LONG NON-CODING RNAS, LEARNING AND MEMORY

描述（由申请人提供）：本提案是对申请R21MH103812-01，“Long non-coding RNAs, learning and memory”的再提交，该申请于2013年11月由MNG研究部门审核。其重点是建立活动依赖性长链非编码rna在皮层中的功能作用。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的基本分子交易的，以及它们是如何促进与恐惧相关的精神疾病的发展的。我们已经努力解决提出的每一个问题，并对拨款进行了实质性修改，以澄清实验方法并包括额外的数据。作为一名年轻的研究者，我真诚地感谢初审时的思考和建设性的批评，我认为修改后的申请比初审时的申请强得多。审稿人1指出，“应该花时间讨论从ChIRP实验中期望得到什么结果以及如何解释这些结果”，“应该提出研究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一问题，包括利用ChIP测定对蛋白编码基因靶TSS周围染色质景观的调查，候选lncrna -染色质修饰复合物相互作用的RIP和lncrna -靶蛋白编码基因启动子相互作用的ChIRP。此外，我们现在描述了它们将如何阐明lncrna在调节与学习相关的基因表达中的作用。审稿人1还指出，“了解lncRNA是否以及如何作为调节蛋白质编码基因表达的诱饵、支架或指南将具有指导意义”。我们相信这是一个很好的问题，尽管它超出了当前探索性R21资助的范围，但它肯定会成为未来R01在支持行为适应的细胞类型特定基因调控背景下应用的主题。最后，审稿人1不清楚PI将与谁合作和/或他们将提供哪些服务来促进项目的成功。UCI发育和细胞生物学系的孙沙博士是lncrna和表观遗传编程方面的专家（Sun et al., 2013），他主动提出作为该项目的合作者（附支持信），在项目进展过程中提供战略建议，并在新发现的实验验证应用方面分享专业知识，以协助有效和成功的检控

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.