転写とRNAの制御
Transcription and RNA regulation
O2-6-4-1
神経特異的RNA結合蛋白質Elavl2の成体脳におけるRNA制御機構の解析
Roles of neuronal RNA binding protein Elavl2 in the brain

○大塚貴文1, 矢野真人1, 吉川昇之1, 岡野栄之1
○Takafumi Ohtsuka1, Masato Yano1, Nobuyuki Yoshikawa1, Hideyuki Okano1
慶應義塾大学 生理学 岡野研究室1
Dept Physio, Keio Univ, Tokyo1

Elav-like (Elavl) family proteins (also called Hu proteins) were identified as autoimmune targets of autoantibodies appearing in the sera of patients associated with paraneoplastic encephalomyelitis. Four family members of Elavl genes have been cloned, identified as neuronal RNA binding proteins(RNA BPs), have highly conservation with Drosophila Elav, and all of them have three conserved RNA recognition motifs (RRM). In vitro analysis suggested that RRM1 and RRM2 of Elavl proteins recognized the urigine-rich region, and RRM3 was supposed to bind A-tail of target transcripts. Their expression patterns are mainly in post-mitotic neurons for the exception of Elavl1 which is expressed ubiquitously. Previous studies reported that neuronal Elavl protiens (Elavl2,3,4, nElavls) have been shown to induce neuronal differentiation in vitro culture model, and involved in making spatial memory or RNA regulation in drug induced seizure brain. And recent advance technology HITS-CLIP, High-throughput sequencing of RNA isolated by crosslinking immunoprecipitation methods, succeeded to identify genome-wide nElavls-RNA interaction map in the mouse brain. Here we focused on Elavl2 protein among neuronal nElavl proteins to understand the comprehensive roles of specific member of nElavl proteins using Elavl2 specific antibody and Elavl2 deficient mouse. Our histological analysis revealed that Elavl2 protein showed unique expression patterns in the adult mouse brain, especially in hippocampus compared with other nElavls. And we found that Elavl2 proteins but not other nElavl are specifically regulated in hippocampus by neuronal activity. In addition, we are now performing transcriptome-wide HITS-CLIP analysis, combined with genetics and bioinformatics to understand comprehensive RNA regulation by Elavl2 in the brain. Lastly, we will discuss the biological function of Elavl2 and how Elavl2-RNAs link to brain complexity.
O2-6-4-2
臨界期を制御するホメオタンパク質Otx2の下流因子の網羅的探索
Comprehensive identification of the downstream targets of Otx2 homeoprotein in cortical plasticity

○酒井晶子1, 中戸隆一郎2, 原範和3, 桑野良三3, 白髭克彦2, 杉山清佳1
○Akiko Sakai1, Nakato Ryuichiro2, Norikazu Hara3, Kuwano Ryozo3, Katushiko Shirahige2, Sayaka Sugiyama1
新潟大院・医歯学総合1, 東大・分生研2, 新潟大・脳研3
Grad Sch Med Dent Sci, Niigata Univ, Niigata1, Inst Mol Cell Biosci, Univ of Tokyo, Tokyo2, Brain Res Inst, Niigata Univ, Niigata3

Juvenile brain experiences a unique time window or 'critical period' when neuronal circuits are intensively remodeled based on experience. For example, binocular vision is established in the primary visual cortex (V1) through an activity-dependent competition between the two eyes. We have previously reported Otx2 as an essential activator of the critical period in V1, promoting maturation of Parvalbumin (PV)-positive interneuron (Sugiyama et al., Cell 134:508, 2008). As a transcription factor, this homeoprotein is expected to regulate downstream gene expressions involved in neuronal circuits rewiring and plasticity. However, direct targets of Otx2 are totally unknown yet. Here, we show the possible binding sites of Otx2 throughout the neuronal genome by ChIP-seq (chromatin immunoprecipitation followed by high-throughput sequencing) methods. Our initial trial revealed approximately 100 genes, bearing Otx2 binding sites within or nearby their gene bodies (~10 kb). Interestingly, factors involved in neural development (8 genes), transcriptional regulations (12 genes) and signal transmission (13 genes) were enriched. Identification of critical factors for the development of PV cells, like TrkB receptor, is consistent with the notion that Otx2 promotes their maturation to activate the plasticity. Thus, our approach would uncover important new factors implicated involved in the critical period regulation.
O2-6-4-3
RNA結合タンパク質は、mRNA processingを介して脳形成過程で何を制御しているのか?
What is the role of RNA-binding proteins in the process of brain formation through regulating mRNA processing ?

○武内章英1, 飯田慶1, 二宮賢介1, 伊藤美佳子2, 和根崎圭子1, 中川真美1, 大野欽司2, 萩原正敏1
○Akihide Takeuchi1, Kei Iida1, Kensuke Ninomiya1, Mikako Ito2, Keiko Wanezaki1, Mami Nakagawa1, Kinji Ohno2, Masatoshi Hagiwara1
京都大学院・医・形態形成1, 名古屋大院・医・神経遺伝情報2
Dept Anat & Dev Biol, Kyoto Univ Grad Sch Med1, Div Neurogenet, Nagoya Univ Grad Sch Med2

Recent high-throughput sequencing transcriptome studies are proving growing evidence for the essential roles of mRNA processing, especially in mammalian nervous system. Processing of mRNA is mediated by the RNA-binding proteins (RBPs) which regulate the expression of many genes co-transcriptionally or post-transcriptionally thorough direct interaction however, relatively little is known about their molecular mechanisms what and how RBPs regulate in neuronal development or function. Here we show that RNA binding protein, Sfpq (Splicing factor, proline/glutamine rich or PSF, Poly-pyrimidine tract binding protein associated Splicing Factor) plays critical roles in developing of brain through regulating neuron specific mRNA expression. Sfpq is specifically expressed in nascent cortical plate, suggesting its specific roles for regulating cortical plate formation and maturation. Transcriptome analysis using Sfpq disrupting cerebral cortex showed significant down-regulation of neuron-specific mRNAs which is categorized in cell-adhesion, cell motion, axon guidance, ion channel activity or receptor related, synaptic vesicle transport and related in Gene Ontology, all of these have specific and essential functions during nervous system development. Transcriptome-wide binding map of Sfpq using iCLIP (individual-nucleotide resolution UV Cross-Linking and ImmunoPrecipitation) reveals Sfpq to have a preference for binding to the long neuron specific pre-mRNA. In vivo analysis of Sfpq knockout mouse brain, proliferation of neural stem cell and their differentiation for neuron were grossly normal by E12.5 however, neuronal apoptosis was observed after E13.5 in which stage Sfpq expression became evident in cortical plate. Finally most part of the cerebral cortex was dropped-off by E18.5. These observations indicate that RNA Binding Protein Sfpq is essential for the development of cerebral cortex through regulating neuron specific long pre-mRNA expression.
O2-6-4-4
神経分化と可塑性を司るRNA制御メカニズムーシナプス形成因子Neurexinのダイナミックな選択的スプライシング制御
RNA-based mechanism underlying neuronal differentiation and plasticity - Dynamic regulation of alternative pre-mRNA splicing of Neurexins

○飯島崇利1, , 飯島陽子1
○Takatoshi Iijima1, Harald Witte1, Yoko Hanno-Iijima1, Timo Glatter1, Peter Scheiffele1
Dept. of Cell and Neurobiol., Biozentrum, Univ. of Basel1

Synapses are highly organized cellular junctions between neurons, which underlie transmission and processing of cellular signals in neuronal networks. Trans-synaptic cell adhesion molecules, such as Neurexins, Neuroligins and LRRTMs, bridge the synaptic cleft and play an important role in synapse formation. Neurexin transcripts are encoded by multiple genes and undergo extensive alternative splicing. Importantly alternative splicing of Neurexin at alternatively spliced segment 4 (AS4) is assumed to underlie an adhesive code for selective protein interactions at synapses as AS4 variants exhibit different affinities for the several binding partners. However the mechanism underlying the control of splice isoform choice remains to be understood.Here we demonstrate that neuronal activity triggers a shift in splice isoform choice of Neurexins at AS4 through Ca2+/calmodulin-dependent protein kinase IV (CaMKIV) signaling. This regulation alters the response to the synaptogenic receptors in cultured cerebellar granule cells. We identified SAM68, an RNA-binding protein which belongs to signal transduction and activation of RNA (STAR) family, as a key mediator for activity-dependent alternative splicing at AS4. We further revealed that other STAR proteins regulate alternative alternative splicing of Neurexins in vitro and iin vivo. Notably these proteins exhibited cell-type-specific expression in adult brains. Therefore STAR proteins regulate alternative splicing of Neurexins in neuronal activity-dependent as well as cell-type specific fashions, and may contribute to synapse specificity, plasticity and remodeling.
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