Deep sequencing and characterization of novel mammalian retinal microRNAs

描述（由申请人提供）：哺乳动物视网膜由具有不同功能作用的细胞类型（例如光感受器和多种类型的中间神经元）的多种混合物组成。不同视网膜细胞类型的特性似乎由不同的潜在基因表达模式指定，但在单个视网膜细胞类型中建立和维持特定基因表达模式的机制仅部分了解。最近在动物中发现了一类丰富的小RNA调节分子，microRNA（miRNAs），为视网膜细胞多样化提供了一种潜在的新的调节机制。尽管已经发现数百种miRNAs在哺乳动物神经系统中表达，但是最近对编码miRNAs的基因的计算机搜索以及对哺乳动物脑中表达的miRNAs的测序表明，许多哺乳动物miRNAs仍有待鉴定。线虫C.线虫已经鉴定出控制特定神经元的特性的miRNA，并且这些miRNA中的一些在非常小的细胞群体中表达。如果在哺乳动物视网膜中存在局限于特定细胞类型的类似miRNA，那么来自不太丰富的细胞类型的那些miRNA将代表所有miRNA的一小部分，因此迄今为止不太可能被鉴定。 该项目的具体目标是建立一个广泛的成年和新生小鼠视网膜中表达的miRNAs谱，以鉴定仅限于特定视网膜细胞类型或亚型的miRNAs，并鉴定视网膜变性1（Pde6brd1）小鼠突变体（一种人类视网膜色素变性模型）中表达改变的视网膜miRNAs。将使用大规模平行测序技术通过广泛或深度测序来鉴定miRNA。将对来自成人和新生儿野生型视网膜以及来自成人Pde6brd1视网膜的数百万短RNA进行测序（比先前的视网膜miRNA分析深1000倍）。将对序列进行计算分析，以鉴定已知和新的miRNA并排除其他RNA。将使用最近开发的miRNA原位杂交技术进一步表征在视网膜中鉴定的miRNA。基于视网膜中不同细胞类型的频率，视网膜miRNAs的深度测序应该允许鉴定限于低丰度视网膜细胞类型的miRNAs，以及产生视网膜中表达的miRNAs的综合谱。拟议的研究旨在为理解哺乳动物视网膜中miRNA的表达和功能提供一个框架。视网膜中细胞类型特异性和其他miRNAs的鉴定将提供新的基因，可以被认为是人类视网膜疾病的候选基因。此外，细胞类型特异性miRNA的表达可能在视网膜疾病中受到调节。

DESCRIPTION (provided by applicant): The mammalian retina is composed of a diverse mixture of cell types with distinct functional roles (e.g. photoreceptors and multiple types of interneurons). The properties of distinct retinal cell types appear to be specified by different underlying patterns of gene expression, but the mechanisms that establish and maintain specific patterns of gene expression in individual retinal cell types are only partially understood. The recent discovery of an abundant class of small RNA regulatory molecules in animals, microRNAs (miRNAs), provides a potential novel regulatory mechanism for retinal cell diversification. Although several hundred miRNAs have been found to be expressed in the mammalian nervous system, recent computational searches for genes that encode miRNAs, as well as sequencing of miRNAs expressed in mammalian brain, suggest that numerous mammalian miRNAs remain to be identified. Genetic studies in the nematode C. elegans have identified miRNAs that control the identity of specific neurons and some of these miRNAs are expressed in very small populations of cells. If similar miRNAs, restricted to specific cell types, exist in the mammalian retina, those miRNAs from less abundant cell types will represent a small fraction of all miRNAs and therefore are unlikely to have been identified to date. The specific goals of the proposed project are to create an extensive profile of miRNAs expressed in the adult and neonatal mouse retina, to identify miRNAs that are restricted to specific retinal cell types or subtypes, and to identify retinal miRNAs with altered expression in the retinal degeneration 1 (Pde6brd1) mouse mutant, a model of human retinitis pigmentosa. miRNAs will be identified by extensive or deep sequencing, using a massively parallel sequencing technology. Several million short RNAs from adult and neonatal wild-type retinas and from adult Pde6brd1 retinas will be sequenced (>1000-fold deeper sequencing than prior analyses of retinal miRNAs). Sequences will be computationally analyzed to identify known and novel miRNAs and to exclude other RNAs. The miRNAs identified in the retina will be further characterized using recently developed miRNA in situ hybridization techniques. Based upon the frequency of different cell types in the retina, the deep sequencing of retinal miRNAs should allow the identification of miRNAs restricted to low abundance retinal cell types, as well as the generation of a comprehensive profile of miRNAs expressed in the retina. The proposed studies are intended to provide a framework for understanding miRNA expression and function in the mammalian retina. Identification of cell-type specific and other miRNAs in the retina will provide new genes that can be considered as candidates for human retinal diseases. In addition, expression of cell-type specific miRNAs may be subject to modulation in retinal diseases.