Transcriptional elongation and splicing in human genes in situ

描述（由申请人提供）：虽然人类基因的平均长度约为10-20 kb，但人类基因组也含有大量更长的基因。有些基因的长度可以超过一百万个碱基对。这些长基因中的许多还含有数百个内含子长度的内含子。这些特征代表了对转录和RNA剪接过程的极端挑战。对于RNA剪接，非常大的内含子存在识别正确剪接位点和外显子的问题，尽管内含子内存在类似“诱饵”序列的背景。目前的剪接信号模型不能正确预测大基因的剪接模式。为了开始解决其中的一些问题，我们最近开发了一些方法来测量大型人类基因在原位染色体位置和正常染色质环境中的RNA聚合酶II（RNAPII）延伸和RNA剪接速率。我们已经证明，转录在大基因中进行得很快，并且无论内含子的长度如何，剪接在合成的几分钟内就以共转录的方式发生。我们现在有证据表明，大内含子是在一个单一的事件剪接，这意味着机制必须存在抑制诱饵剪接位点的使用。我们建议使用我们以前的实验的变化，以调查剪接因子结合到长内含子的时间顺序，以测试目前的外显子定义的理论。除了包含在前mRNA中的剪接信号之外，剪接信息也可能沿着沿着基因编码在染色质结构中。为了支持这一观点，我们已经表明，外显子是丰富的核小体相对于相邻的内含子序列，这些外显子核小体也富含特定的组蛋白甲基标记。我们建议通过敲低或过表达特定的甲基转移酶来选择性地去除或增强这些甲基标记，以确定其功能。我们将通过ChIP分析确认这些改变，然后我们将测量RNA剪接的速率和保真度。将通过转录组分析检测可变剪接的变化。RNAPII转录延长的许多辅助因子已在体外和体内研究中被鉴定。然而，这些中的很少（如果有的话）已显示为在哺乳动物细胞中RNAPII的体内延伸所需。特别是大基因应该依赖于RNAPII的最佳延伸，从而使这些基因潜在地可用于分析这些因素。我们建议检查这些利率修改后的基因表达机制。首先，我们将使用RNAi敲低延伸因子，以确定它们在长基因转录中的体内作用。我们还将使用RNAPII的突变版本，其中包含重要的C-末端结构域的截短和修饰，以解决该结构域在大基因转录和剪接中的作用。这些研究将促进我们对人类基因表达的理解，这与正常和病理细胞的生长和发育都有重要关系。 公共卫生相关性：基因的调节表达是人类生长、发育、正常和病理功能以及身体对内外环境变化的反应的核心。该提案旨在了解它们在自然染色体环境中如何在人类细胞中正确表达。特别是，我们提出的实验，探测基因的表达机制，大大大于平均水平。这些大基因包括在许多人类癌症中失活的几种肿瘤抑制基因。我们希望了解这些规则并确定在大基因表达中起作用的因素，以便了解，预测并预防病理条件下基因的异常表达。

DESCRIPTION (provided by applicant): While the average human gene is on the order of 10-20 kb in length, the human genome also contains a significant number of genes which are much longer. Some genes can exceed one million base pairs in length. Many of these long genes also contain introns of hundreds of kilobases in length. These features represent an extreme challenge to the processes of transcription and RNA splicing. For RNA splicing, very large introns present the problem of identifying the correct splice sites and exons in spite of a background of similar "decoy" sequences present within the introns. The current models of splicing signals cannot properly predict the splicing pattern of large genes. To begin to address some of these problems, we have recently developed methods to measure the rate of RNA polymerase II (RNAPII) elongation and RNA splicing in large human genes in their in situ chromosomal locations and normal chromatin environments. We have shown that transcription proceeds rapidly in large genes and that splicing occurs co-transcriptionally within minutes of synthesis regardless of the length of the intron. We now have evidence that large introns are spliced in a single event implying that mechanisms must exist to suppress the use of decoy splice sites. We propose to use a variation of our previous experiment to investigate the temporal order of splicing factor binding to long introns in order to test current theories of exon definition. In addition to the splicing signals contained in the pre-mRNA, it is possible that splicing information could also be encoded in the structure of chromatin along genes. To support this idea, we have shown that exons are enriched in nucleosomes relative to adjacent intron sequences and that these exonic nucleosomes are also enriched in specific histone methyl marks. We propose to determine the function of these methyl marks by selectively removing or enhancing them by knocking down or over-expressing the specific methyltransferases. We will confirm these alterations by ChIP analysis and then we will measure the rate and fidelity of RNA splicing. Changes in alternative splicing will be detected by transcriptome analysis. Many accessory factors for RNAPII transcription elongation have been identified in in vitro and in vivo studies. However, few if any of these have been shown to be required for elongation of RNAPII in vivo in mammalian cells. Large genes in particular should be dependent on optimum elongation of RNAPII thus making these genes potentially useful for the analysis of these factors. We propose to examine these rates following modification of the gene expression machinery. First, we will use RNAi knockdowns of elongation factors to determine their in vivo roles in the transcription of long genes. We will also use mutant versions of RNAPII containing truncations and modifications of the important C-terminal domain to address the roles of this domain in transcription and splicing in large genes. These studies will advance our understanding of human gene expression which is of major relevance to both normal and pathological cell growth and development. PUBLIC HEALTH RELEVANCE: The regulated expression of genes is central to human growth, development, normal and pathological functioning and the response of the body to changes in the internal and external environment. This proposal is designed to understand how in their natural chromosomal environment are correctly expressed in human cells. In particular, we propose experiments that probe expression mechanisms in genes that are substantially larger than average. Such large genes include several tumor suppressor genes which are inactivated in many human cancers. We hope to learn the rules and identify the factors that play roles in the expression of large genes in order to understand, predict and perhaps prevent the aberrrant expression of genes in pathological conditions.