LONG NON-CODING RNAS, LEARNING AND MEMORY

长非编码 RNA、学习和记忆

• 批准号: 8974443
• 负责人: Timothy W Bredy
• 金额: $ 19.31万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-12-01 至 2016-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8974443
• 关键词: Adaptive Behaviors; Address; Antisense Oligonucleotides; Behavior; Behavioral; Brain; CRISPR/Cas technology; Chromatin; Code; Cognition; Complex; Cultured Cells; DNA Methylation; Data; Development; Developmental Cell Biology; Epigenetic Process; Event; Fright; Future; Gene Expression; Gene Expression Regulation; Gene Targeting; Genes; Genetic Transcription; Genome; Genomics; Grant; Health; Immunoprecipitation; In Vitro; Infusion procedures; Knock-out; Lead; Learning; Letters; Light; Mediating; Memory; Mental disorders; Molecular; Nature; Neurons; Nuclear; Positioning Attribute; Production; Proteins; RNA; RNA purification; Research Personnel; Risk; Role; Services; Site; Stimulus; Study Section; Technology; Testing; The Sun; Therapeutic Intervention; Time; Tissues; Transcript; Transcriptional Regulation; Untranslated RNA; Validation; Variant; anxiety-related disorders; cell type; chromatin immunoprecipitation; cognitive function; design; epigenetic regulation; experience; fear memory; histone modification; in vivo; insight; interest; knock-down; lentiviral-mediated; long term memory; mammalian genome; neuropsychiatric disorder; next generation sequencing; novel; overexpression; programs; promoter; protein expression; research study; response; scaffold; spatiotemporal; success; targeted treatment; transcriptome sequencing

DESCRIPTION (provided by applicant): This proposal is a resubmission of application R21MH103812-01, "Long non-coding RNAs, learning and memory", which was reviewed in November 2013 by the MNG study section. Its focus is to establish a functional role for activity-dependent long non-coding RNAs in cortex. We are particularly interested in how non-coding RNAs may mediate epigenetic regulation of the gene expression underlying behavior and cognition. Eventually, we hope to examine how these enigmatic transcripts are involved in the fundamental molecular transactions underlying memory, and how they contribute to the development of fear- related psychiatric disorders. We have made an effort to address every issue that has been raised, and the grant has been substantially revised both to clarify the experimental approach and to include additional data. As a young investigator, I sincerely appreciate the thought and constructive criticism that went into the initial review, and I believe that the revised application is significantly stronger than the initial submission. Reviewer 1 indicated that, "time should have been spent discussing what results would be expected from the ChIRP experiments and how they would be interpreted", and "to propose to study how interaction of the lncRNA with the promoter of the protein coding genes leads to transcription regulation". We have now addressed this issue by including experiments utilizing ChIP assay for interrogation of the chromatin landscape surrounding TSS of protein coding gene targets, RIP for candidate lncRNA-chromatin modifying complex interactions and ChIRP for lncRNA-target protein coding gene promoter interactions. Moreover, we now describe how they will shed light on the role of lncRNAs in regulating gene expression associated with learning. Reviewer 1 also stated that, "it would be instructive to know if and how the lncRNA acts as a decoy, a scaffold or a guide for regulating expression of the protein coding genes". We believe this is a great question and, although it is beyond the scope of the current exploratory R21 grant, it will definitely be subject of a future R01 application within the context of cell-type specific gene regulation supporting behavioral adaptation. Finally, it was unclear to reviewer 1 with whom the PI would collaborate and/or what services they will provide that will facilitate success of this project. Dr. Sha Sun in the Department of Developmental and Cell Biology at UCI is an expert in both lncRNAs and epigenetic programming (Sun et al., 2013) and has offered to serve as a collaborator on this project (letter of support attached) by providing strategic advice on the project as it progresses, and practical support in sharing expertise in the application of experimental validation of the novel findings to assist in the efficient and successful prosecution of this project. Both Reviewer 2 and 3 expressed concern that, "the ASO technology may not be efficient at knocking down lncRNA", and that no other alternative approach was mentioned. It is important to note that we already have successfully manipulated Gomafu in primary cortical neurons in culture (Barry et al., 2013) and preliminary data, Aim 2). In the event that we can't reduce Gomafu or other lncRNAs in vivo using ASO, we propose to use CRISPR/Cas9 technology to knockdown these lncRNA. However, we also acknowledge that this approach carries some degree of risk, due to the permanent nature of the knockout, which may lead to compensatory effects on the expression of genes that were under inhibitory control by the target lncRNA. Finally, we have a Gomafu lncRNA overexpression construct that will also be tested in Aim 3. Reviewer 3 indicated that, "it would also be important to determine whether there is indeed a functional relationship between lncRNA and its protein-coding targets. E.g. can the effect of manipulating lncRNA be counter-acted by the manipulation of protein-coding target?" We completely agree with this reviewer; however, due to time limitations for the R21 project, these experiments will be included in a future R01 designed to explore in greater detail the functional relationship between lncRNA-mediated epigenetic regulation of gene expression, once a role for candidate lncRNAs in regulating learning and memory have been firmly established.

描述（由申请人提供）：本提案是申请R21 MH 103812 -01“长非编码RNA，学习和记忆”的重新提交，MNG研究部门于2013年11月对该申请进行了审查。它的重点是建立一个功能的作用，在皮层的活动依赖性长非编码RNA。我们对非编码RNA如何介导行为和认知基础基因表达的表观遗传调控特别感兴趣。最终，我们希望研究这些神秘的转录本是如何参与记忆基础的基本分子交易的，以及它们是如何促成与恐惧相关的精神障碍的发展的。我们已经努力解决提出的每一个问题，并对赠款进行了大幅修改，以澄清实验方法并纳入更多数据。作为一名年轻的研究员，我真诚地感谢初步审查中的想法和建设性批评，我相信修改后的申请比最初提交的申请明显更强大。评审员1指出，“应该花时间讨论ChIRP实验预期的结果以及如何解释这些结果”，以及“建议研究lncRNA与蛋白质编码基因启动子的相互作用如何导致转录调控”。我们现在已经解决了这个问题，包括实验利用ChIP测定询问周围的蛋白质编码基因靶TSS的染色质景观，RIP的候选lncRNA-染色质修饰复合物的相互作用和ChIRP的lncRNA-靶蛋白编码基因启动子的相互作用。此外，我们现在描述他们将如何阐明lncRNA在调节与学习相关的基因表达中的作用。评审员1还指出，“了解lncRNA是否以及如何作为诱饵、支架或指导来调节蛋白质编码基因的表达将是有益的”。我们相信这是一个很大的问题，虽然它超出了目前探索性R21资助的范围，但它肯定会成为未来R 01在支持行为适应的细胞类型特异性基因调控背景下应用的主题。最后，审查人1不清楚PI将与谁合作和/或他们将提供哪些服务以促进该项目的成功。UCI发育和细胞生物学系的Sha Sun博士是lncRNA和表观遗传编程的专家（Sun et al.，2013年），并表示愿意担任该项目的合作者（随附支持函），在项目进展过程中提供战略建议，并提供实际支持，分享应用实验验证新发现的专业知识，以协助有效和成功地起诉 of this project项目. 2号和3号评审员均表示担心，“阿索技术可能无法有效敲除lncRNA”，并且未提及其他替代方法。值得注意的是，我们已经成功地在培养的原代皮层神经元中操纵了Gomafu（巴里等人，2013年）和初步数据，目标2）。如果我们不能使用阿索在体内减少Gomafu或其他lncRNA，我们建议使用CRISPR/Cas9技术来敲低这些lncRNA。然而，我们也承认，这种方法具有一定程度的风险，由于敲除的永久性，这可能会导致对受靶lncRNA抑制控制的基因表达的补偿效应。最后，我们有一个Gomafu lncRNA过表达构建体，也将在目标3中进行测试。Reviewer 3指出，“确定lncRNA与其蛋白质编码靶标之间是否确实存在功能关系也很重要。例如，操纵lncRNA的效果是否可以通过操纵蛋白质编码靶标来抵消？“我们完全同意这位评论家的观点;然而，由于R21项目的时间限制，这些实验将被纳入未来的R 01中，该R 01旨在更详细地探索lncRNA介导的基因表达表观遗传调控之间的功能关系，一旦候选lncRNA在调节学习和记忆中的作用已经确立。

项目成果

Long Noncoding RNA-Directed Epigenetic Regulation of Gene Expression Is Associated With Anxiety-like Behavior in Mice.

• DOI: 10.1016/j.biopsych.2015.02.004
• 发表时间: 2015-12-15
• 期刊: Biological psychiatry
• 影响因子: 10.6
• 作者: Spadaro PA;Flavell CR;Widagdo J;Ratnu VS;Troup M;Ragan C;Mattick JS;Bredy TW
• 通讯作者: Bredy TW