGCs) are set aside from somatic cells and acquire a unique gene-expression program [1].The mechanisms underlying germline-specific gene expression are largely unknown. Nanos expression is required during germline development [2-5] and is posttranscriptionally restricted to PGCs [4,6-8]. Here we report that the microRNA miR-430 targets the 3' untranslated region (UTR) of nanos1 during zebrafish embryogenesis. A miR430 target site within the nanos1 3' UTR reduces poly(A) tail length, mRNA stability, and translation. Repression is disrupted in maternal-zygotic dicer mutants (MZdicer), which lack mature miRNAs [9], and is restored by injection of processed miR-430. Although miR-430 represses other genes equally in germline and soma, specific regions in the nanos1 31 UTR compensate for microRNA-mediated repression in PGCs and allow germline-specific expression. We show that the 3' UTR of an additional PGC-specific gene, TDRD7, is also targeted by miR-430. These results indicate that miR-430 targets the 3' UTRs of germline genes and suggest that differential susceptibility to microRNAs contributes to tissue-specific gene expression.

Recycling the post-termination ribosomal complex requires the co-ordinated effort of the ribosome, ribosome recycling factor (RRF) and elongation factor EF-G. Although Aquifex aeolicus RRF (aaRRF) binds Escherichia coli ribosomes as efficiently as E. coli RRF, the resulting complex is non-functional and dominant lethal in E. coli, even in the presence of homologous A. aeolicus EF-G. These findings suggest that the E. coli post-termination ribosomal complex with aaRRF lacks functional co-ordination with EF-G required for ribosome recycling. A chimeric EF-G (E. coli domains I-III, A. aeolicus domains IV-V) or an A. aeolicus EF-G with distinct mutations in the domain I-II interface could activate aaRRF. Furthermore, novel mutations that localize to one surface of the L-shape structure of aaRRF restored activity in E. coli. These aaRRF mutations are spatially distinct from mutations previously described and suggest a novel active centre for coupling EF-G's G domain motor action to ribosome disassembly.

Ribosome recycling factor (RRF) disassembles post-termination ribosomal complexes in concert with elongation factor EF-G freeing the ribosome for a new round of polypeptide synthesis. How RRF interacts with EF-G and disassembles post-termination ribosomes is unknown. RRF is structurally similar to tRNA and is therefore thought to bind to the ribosomal A site and be translocated by EF-G during ribosome disassembly as a mimic of tRNA. However, EF-G variants that remain active in GTP hydrolysis but are defective in tRNA translocation fully activate RRF function in vivo and in vitro. Furthermore, RRF and the GTP form of EF-G do not co-occupy the terminating ribosome in vitro; RRF is ejected by EF-G from the preformed complex. These findings suggest that RRF is not a functional mimic of tRNA and disassembles the post-termination ribosomal complex independently of the translocation activity of EF-G.

RBD-1 is the Caenorhabditis elegans homolog of Mrd1p, which was recently shown to be required for 18S ribosomal RNA (rRNA) processing in yeast. To gain insights into the relationship between ribosome biogenesis and the development of multicellular organisms, we examined the expression and function of RBD-1. Maternal RBD-1 in the fertilized egg disappears immediately after cleavage starts, whereas zygotic RBD-1 first appears in late embryos and is localized in the nucleolus in most cells, although zygotic transcription of pre-rRNA is known to be initiated as early as the one-cell stage. RNA interference of the rbd-1gene severely inhibits the processing of 18S rRNA in association with various developmental abnormalities, indicating its essential role in pre-rRNA processing and development in C. elegans. These results provide evidence for the linkage between ribosome biogenesis and the control of development and imply unexpected uncoupling of transcription and processing of pre-rRNA in early C. elegans embryos.

Elongation factor G (EF-G) is a G protein with motor function that drives two target molecules, a tRNA in the translating ribosome and the ribosome recycling factor (RRF) in the posttermination complex. How G protein motor action is transmitted to RRF is unknown. Thermus thermophilus RRF is nonfunctional in Escherichia coli. It became functional upon introducing a plasmid expressing E. coli EF-G with surface changes in its tRNA-mimic domain or by replacing the E. coli EF-G tRNA-mimic domain by the Thermus domain. Thermus RRF could also be activated by introducing surface substitutions in its anticodon arm-mimic region. These gain-of-function phenotypes depend on the combination of heterologous EF-G and RRF alleles. These mutational studies suggest that EF-G motor action is transmitted to RRF by specific surface contacts between the domains that mimic the anticodon arm.

lncRNAであるXistは、遺伝子量補正において、染色体レベルでのグローバルな転写抑制を行う。この転写抑制の仕組みを理解するため、鍵となる分子間相互作用の構造基盤の確立を目指した。特にXistとSHARPとの複合体の立体構造の解析にフォーカスし、多次元NMR、X線結晶構造解析、生化学的解析、リボプロテオミクスといった多面的な手法を用いて解析を行った。安定同位体標識タンパク質を調製し、RRM23部分の良好なNMRスペクトルを取得し信号の帰属を完了した。またXistのNMR測定では、 RNA-repeat間のダイナミクスにより信号が消失する等の困難さが生じたが、部分同位体標識しにより解決した。

miRNAは、Agoタンパク質と複合体（miRISC）を形成し、標的mRNAのpoly(A)鎖の分解と翻訳抑制という二つの経路を介して、遺伝子発現を抑制する。我々は、miRNAによる翻訳開始抑制時に、翻訳開始過程のどのステップが標的とされているのか、またどのような因子を標的としているのかを、ヒト培養細胞抽出液を用いた独自のin vitro翻訳系およびmRNA-タンパク質複合体（mRNP）pull-down法を用いて解析した。そして、miRNA存在下においては、翻訳開始複合体eIF4Fの構成因子であるeIF4A（eIF4AI、eIF4AII）が、eIF4F複合体から除外されていることを直接的に証明している。しかしながら、miRNA反応マシナリーがどのようにしてeIF4Aを翻訳開始複合体より解離させているのかについては依然不明である。 我々はこれまでに、CCR4-NOT複合体の構成因子がmiRNAに誘導される翻訳開始抑制にどのように寄与するかを、各構成因子のノックアウトMEF（Mouse Embryonic Fibroblast）細胞を用いて解析を進めてきた。その結果、TNRC6タンパク質のmiRNAによる翻訳抑制への寄与をはじめ、これまで報告されてきたCCR4-NOT複合体を中心とする分子機構では説明がつかない結果を得たので報告する。さらに、ヒト培養細胞抽出液によるin vitro翻訳系を用いたmRNA pull-down法により、miRISCが形成された標的mRNA上のみに存在するタンパク質を精製し、質量分析によりmiRISCによる翻訳抑制に必要であると考えられる因子Xを同定した。

真菌のキャッピング酵素Ceg1、Cet1および、Ceg1と結合できないCet1変異体を用いて、核移行シグナルはCet1のみに存在し、Ceg1はCet１に結合しないと核移行できないこと、細胞質でCet1とCeg1が結合し、Cet1-Ceg1複合体が核に移行することを明らかにしている。そして、酵母を用いた遺伝学的研究により、Cet1とCeg1の結合が真菌の生育に必須であることが示された。このことから、Cet1-Ceg1結合阻害が新規抗真菌薬の作用機序となりうる事が示された。そして、これまでにモデルとした出芽酵母のcapping反応を選択的に阻害するアプタマーを取得している。

ARE結合タンパク質であるZFP36LやZFL36L1は、標的mRNA上にCCR4-NOT複合体をリクルートし、脱アデニル化を介したmRNA分解を惹起し、遺伝子発現を負に調節する。本研究では、AREを有するInterleukin-6 (IL-6) の3'-UTRを組み込んだレポーターmRNAとZFL36L1を過剰発現させた細胞抽出液を用いたin vitro実験系を構築し、解析を行なった。その結果、ZFL36L1がmiRISC同様に脱アデニル化非依存的に翻訳を抑制すること、さらにmiRISCと異なり翻訳開始複合体の形成は阻害せず翻訳を抑制することが明らかとなった。

長鎖ノンコーディング（lnc）RNAのひとつであるSRA（steroid hormone receptor RNA activator）は、転写されたRNA分子自身が多くの因子と相互作用し、転写を調節する足場分子として機能する。SHARPタンパク質はSRAと直接相互作用し、HDACをリクルートすることで核内受容体による転写を抑制的に制御する。本研究では、SHARPによる転写調節の分子機構の解明を目指し、SHARP・SRA複合体の構造解析、またCLIP等の生化学的手法により解析を行った。SHARPのホモログRBM15や、カウンターパートとしてlncXISTの解析も行った。

胆道がん症例から採取した胆汁の酸化還元動態、胆汁酸組成、変異原性および胆汁中のエクソソームに含まれるmiRNAを解析した。胆道癌症例の胆汁には酸化型グルタチオンが有意に多く、二次胆汁酸の割合が高いことが判明した。各種病態より採取された胆汁の変異原性試験は陰性であった。胆汁中エクソソームに含まれるmiRNAは既法では安定して採取できなかったため、新規の回収法を開発し、胆汁中エクソソームに含まれる胆管癌特異的miRNA候補を検出することができた。

本研究は、遺伝学的と薬理学を融合した独自のケミカルゲノミクスを展開し、その成果として、細胞増殖とがん化の鍵を握るマップキナーゼ（MAPK）シグナルの調節機構を明らかにし、MAPKシグナルを標的とした創薬を展開した。具体的には、MAPKの活性化に関わる遺伝子群や標的遺伝子の同定に加え、MAPKシグナルを制御できる化合物を発見した。さらに、免疫抑制薬であるFK506, RapamycinやFTY720の感受性を決定する遺伝子群を同定し、これらの遺伝子とMAPK経路のクロストーク機構を明らかにした。これらの成果は、MAPKシグナルの破綻が引き起こすがんを始めとした疾患治療法開発に貢献する。

luciferase mRNAにmiRNAの標的配列を付加したレポーターRNAを用いて、miRNAの存在下および非存在下でのレポーターの発現をHEK293細胞の抽出液を用いたin vitro翻訳系で検証することに成功した。そこで、このin vitro解析系を利用し、RNA結合蛋白質HuDによる翻訳活性化とmiRNAによる翻訳抑制との関係を検証した。その結果、HUDはmiRNAによる翻訳抑制に対し阻害的に働くことを明らかにした。次にどの過程・因子がmiRNAマシナリーの標的となっているかという命題を解決するため、mRNA pull-down法によりmiRNAマシナリーに関わる因子を同定することを試みた。その結果、ある翻訳開始因子が重要な役割を果たすことを明らかに、これらの内容は現在Molecular Cell誌に投稿中である。

スフィンゴシン1-リン酸（S1P）は生体膜構成成分スフィンゴ脂質の代謝産物であり、細胞表面に発現しているスフィンゴシン1-リン酸受容体（S1PR）と結合し、細胞遊走などの生理活性を発揮する。S1PRはGタンパク質共役型受容体(GPCR)であり、これまでに５つのサブタイプが同定され、それぞれ組織特異的に発現している。そして、近年S1PRがガンの悪性化に深く関わることが明らかになってきており、腫瘍マーカーとしての位置づけも重要となってきた。現在、受容体を標的とする腫瘍イメージングは、RI標識したリガンドおよびリガンドアナログを用いて行われている。また、抗腫瘍作用を有するリガンドの開発も盛んに行われている。しかしながら、従来の治療・診断用物質は、標的となる腫瘍細胞表面の受容体のみならず、正常細胞に発現している受容体とも交差反応するために、診断精度・治療上の安全性に問題が指摘されている。そのため、サブタイプ特異的作動薬および診断薬の開発が期待されている。そこで、S1PRの各サブタイプと特異的に結合できる人工リガンドを「RNA」という高分子マテリアルを利用し、創製することを試みている。本年度はヒトS1PRを標的とするRNAアプタマーを、SELEX法で創製するために必要な組換えタンパク質およびS1PR発現細胞の作成に取りかかった。具体的にはS1PRのうち、細胞表面に出ているペプチド部分について各S1PRのサブタイプについて大腸菌を用いた組換えタンパク質として調製した。一方で、S1PRを発現していないCHO-K1 細胞にレンチウィルス発現系を用いてS1PRの各サブタイプを恒常的に発現するstable cell lineを構築し、この細胞を用いたCell-base SELEXを実施を開始した。

cap構造の認識から開始される翻訳開始複合体の形成は翻訳の律速段階であり、タンパク質合成を調節するうえでこの過程を制御することは効率がよく、翻訳開始因子群のリン酸化を担うシグナル伝達因子を中心に実例も数多くある。そしてこれまでにmiRNAによる翻訳抑制の標的は翻訳の律速段階である開始であることが強く示唆されている。したがって翻訳を下方修正する手段としてmiRNAによる翻訳開始の抑制は効率的であるといえる。しかしながら、高等真核生物におけるmiRNAによる翻訳開始過程の抑制の素過程は、精力的に研究されているmiRNA が触媒するmRNAの分解過程とは異なりその詳細が明らかにされていない。そこで、miRNAによる翻訳開始抑制時に、翻訳開始過程のどのステップが標的とされているのか、またどのような因子を標的としているのかを申請者が有する生化学的技法を駆使して、明らかにすることを試みた。その結果、miRNAによる翻訳抑制機構は、翻訳開始因子の1つを標的としていることを明らかにした。

神経細胞の分化や機能発現を、翻訳調節ネットワークという視点から解明することを目的として、神経特異的なRNA結合タンパク質HuDの働きがmicroRNAマシナリーとどのように協調あるいは拮抗して働くかを研究する。領域代表・泊博士および領域内研究者である神戸大学・三嶋博士との共同研究を実施し、翻訳開始機構およびmiRNAによる翻訳抑制との相関関係について研究を行った。具体的には、miRNA存在、非存在下における翻訳開始複合体形成およびPABP枯渇状態でのpolysome形成状態をショ糖密度勾配遠心により解析を行った。その結果、miRNAによる標的mRNAの抑制には脱アデニル化・mRNA分解およびPABPに依存しない「直接的な翻訳抑制」が関与することを明らかにし、PNAS誌上で発表した。また、HuDがどのように局所翻訳に寄与しているかを、微小管との結合を足掛かりに解析した。これまでの神経特異的Huタンパク質がどのように微小管上に結合しているのかは不明であった。そして、HuDは微小管結合タンパク質であるLC1を介して微小管に結合し、標的mRNAの神経突起内における輸送などに関与する可能性が示唆された。さらに、RNA結合タンパク質HuDがシグナル伝達因子Aktの活性型と特異的に結合すること、さらにAkt1との結合がHuDの分化誘導能に必須であることを証明し、NAR誌上で発表した。また、これまで機能未知であったeIF5 mimic proteinの機能解析をカンサス州立大学・浅野桂博士との共同研究で行い、翻訳抑制機能を持つことを明らかにした。さらに、弘前大学・牛田千里准教授との共同研究により、藤原が有する独自の生化学系を用いてこれまでに報告がなかったC.elegansのU8 snoRNAを発見している。

線虫では未同定であったU3 snoRNAを同定した。その5'末端にはキャップ構造(恐らくトリメチルGキャップ:TMG)が存在することを確認した。そして、線虫個体におけるその発現解析を行ってきた。胚発生期における発現様式をin situ hybridization法で解析したところ以下のような知見を得ている。i)初期胚では核小体は形成されていないという報告があるものの、U3 snoRNAの存在を示すシグナルは局所的な強いフォーカスとして観察された。ii)体細胞型の細胞周期に移行するMBTになると核小体が出現し、かつU3 snoRNAは核小体に局在する。線虫の場合、核小体は胚発生の過程で100細胞期あたりから形成されてくる。したがって、核小体に局在する低分子RNAの集合もこの時期から始まることが期待された。しかし、U3 snoRNA、U18 snoRNA (rRNAの修飾をガイドするC/D snoRNAの一つ)のそれぞれと相補的な配列を持つプローブを用いたRNA-FISHの結果は予想に反し、雌性前核と雄性前核融合の前までに、これらが局所的に集合することを示した。また、受精直後から雌性前核と雄性前核の融合に至るまでの時期における、これら核小体RNAの挙動には微妙な違いが観察され、RNAの種類によって集合の過程に違いがあることが示唆された。現在これに関する論文を投稿準備中である。