Comprehensive Characterization and Classification of the Human Transcriptome

描述（由申请人提供）：本提案的目标是全面鉴定与转录序列相关的所有基于序列的功能元件，包括蛋白质编码和非蛋白质编码序列，表征基因结构，包括转录起始位点（TSS）多聚腺苷酸化位点和在代表性和多样化的人类细胞和组织组中检测到的替代转录物。基于本提案中发现的检测到的转录本的经验确定的特征，将建立人类转录组的转录蛋白质编码和非蛋白质编码部分的分类系统。 我们的目标包括首先从所研究的每种细胞类型中产生一组全面的亚细胞区室特异性长（>200个核苷酸，nts）和短（<200 nts）聚腺苷酸化（polyA+）和非聚腺苷酸化（polyA-）RNA样品。将使用以下方法分析这些RNA样本：a）高密度平铺阵列（长和短RNA的5个核苷酸[nt]询问分辨率），B）测序（焦磷酸测序[454]和短RNA的克隆单分子测序[Solexa]），c）测序的用于polyA+转录物的5' TSS和3'终止位置的配对末端双标签（PET）和d）基因表达的测序帽分析（CAGE）polyA-RNA的5' TSS标签。全长亚细胞区室特异性转录物的表征也将使用：1）cDNA末端快速扩增（RACE）、RT-PCR和测序的组合，2）RNA免疫沉淀（RIP）和3）原位免疫组织化学。这些表征步骤将提供关于发现与已知功能性、区室特异性蛋白质相关的注释和未注释RNA及其在已知功能的亚细胞器中的定位的额外信息。 研究和卫生保健界处于有利地位，可以利用人类基因组中分类转录区域的详细目录。例如，数百万个单核苷酸多态性（SNP）的鉴定和通过小抑制性（si-）和微（mi-）RNA遗传改变特异性转录物表达的能力对于与转录区域相关的疾病的分子表征是非常有用的。然而，这些和其他基因组资源的效用依赖于具有完整和高质量的转录区域目录。

DESCRIPTION (provided by applicant): The goal of this proposal is to comprehensively identify all sequence-based functional elements associated with transcribed sequences including both protein coding and non-protein coding sequences, characterizing gene structures including transcription start sites (TSS) polyadenylation sites and alternative transcripts detected in a representative and diverse panel of human cells and tissues. Based on the empirically determined characteristics of the detected transcripts uncovered in this proposal, a classification system for transcribed protein coding and non-protein coding portions of the human transcriptome will be established. Our aims include first to generate a comprehensive set of subcellular compartment-specific long (>200 nucleotides, nts) and short (<200 nts) polyadenylated (polyA+) and non-polyadenylated (polyA-) RNA samples from each of the cell types studied. These RNA samples will be analyzed using: a) high density tiling arrays (5 nucleotides [nt] interrogation resolution for long and short RNAs), b) sequencing (pyrosequencing [454] and clonal single molecule sequencing for short RNAs [Solexa]), c) sequenced paired-end ditags (PETs) for 5' TSS and 3' termination locations for polyA+ transcripts and d) sequenced cap analysis of gene expression (CAGE) tags for 5' TSS of polyA- RNAs. Characterization of full length subcellular compartment-specific transcripts will also be carried out using: 1) a combination of rapid amplification of cDNA ends (RACE), RT-PCR and sequencing, 2) RNA immunoprecipitation (RIP) and 3) in situ immunohistochemistry. These characterization steps will provide additional information concerning the annotated and unannotated RNAs found to be associated with known functional, compartment-specific proteins and their localization in subcellular organelles of known function. The research and health-care community are well positioned to take advantage of a detailed catalog of classified transcribed regions in the human genome. For example, the identification of millions of single nucleotide polymorphism (SNPs) and the ability to genetically alter specific transcript expression by small inhibitory (si-) and micro (mi-) RNAs are highly useful for the molecular characterization of diseases associated with the transcribed regions. However, the utility of these and other genomic resources are dependent upon having a complete and high quality catalogue of transcribed regions.