Transcription and processing of very large genes

描述(由申请人提供)： 虽然“平均”人类基因包含的内含子序列大约是外显子序列的十倍，但由于存在较大的内含子，许多基因的长度要大得多。在这个大小范围的极端末端是大于1000kb的基因，它们编码几kb的mRNAs。这些基因含有长度经常超过100kb的内含子。两个这种类型的基因，FHIT和WWOX，被怀疑是肿瘤抑制基因，它们的大内含子中也包含常见的染色体脆性部位。虽然肿瘤经常表现出这些基因的异常形式的表达，但产生这些基因的机制尚不清楚。事实上，尽管这些大基因对转录和RNA加工的保真度提出了极大的挑战，但人们对它们的表达总体上还没有很好的了解。本申请为这些基因的表达提出了一个不连续的转录模型。该模型的两个变体被提出，它们要么调用RNA聚合酶跳过的机制，其中大部分内含子不被转录，要么调用来自多个内部启动子的多个转录本的反式剪接。提出了一些实验，这些实验将解决这些基因在表达它们的细胞间期核中的组织问题。首先，将测量在细胞分裂中合成如此大的基因所需的时间。初步数据显示，这一时间明显短于预期。其次，将使用RT-PCR来确定实际转录的大基因的区域。第三，外显子和内含子区域的RNA聚合酶的密度将通过染色质免疫沉淀实验来确定。最后，将使用荧光原位杂交以及最近发展的两种方法-RNA陷阱和染色体构象捕获-来测量间期核内外显子和内含子之间的物理距离。

DESCRIPTION (provided by applicant): While the "average" human gene contains about ten times more intron sequence than exon sequence, there are a number of genes that are considerably larger due to the presence of large introns. At the extreme end of this size range are genes of greater than 1,000 kb which encode mRNAs of a few kb. These genes contain introns which often exceed 100 kb in length. Two genes of this type, FHIT and WWOX, are suspected tumor suppressor genes which also contain common chromosomal fragile sites within their large introns. While tumors frequently show expression of aberrant forms of these genes, the mechanism by which these are produced is unclear. Indeed, the expression of such large genes in general is not well understood although they present an extreme challenge to the fidelity of transcription and RNA processing. This application proposes a discontinuous transcription model for the expression of such genes. Two variants of this model are presented which invoke either a mechanism of RNA potymerase skipping, in which large regions of the introns are not transcribed, or trans splicing of multiple transcripts arising from multiple internal promoters. Experiments are proposed which will address the organization of such genes in the interphase nuclei of cells in which they are expressed. First, the time required to synthesize such large genes in dividing cells will be measured. Preliminary data suggests that this time is significantly shorter than expected. Second, the regions of large genes that are actually transcribed will be determined using RT-PCR. Third, the density of RNA polymerases within exonic and intronic regions will be determined using chromatin immunoprecipitation experiments. Finally, the physical distance between exons and introns within interphase nuclei will be measured using fluorescence in situ hybridization as well as two recently developed methods known as RNA-TRAP and chromosome conformation capture.