RNA の制御と機能、翻訳制御 Regulation and Function of RNA, Translational Regulation
P1-2-26 TDP-43とFUSに共有されるRNA制御能の網羅的解析 Comprehensive analysis of the roles of TDP-43 and FUS in RNA processing ○李全1, 山本宗隆1, 瀬尾茂人2, 松田秀雄2, 鈴木穣3, 河原行郎1 ○Quan Li1, Munetaka Yamamoto1, Shigeto Seno2, Hideo Matsuda2, Yutaka Suzuki3, Yukio Kawahara1 大阪大院・医・遺伝子機能制御学1, 大阪大院・情報科学研究科・バイオ情報工学専攻2, 東京大院・新領域創成科学研究科・メディカルゲノム専攻3 Lab. of RNA Function, Grad School of Med, Osaka Univ, Osaka1, Dept Bioinformatic Engineering, Grad School of Information Science and Technology, Osaka Univ, Osaka2, Dept Medical Genome Sciences, Grad School of Frontier Sciences, The Univ of Tokyo, Tokyo3 Accumulated lines of evidence have revealed that abnormal RNA processing through RNA binding proteins (RBPs) in specific neuronal cells is involved in the pathogenesis of some neurodegenerative diseases. Therefore, understanding of the physiological functions of disease-linked RBPs is crucial for elucidating disease mechanisms. Here, we report a fundamental analysis of the RNAs bound to RBPs that are involved in a progressive neurodegenerative disease termed amyotrophic lateral sclerosis (ALS). Recently, genetic mutations in disease-linked RBPs, TAR-DNA binding protein (TDP-43) and fused in sarcoma (FUS), have been identified in both sporadic and familial types of ALS patients. Since both TDP43 and FUS are predominantly localized in the nuclear and form a complex, identification of the RNA targets regulated by both RBPs is critical to understand the common disease mechanism in ALS. In this study, we have investigated the RNA targets of TDP-43 and FUS using a deep sequence-based analysis coupled with PAR-CLIP (Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation). PAR-CLIP method relies on the intensive cross-linking of RBPs with RNA using 4-thiouridine (4-SU), thereby resulting in a high frequency of T to C mutations at the direct binding site. Using mutation-based bioinformatic analysis, we found that about 50% of all sequence reads contained at least one T-to-C mutation compared to the human genome reference, warranting well establishment of cDNA library for both TDP-43 and FUS. The majority of the binding sites of both RBPs are situated within introns, suggesting that TDP-43 and FUS are involved in splicing regulation. Comparative analysis of TDP-43 and FUS showed some common RNA targets, predicting the possibility for the presence of complex-derived RNAs. Further analysis of the nature of these RNAs can provide a new avenue for research in understanding the pathogenesis of ALS.	P1-2-27 げっ歯類BdnfアンチセンスRNAの機能解析 Molecular and functional characterization of rodent Bdnf antisense RNAs ○熊ノ郷晴子1,2,3, 水井利幸1,2, 清末和之1,2, 高橋正身2,3, 小島正己1,2 ○Haruko Kumanogoh1,2,3, Toshiyuki Mizui1,2, Kazuyuki Kiyosue1,2, Masami Takahashi2,3, Masami Kojima1,2 産総研・健康工学・バイオインターフェース1, 科学技術振興機構 戦略的創造研究推進事業2, 北里大・医・生化学3 Bio-interface Research Group, Health Research Inst, AIST, Osaka1, CREST, JST, Kawaguchi2, Dept Biochem, Kitasato Univ, Kanagawa3 Although there is increasing report indicating that non-coding natural antisense RNA controls cell functions, the biological role of non-coding RNA in brain is ill-defined. In the present study, we investigated whether the expression of BDNF (brain-derived neurotrophic factor) antisense RNA affected the molecular and cellular function of BDNF, which exerts the control of neuronal survival, development, differentiation and maintenance of neuronal cells, was affected by the expression of its antisense RNA. The human BDNF gene is composed of 11 exons, and the spatial and temporal expression of BDNF mRNA is controlled under 9 promoters. Recently, it was reported that non-coding natural antisense RNAs with complex splicing and expression patterns, were transcribed in the BDNF gene locus from the BDNF-AS (antisense) gene. Recently, we identified eight Bdnf antisense cDNAs with rat neuronal samples, and the rodent antisense RNAs displayed complex splicing pattern similarly to that of human. However, the architectures were quite different from that of human BDNF-AS transcripts. As the expression levels of Bdnf sense RNA are up-regulated by neuronal activity, we first quantitate the expression levels of antisense RNAs in 14-day cultured hippocampal neurons after the treatment with NMDA. We found that one of the antisense RNAs, AS9a, raised its expression level in the NMDA-treated neurons. Moreover, double fluorescent in situ hybridization showed that both of AS9a RNA and Bdnf sense RNA increased the expression levels in the same neurons. These data suggest that the expression of AS9a RNA and Bdnf mRNA is controlled coordinately. We have now assayed the biological roles of the Bdnf-AS RNAs (AS9a and others) using primary cultured neurons and, to understand the physiological and pathological roles, several model animals will be studied.
P1-2-28 ラット培養大脳皮質ニューロンにおけるBDNF mRNA発現へのフェンサイクリジンの影響 The Effect of Phencyclidine on BDNF mRNA Expression in Rat Cultured Cortical Neurons ○片沼佑介1,2, 沼川忠広2,3, 安達直樹2,3, 井上貴文1, 功刀浩2,3 ○Yusuke Katanuma1,2, Tadahiro Numakawa2,3, Naoki Adachi2,3, Takafumi Inoue1, Hiroshi Kunugi2,3 早稲田大学　先進理工学部　生命医科学科1, 国立精神・神経医療研究センター　神経研究所　疾病研究第三部2, 戦略的創造研究推進事業、科学技術振興機構3 Dept Life Sci Medi Biosci, Univ of Waseda, Tokyo, Japan1, Department of Mental Disorder Research, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Tokyo, Japan2, CREST, JST, Saitama, Japan3 Phencyclidine (PCP) causes schizophrenia-like symptoms, so it is widely used for inducing biological model for schizophrenia research. In this study, we focused on brain-derived neurotrophic factor (BDNF) which has been implicated in the onset of schizophrenia, determining the effect of PCP on BDNF mRNA levels in cultured cortical neurons. We found that PCP dramatically reduced the amount of total BDNF mRNA in dose- and time-dependent manners. An experiment of co-application with a transcription inhibitor actinomycin D (ActD) suggested that PCP effect on BDNF mRNA is via inhibiting transcription. Rat BDNF gene consists of eight untranslated 5´-exons that possess identical promoters and one translated 3´-exon IX that encodes BDNF protein. Thus, we performed analysis targeting exon-specific BDNF mRNA transcripts (exon I, IV, and VI). Cortical neurons dominantly express exon IV-containing BDNF mRNA, and PCP remarkably reduced exon IV-containing BDNF transcript while ActD decreased exon I, IV, and VI-containing ones uniformly. It is possible that PCP reduced BDNF mRNA levels mainly by suppressing transcription from BDNF exon IV.	P1-2-29 p21 mRNA における hnRNP K の結合部位の新規ソフトウェアを用いた in silico 解析 In silico analysis of hnRNP K binding sites in p21 mRNA and its in vitro examination with a new search engine ○五十嵐真奈1, 青井久2, 岡野洋尚3, 岡野栄之1 ○Mana Igarashi1, Hisashi Aoi2, Hirotaka J. Okano3, Hideyuki Okano1 慶大・医・生理1, 立命館大・理工・数理2, 慈恵医大・医・再生医学3 Dept Physiol, Keio Univ Sch Med, Tokyo, Japan1, Dept Math, Ritsumeikan Univ, Shiga, Japan2, Div Reg Med, Jikei Univ Sch Med, Tokyo, Japan3 Neuronal RNA binding protein family Hu is homolog of Drosophila ELAV protein, which is essential for differentiation and maintenance of the nervous system. In mammals, Hu proteins are expressed in both early postmitotic and mature neurons and are shown to induce neuronal differentiation by binding to the UTR sequences of specific target mRNAs.To understand the molecular mechanism of the function of Hu, we purified HuB associated complexes and identified the components. One of them, hnRNP K, a single strand RNA binding protein which is directly associated with Hu and also interacts with 3'UTR of p21 mRNA which is also known as a target of Hu. Previously, we have shown that HuB binds AU-rich elements in 3'UTR of p21 mRNA and up-regulates p21 expression. We have also demonstrated that hnRNP K antagonizes the action of Hu by binding CU-rich elements in 3'UTR of p21. Now we further investigated the hnRNP K binding sites in p21 mRNA 3'UTR by using a novel in silico methodology to analyze the sequence and secondary structure of RNAs. To find specified sequences in enormous databases, we construct a newsearch engine on our Web server. We brush up our search engine with BioPython --- a tools for biological computation written in Python. We also improve our interface by JSON(JavaScript Object Notation).This new method might be able to determine whether hnRNP K binds to 3' UTR by recognizing merely the sequence of the target or also the corresponding structure in addition.We will also present results of in vitro examinations of in silico predictions. We discovered interesting sequences in some genes, which are highly related to the development of the nervous system.

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