Synonymous codon usage controls global gene expression in both prokaryotic and eukaryotic species. Nonoptimal codons are known to induce mRNA decay; however, the underlying molecular mechanism remains poorly understood in human cells. Through genome-wide CRISPR screening, we identified the RNA-binding protein DHX29 as a critical regulator of codon-dependent gene expression. Cryogenic electron microscopy and selective ribosome profiling demonstrated that DHX29 directly interacts with the A-site entrance of the translating 80S ribosome, the binding site for the eEF1A•GTP•aminoacyl-tRNA ternary complex, suggesting a role in monitoring aminoacyl-tRNA sampling. Proteomic analysis further revealed that DHX29 recruits the GIGYF2•4EHP complex to mediate global suppression of nonoptimal mRNAs. These findings establish a mechanistic link between synonymous codon usage and the regulation of gene expression.

The neuronal ELAV protein HuD is known to promote neuronal differentiation and stimulate cap-dependent translation, but the underlying mechanism remains incompletely understood. Here, we identify a functional domain within the HuD linker that governs interaction with the nuclear export factor TAP/NXF1 and stimulates translation. Using a series of deletion mutants, we show that TAP/NXF1 binding is functionally separable from cytoplasmic localization, and that loss of this interaction correlates with impaired translation and neurite outgrowth. These findings reveal a critical subdomain within HuD that recruits TAP/NXF1 to promote translation and neuronal differentiation, establishing a mechanistic link between RNA export factors and ELAV-mediated translational control.

Transducer of ErbB2 (TOB) proteins have been shown to promote mRNA decay through interactions with the CCR4-NOT complex and poly(A)-binding protein (PABP). While their role in deadenylation-mediated mRNA degradation is well established, their potential function in translational control remains to be elucidated. Here, we employed an in vitro translation system combined with an RNA tethering strategy to examine the function of TOB1 and TOB2 in translation. Our results demonstrate that TOB1 and TOB2 act as repressors of translation initiation, independent of deadenylation. Notably, this translational repression selectively targets eIF4A-dependent translation, while translation driven by eIF4A-independent IRES elements remains unaffected. While the interaction between TOB proteins and PABP appears to be dispensable, as disruption of this interaction only partially reduces translational repression, the knockdown of CNOT1, the scaffold of the CCR4-NOT complex, substantially relieves this repression, highlighting its indispensable role in the mechanism. Collectively, our findings uncover a previously unrecognized function of TOB proteins as direct repressors of translation initiation, independent of mRNA decay, and highlight a specific reliance on eIF4A activity and CCR4-NOT complex integrity.

The migration of gonadal distal tip cells (DTCs) in Caenorhabditis elegans serves as an excellent model for studying the migration of epithelial tubes during organogenesis. Mutations in the mig-17/ADAMTS gene cause misdirected DTC migration during gonad formation, resulting in deformed gonad arms. An amino acid substitution in RPL-20, the ortholog of mammalian RPL18a/eL20, a component of the 60 S ribosomal large subunit, exhibited a slow-growth phenotype and strongly suppressed the mig-17 gonadal defects. Slow-growing mutations clk-1 and clk-2 also suppressed mig-17. Intestine-specific overexpression of mutant RPL-20 protein resulted in a slow-growth phenotype and suppressed the mig-17 gonadal defects, but these effects were much weaker when wild-type RPL-20 was overexpressed, suggesting that the mutant RPL-20 protein acquired a novel function. Analysis of ribosome profiles revealed reduced biogenesis of the 60 S subunit, leading to a reduction of 80 S ribosomes in the rpl-20 mutant. These results suggest that DTC migration defects in mig-17/ADAMTS mutants can be partly suppressed by growth retardation caused by the rpl-20 mutation. While defective ribosome biogenesis may contribute to the observed growth retardation, further investigation is needed to clarify the molecular basis of this phenomenon.

Stress granules (SGs) are dense aggregates of RNA and proteins that form in response to various cellular stresses. Virus-induced SGs, known as antiviral SGs (avSGs), play a crucial role in regulating retinoic acid-inducible gene I-like receptors (RLRs)-mediated antiviral innate immunity. However, the regulation of avSG formation remains not fully understood. In this study, we demonstrate that TAR-RNA binding protein (TRBP), an RNA silencing regulator, negatively regulates type I interferon (IFN) expression by inhibiting avSG formation in response to RNA virus infection. Overexpression of TRBP inhibits both IFN-β promoter activity and avSG formation following viral infection or the viral RNA mimic, polyinosinic-polycytidylic acid transfection. TRBP knockout cells exhibit enhanced phosphorylation and activation of IFN regulatory factor-3 (IRF-3) and increased IFN-β mRNA expression compared to wild-type cells. Additionally, depletion of G3BP1 and G3BP2, which are essential for SG formation, abolishes the inhibitory effect of TRBP on IRF-3 phosphorylation. Mechanistically, TRBP physically interacts with double-stranded RNA (dsRNA)-dependent protein kinase R (PKR), a key kinase involved in avSG formation, via its dsRNA-binding domains, and inhibits PKR activation. In summary, our findings reveal a novel function for TRBP as a negative regulator of RLR-mediated signaling through PKR-dependent inhibition of avSG formation.

The precursor rRNA (pre-rRNA) processing pathway in Caenorhabditis elegans remains unidentified. We have determined the cleavage site within the internal transcribed spacer 2, which generates pre-rRNA f of 5.8S rRNA and pre-rRNA g of 26S rRNA. Ribosome biogenesis is the most energetically demanding process of the cell. Under starved conditions, cells conserve energy by reconfiguring ribosome biogenesis to sustain proper growth. We investigated the short-term effects of starvation on the ribosomal RNA (rRNA) maturation in C. elegans. Northern blot analysis revealed significant changes in both small and large pre-rRNAs, with a substantial decrease in pre-rRNA c'1/27SA2 of the large subunit and an increase in pre-rRNA d of the small subunit. Additionally, most pre-rRNAs accumulated in the nucleolar center, while the rRNA maturation factor FIB-1 accumulated at the nucleolar periphery. Nutritional replenishment restored the pre-rRNA accumulation pattern and the distribution of pre-rRNAs in the nucleolus to pre-starvation conditions.

BACKGROUND: Current L1-based human papillomavirus (HPV) vaccines provide type-specific protection but offer limited cross-protection against non-vaccine HPV types. Therefore, developing a broad-spectrum HPV vaccine is highly desirable. METHODS: In this study, we optimized mRNA constructs and developed a multivalent L2-based mRNA vaccine encoding L2 aa 2-130, which includes all known neutralizing epitopes from four prevalent HPV types (HPV-6, -11, -16, and -18). We evaluated its immunogenicity in a mouse model and compared the efficacy of a commercially available mRNA delivery reagent with a custom-synthesized lipid nanoparticle (LNP) formulation. RESULTS: We identified that a construct containing E01 (a 5'-untranslated region) and SL2.7 (a poly(A) polymerase recruitment sequence) significantly increased protein expression. The L2-based mRNA vaccine induced robust and long-lasting humoral immune responses, with significant titers of cross-reactive serum IgG antibodies against L2 epitopes. Notably, the vaccine elicited cross-neutralizing antibodies and conferred cross-protective immunity not only against vaccine-targeted HPV types but also against non-vaccine HPV types, following intravaginal challenge in mice. We also found that LNP delivered mRNA more effectively in vivo. CONCLUSIONS: The L2-based mRNA vaccine developed in this study shows significant potential for broad-spectrum protection against multiple HPV types. This approach offers a promising strategy for reducing the global burden of HPV-associated cancers.

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.

The mammalian Ccr4-Not complex, carbon catabolite repression 4 (Ccr4)-negative on TATA-less (Not), is a large, highly conserved, multifunctional assembly of proteins that acts at different cellular levels to regulate gene expression. It is involved in the control of the cell cycle, chromatin modification, activation and inhibition of transcription initiation, control of transcription elongation, RNA export, and nuclear RNA surveillance; the Ccr4-Not complex also plays a central role in the regulation of mRNA decay. Growing evidence suggests that gene transcription has a vital role in shaping the landscape of genome replication and is also a potent source of replication stress and genome instability. Here, we have examined the effects of the inactivation of the Ccr4-Not complex, via the depletion of the scaffold subunit CNOT1, on DNA replication and genome integrity in mammalian cells. In CNOT1-depleted cells, the elevated expression of the general transcription factor TATA-box binding protein (TBP) leads to increased RNA synthesis, which, together with R-loop accumulation, results in replication fork slowing, DNA damage, and senescence. Furthermore, we have shown that the stability of TBP mRNA increases in the absence of CNOT1, which may explain its elevated protein expression in CNOT1-depleted cells. Finally, we have shown the activation of mitogen-activated protein kinase signalling as evidenced by ERK1/2 phosphorylation in the absence of CNOT1, which may be responsible for the observed cell cycle arrest at the border of G1/S.

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.

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.

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 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.

昨年度の研究により、リボソーム結合因子が細胞・組織特異的にIRESの配列情報を識別するために必須であることが示されたため、本年度は「3.結合因子によって機能が特化したリボソームにより翻訳指向性が特徴付けられる新規配列を見いだす。」ことに着手した。足がかりとして簡便に分化誘導可能なマウス筋細胞を用いて、ショ糖密度勾配遠心による分画後にポリソームから抽出したRNAの配列情報を分化前後で比較解析を実施した。その結果、細胞の分化前後でmRNAの発現量に変化がなくポリソーム画分への局在にのみ変化のあった遺伝子が複数同定された。さらに、同定された遺伝子の5’非翻訳領域を挿入したレポーターmRNAを作成し、分化前および分化後の細胞抽出液を用いたin vitro翻訳解析を実施した。そして、分化前後で生じる5’非翻訳領域の配列依存的な翻訳活性の変化をin vitroで再現することに成功した。同様の比較解析を野生型および因子XをKOした神経細胞において行なっている。分担者の三嶋は、昨年度の条件検討結果をもとに、ヒト培養細胞においてRNA-Seqとリボソームプロファイリングによって翻訳効率とRNA制御配列を統合的に解析するための予備実験をおこなった。また、リボソーム衝突に由来するDisomeのプロファイリングの結果を解析に含めるためのパイプラインを構築した。さらに分担者の伊藤は、翻訳開始因子eIF3の変異体をヒト培養細胞を用いて精製する方法を確立し、再構成型試験管内翻訳系に使用可能であることを確認した。

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。