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在10-20 kb左右，但人类基因组也包含大量比人类基因组长得多的基因。有些基因的长度可以超过一百万个碱基对。许多这些长基因也含有数百个千碱基长度的内含子。这些特征对转录和RNA剪接过程构成了极大的挑战。对于RNA剪接，尽管在内含子中存在类似的“诱饵”序列，但非常大的内含子存在识别正确剪接位点和外显子的问题。目前的剪接信号模型不能很好地预测大基因的剪接模式。为了解决这些问题，我们最近开发了一些方法来测量RNA聚合酶II （RNAPII）在原位染色体位置和正常染色质环境下的大人类基因的延伸率和RNA剪接率。我们已经证明，转录在大基因中进行得很快，剪接在合成后几分钟内发生共转录，无论内含子的长度如何。我们现在有证据表明，大内含子是在一次事件中拼接的，这意味着必须存在抑制诱饵剪接位点使用的机制。我们建议使用先前实验的一个变体来研究剪接因子与长内含子结合的时间顺序，以测试当前的外显子定义理论。除了pre-mRNA中包含的剪接信号外，剪接信息也可能沿着基因编码在染色质结构中。为了支持这一观点，我们已经证明外显子相对于邻近的内含子序列在核小体中富集，并且这些外显子核小体也在特定的组蛋白甲基标记中富集。我们建议通过敲除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的功能。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。选择性剪接的变化将通过转录组分析来检测。许多辅助因素RNAPII转录延伸已确定在体外和体内研究。然而，在哺乳动物细胞中，很少有这些被证明是RNAPII延长所必需的。特别是大基因应该依赖于RNAPII的最佳伸长，从而使这些基因对这些因素的分析可能有用。我们建议在修改基因表达机制后检查这些速率。首先，我们将使用RNAi敲低延伸因子来确定它们在长基因转录中的体内作用。我们还将使用含有截断和修饰重要c端结构域的RNAPII突变版本来解决该结构域在大型基因的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的关系。

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.