Deep sequencing and characterization of novel mammalian retinal microRNAs

描述(申请人提供)：哺乳动物的视网膜由不同类型的细胞组成，具有不同的功能(例如，光感受器和多种类型的中间神经元)。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所指定，但在个别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被部分了解。最近在动物体内发现了一类丰富的小RNA调控分子--microRNAs(MiRNAs)，它为视网膜细胞的多样化提供了一种潜在的新的调控机制。虽然已发现数百个miRNAs在哺乳动物的神经系统中表达，但最近对编码miRNAs的基因的计算搜索以及对哺乳动物大脑中表达的miRNAs的测序表明，仍有大量的哺乳动物miRNAs有待识别。对线虫的遗传学研究已经发现了控制特定神经元身份的miRNAs，其中一些miRNAs在非常小的细胞群中表达。如果哺乳动物视网膜中存在类似的miRNAs，仅限于特定的细胞类型，那么那些来自不太丰富的细胞类型的miRNAs将只占所有miRNAs的一小部分，因此到目前为止不太可能被发现。 该项目的具体目标是建立在成年和新生小鼠视网膜中表达的miRNAs的广泛图谱，鉴定仅限于特定视网膜细胞类型或亚型的miRNAs，并鉴定在视网膜变性1(Pde6brd1)小鼠突变体中表达改变的视网膜miRNAs，这是人类视网膜色素变性的模型。MiRNAs将通过广泛或深度测序，使用大规模并行测序技术进行识别。将对来自成人和新生野生型视网膜以及成年Pde6brd1视网膜的数百万个短RNA进行测序(测序深度是之前对视网膜miRNAs分析的1000倍)。将对序列进行计算分析，以识别已知和新的miRNAs并排除其他RNAs。在视网膜中发现的miRNAs将使用最近发展的miRNA原位杂交技术进一步表征。基于视网膜中不同细胞类型的频率，视网膜miRNAs的深度测序应该能够识别仅限于低丰度视网膜细胞类型的miRNAs，以及产生在视网膜中表达的全面的miRNAs谱。这些研究旨在为了解miRNA在哺乳动物视网膜中的表达和功能提供一个框架。识别视网膜中的细胞类型特异性和其他miRNAs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，在视网膜疾病中，细胞类型特异性miRNAs的表达可能受到调节。

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.