Transcriptional elongation and splicing in human genes in situ

人类基因的转录延伸和原位剪接

• 批准号: 8307820
• 负责人: RICHARD A PADGETT
• 金额: $ 29.53万
• 依托单位: CLEVELAND CLINIC LERNER COM-CWRU
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-24 至 2014-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8307820
• 关键词: Address; Affect; Alternative Splicing; Base Pairing; Binding; C-terminal; Cells; Chromatin; Chromatin Structure; Data; Elongation Factor; Environment; Event; Exons; Factor Analysis; Feedback; Gene Expression; Gene Expression Profile; Genes; Genetic Transcription; Growth and Development function; Histones; Human; Human Genome; ITPR1 gene; In Situ; In Vitro; Introns; Invertebrates; Kinetics; Knowledge; Learning; Length; Location; Malignant Neoplasms; Mammalian Cell; Measures; Methods; Methylation; Methyltransferase; Modeling; Modification; Molecular; Monitor; Nature; Nucleosomes; Nucleotides; Pattern; Phylogeny; Play; Process; Publishing; RNA; RNA Interference; RNA Polymerase II; RNA Splicing; Reaction; Relative (related person); Role; Signal Transduction; Site; Spliced Genes; Structure; System; Techniques; Testing; To specify; Transcription Elongation; Transcription Process; Transfection; Tumor Suppressor Genes; Ursidae Family; Variant; Work; base; cell growth; coping; demethylation; design; follow-up; gene conservation; genome-wide; histone modification; in vivo; insight; interest; knock-down; mRNA Precursor; mutant; prevent; research study; response; theories

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.

虽然人类基因的平均长度约为10-20kb，但人类基因组也包含一个 相当数量的长得多的基因。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千碱基的内含子。这些功能代表一种 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 尽管有类似的“诱饵”背景，但仍然存在识别正确的剪接位点和外显子的问题。 内含子中存在的序列。目前的信号拼接模型不能正确地预测 大基因的剪接模式。为了开始解决其中一些问题，我们最近开发了 方法测定大块头人体RNA聚合酶II(RNAPII)的伸长率和RNA剪接率 基因在其原位染色体位置和正常染色质环境中。我们已经证明了 转录在大基因中快速进行，剪接在几分钟内以共转录方式发生 合成与内含子的长度无关。我们现在有证据表明大的内含子拼接在一个 单一事件意味着必须存在抑制诱骗剪接位点使用的机制。我们建议 使用我们先前实验的一个变体来研究剪接因子与Long结合的时间顺序 以检验当前外显子定义的理论。除了包含在 在mRNA之前，剪接信息也可能编码在染色质的结构中 基因。为了支持这一观点，我们已经证明外显子相对于相邻的外显子在核小体中是丰富的。 内含子序列和这些外显子核小体也富含特定的组蛋白甲基标记。我们 建议通过有选择地移除或增强这些甲基标记来确定它们的功能 击倒或过度表达特定的甲基转移酶。我们将通过芯片确认这些更改 分析，然后我们将测量RNA剪接的速度和保真度。替代剪接的变化将是 通过转录组分析检测到。RNAPII转录延长的许多辅助因子已经被 在体外和体内研究中都得到了证实。然而，其中很少(如果有的话)被证明是 RNAPII在哺乳动物细胞中的体内延伸。大基因尤其应该依赖于最适基因 RNAPII的延长因此使这些基因对这些因子的分析有潜在的帮助。我们建议 在基因表达机制改变后检查这些比率。首先，我们将使用RNAi 敲除延长因子以确定它们在长基因转录中的活体作用。我们会 也使用包含重要C-末端的截断和修饰的RNAPII的突变版本 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，它与正常和 病理性细胞的生长和发育。