Transcriptional elongation and splicing in human genes in situ

虽然人类基因的平均长度为10-20 kb，但人类基因组还包含一个 有相当数量的基因，它们更长。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剪接过程的极大挑战。对于RNA剪接，非常大的内含子 尽管有类似的“诱饵”背景， 内含子内的序列。目前的剪接信号模型不能正确地预测 大基因的剪接模式。为了开始解决这些问题，我们最近开发了 方法测量RNA聚合酶II（RNAPII）延长和RNA剪接的速率在大的人类 基因在其原位染色体位置和正常染色质环境中。我们已经证明 转录在大基因中快速进行，并且剪接在转录后几分钟内以共转录的方式发生。 无论内含子的长度如何，都可以合成。我们现在有证据表明，大内含子是在一个 单个事件意味着必须存在抑制诱饵剪接位点使用的机制。我们建议 使用我们以前实验的一个变体来研究剪接因子与长链DNA结合的时间顺序。 内含子，以测试当前的外显子定义理论。除了包含在 前mRNA，剪接信息也可能编码在染色质结构中，沿着 基因.为了支持这一观点，我们已经证明，相对于相邻的外显子， 内含子序列，并且这些外显子核小体也富含特异性组蛋白甲基标记。我们 建议通过选择性地去除或增强这些甲基标记来确定它们的功能， 敲低或过表达特异性甲基转移酶。我们将通过ChIP确认这些变更 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是 通过转录组分析检测。RNAPII转录延伸的许多辅助因子已经被发现， 在体外和体内研究中鉴定。然而，很少，如果有的话，这些已被证明是必要的， RNAPII在哺乳动物细胞中的体内延伸。特别是大基因应该依赖于最佳的 RNAPII的延伸，从而使这些基因潜在地可用于这些因子的分析。我们提出 以检查基因表达机制修饰后的这些速率。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。我们将 还使用含有重要的C-末端的截短和修饰的RNAPII的突变形式 结构域，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 病理性细胞生长和发育。

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.