神経系におけるRNA研究の新たな展開：RNAの機能、代謝、病態への関与 RNA Function, RNA Metabolism and Neurological Diseases
S2-2-2-1 プロモーターノンコーディングRNAによるほ乳類脳エピゲノム形成 Epigenome formation in the mammalian brain mediated by promoter-associated noncoding RNA ○今村拓也1 ○Takuya Imamura1 京都大学 理学研究科1 Graduate School of Science, Kyoto University1 The majority of non-coding RNAs (ncRNAs) in mammals have been believed to downregulate the corresponding mRNA expression level in a pre- or post-transcriptional manner by forming short or long ncRNA-mRNA duplex structures. Information on non-duplex-forming long ncRNAs is now also rapidly accumulating. To examine the directional properties of transcription at the whole-genome level, I performed directional RNA-seq analysis of mouse and chimpanzee tissue samples including the brain. I found that there is only a small fraction (about 1%) of the genome where both the top and bottom strands are utilized for transcription, suggesting that RNA-RNA duplexes are not abundantly formed in the tissues examined. Focusing on transcription start sites (TSSs) of protein- coding genes revealed that a significant fraction contain switching-points that separate antisense- and sense-biased transcription, suggesting that head-to-head transcription is more prevalent than previously thought. More than 90% of head-to-head type promoters contain CpG islands. Moreover, CCG and CGG repeats are significantly enriched in the upstream regions and downstream regions, respectively, of TSSs located in head-to-head type promoters. Genes with tissue-specific promoter- associated ncRNAs (pancRNAs) show a positive correlation between their pancRNA and mRNA expression, which is in accord with the proposed role for pancRNA in facultative gene activation, whereas genes with constitutive expression generally lack an association with pancRNA. Here, I propose that single-stranded ncRNA resulting from head-to-head transcription at GC-rich sequences regulates tissue-specific gene expression. In this talk, I will also provide data on the functional significance of pancRNA in the brain for the regulation of cell- to species-level epigenetic setting at specific gene loci.	S2-2-2-2 Ataxin-2の生理的機能の解明を目指して Toward a comprehensive functional analysis of Ataxin-2 for the understanding of a common mechanism underlying neurodegeneration ○河原行郎1 ○Yukio Kawahara1 大阪大学大学院 医学系研究科1 Graduate School of Medicine, Osaka University1 Spinocerebellar ataxia type 2 (SCA2) is an autosomal dominant neurodegenerative disease. The disorder is caused by abnormal CAG repeat expansion in the coding region of the Ataxin-2 gene (ATXN2). Recently, moderate CAG repeat expansion in ATXN2 was identified as a risk factor for amyotrophic lateral sclerosis (ALS). This finding suggests a common pathogenic role of Ataxin-2 in these neurodegenerative diseases. Therefore, a comprehensive understanding of the physiological functions of Ataxin-2, especially related to disease pathogenesis, is necessary to elucidate the mechanism underlying Ataxin-2-mediated neurodegeneration.Ataxin-2 is known to directly bind to polyadenylate-binding protein 1 (PABPC1) and RNA helicase DDX6, which suggests that Ataxin-2 is likely involved in RNA metabolism. However, the functions of Ataxin-2 have not been characterized in detail. In this symposium, we show that Ataxin-2 promotes the translation of mRNA mainly through the recruitment of polysomes but not through the increase of RNA stability. Interaction with PABPC1 partially contributes to the promotion of translation; however, we identified a previously uncharacterized domain that is indispensable for this function. Furthermore, abnormal expansion of the polyglutamine stretch in Ataxin-2 significantly reduced the efficiency of translation, which may contribute to the pathogenesis of neurodegenerative diseases. Based on these findings, we are currently attempting to elucidate how globally Ataxin-2 regulates translation, and to identify the key genes that are associated with neurodegeneration among Ataxin-2-regulating mRNAs.
S2-2-2-3 脳におけるRNAを標的とした新規オートファジー経路 A novel type of autophagy targeting RNA in brain ○藤原悠紀1,2, 和田圭司1, 株田智弘1 ○Yuuki Fujiwara1,2, Keiji Wada1, Tomohiro Kabuta1 国立精神・神経セ・神経研・疾病四部1, 早大院・先進理工・電生2 Dept. of Degener. Neurol. Dis., Natl. Inst. of Neurosci., NCNP1, Dept. of Elec. Eng. and Biosci., Grad. Sch. of Advanced Sci. and Eng., Waseda Univ., Tokyo, Japan2 Degradation of cellular components by lysosomes is a fundamental event for biological homeostasis in many tissues including brain. Lysosomes are characterized by various hydrolases and by abundance of membrane glycoproteins. LAMP-2 is a major lysosomal membrane protein with a single transmembrane region. Each of three splice variants of LAMP-2, LAMP-2A, 2B and 2C, possesses an identical lumenal region and different cytosolic tails. LAMP-2A serves as a receptor for chaperone-mediated autophagy (CMA). In CMA, substrate proteins are directly imported into the lysosomal lumen. However, the specific functions of LAMP-2B and LAMP-2C remain unknown. In the present study, we found that the expression level of LAMP-2C is especially high in the brain. We found that the cytosolic sequence of LAMP-2C can specifically bind to almost all total RNA derived from mouse brain. The cytosolic sequence of LAMP-2C and its affinity for RNA are evolutionarily conserved among flies, nematodes and humans. Considering the relationship between LAMP-2A and CMA, we hypothesized a novel autophagic pathway that directly imports RNA into lysosomes via LAMP-2C. Using isolated lysosomes derived from mouse brains, we identified a novel autophagic pathway that directly imports RNA into lysosomes for degradation. We showed that this pathway, which we term "RNautophagy", is ATP dependent, and unlike CMA, is independent of Hsc70. We demonstrated that LAMP-2C functions as a receptor for RNautophagy, using lysosomes isolated from cells overexpressing LAMP-2C and LAMP-2-deficient mouse brains. Moreover, cells overexpressing LAMP-2C exhibited a significant enhancement in the overall levels of RNA degradation. A significant accumulation of total RNA was observed in the brains of LAMP-2 knockout mice. Our findings shed light on the mechanisms underlying RNA homeostasis in brain.	S2-2-2-4 神経疾患におけるタンパク質・RNA凝集体の意義 Protein/RNA aggregation in neurological disorders ○紀嘉浩1,2, 貫名信行2 ○Yoshihiro Kino1,2, Nobuyuki Nukina2 理化学研究所 科学総合研究センター1, 順天堂大院・医・神経変性疾患病態治療探索講座2 RIKEN Brain Science Institute1, Therapeutic research lab for neurodegenerative diseases, Grad. Sch. of Med., Juntendo Univ., Tokyo, Japan2 Recently, RNA-mediated pathogenesis has gained attention in subsets of neurological diseases. On one hand, mutant RNA containing an expanded repeat tract forms intranuclear inclusions that sequester RNA-biding proteins and misregulate RNA processing in repeat expansion diseases such as myotonic dystrophy, spinocerebellar ataxia type 8, and Huntington's disease-like 2. On the other hand, mutations or aggregation of RNA-binding proteins, TDP43 and FUS/TLS, are associated with amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). These proteins are prone to aggregate and their mislocalization may cause aberrant RNA metabolisms including misregulation of nuclear RNA processing and formation of RNA-containing granules in the cytoplasm. These two classes of diseases are related to each other. For example, a non-coding repeat expansion was identified as a cause of ALS. In addition, the repeat length of polyglutamine tract of Ataxin2 act as a risk factor of ALS. Furthermore, our group previously identified Sqstm1/p62, UBQLN2, and FUS/TLS proteins as components of polyglutamine aggregates. All these three proteins are genetically or pathologically associated with ALS/FTLD. These observations suggest molecular and pathological links among factors of repeat expansion diseases and ALS/FTLD. Furthermore, above factors highlight two key aspects of these diseases, RNA metabolisms and protein misfolding/clearance. However, the roles of RNA/protein inclusion and the consequences of abnormalities in RNA-binding proteins are still elusive. We will present our recent data on these issues and discuss the significance of protein/RNA aggregate formation in neurological disorders.

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