Regulation and Function of Alternative mRNA Isoform Expression in Mammals

摘要 大多数人类基因都含有内含子，而内含子的存在往往会增加宿主基因的表达 这种现象称为内含子介导的增强(IME)。在不同的基因中观察到了IME 动物、植物和真菌，通常在不同的内含子上大小不同。然而，人们对此知之甚少。 内含子影响表达或什么内含子特征调节输入法活性。最近，我们描述了一部小说 我们称之为外显子介导的转录启动激活(EMATS)的现象，在这种现象中， 内部外显子影响启动子的使用范围和基因的表达水平。EMATS在 最大几kb的距离，可以改变基因表达至少几倍，并在 某些推动者--尤其是本质上较弱的推动者。详细的顺序要求和模式 EMAT的行动尚不清楚。这项提议旨在了解管理IME和 EMATS，以改进基因表达的预测，并使通过以下方式调节基因表达的方法成为可能 更改拼接。它围绕以下目标进行组织。 SA1.确定内含子介导的增强的序列依赖性。 SA2.探索EMATS监管的范围和规则。 在目标1中，我们将生成一个包含数千个不同随机序列的文库，这些序列插入到 染色体整合到人类细胞中的双荧光报告系统。这一设计将使 高通量测量每个内含子对新生RNA、成熟RNA和蛋白质水平的影响， 这些数据将被用来识别增强或沉默剪接依赖表达的基序 来自内含子位置的方式。在目标2中，我们将系统地推导和测试EMATS如何 调控取决于内部外显子的位置和序列以及所涉及的属性 推动者。最后，我们将使用所学到的关于输入法和EMATS的信息来改进基因预测 来自主序列的表达。总之，这些目标中描述的研究将建立规则 控制剪接如何影响哺乳动物基因组中的基因表达。识别起作用的基序 作为剪接依赖的表达激活剂或沉默剂可用于提高表达的预测 并且可以检测改变表达的内含子变异体。理解 剪接如何影响表达也可能使基因表达调控的新方法成为可能。

Summary Most human genes contain introns, and presence of introns often increases the expression of the host gene, a phenomenon known as intron-mediated enhancement (IME). IME has been observed in diverse genes in animals, plants and fungi and often varies in magnitude across introns. However, little is known about how introns impact expression or what intron features modulate IME activity. Recently, we have described a novel phenomenon that we call exon-mediated activation of transcription starts (EMATS), in which the splicing of internal exons impacts the spectrum of promoters used and expression level of the gene. EMATS acts at a distance of up to a few kb, can alter gene expression by at least severalfold, and appears more active at certain promoters – especially intrinsically weak promoters. The detailed sequence requirements and mode of action of EMATS are not yet known. This proposal is seeks to understand the rules that govern IME and EMATS, to improve the prediction of gene expression and to enable methods to modulate gene expression by altering splicing. It is organized around the following aims. SA1. Determine the sequence dependence of intron-mediated enhancement. SA2. Explore the scope and rules for EMATS regulation. In Aim 1, we will generate a library of many thousands of distinct random sequences inserted into an intron in a dual fluorescent reporter system that is chromosomally integrated into human cells. This design will enable high-throughput measurement of the effects of each intron on nascent RNA, mature RNA and protein levels, and these data will be used to identify motifs that enhance or silence expression in a splicing-dependent manner from an intronic location. In aim 2, we will systematically derive and test rules for how EMATS regulation depends on the location and sequence of the internal exon and on properties of the involved promoter. Finally, we will use the information learned about IME and EMATS to improve predictions of gene expression from primary sequence. Together, the research described in these aims will establish rules governing how splicing impacts gene expression in mammalian genomes. Identification of motifs that function as splicing-dependent activators or silencers of expression can be used to improve prediction of expression from genome sequence and may enable detection of intronic variants that alter expression. Understanding how splicing impacts expression may also enable new approaches for gene expression modulation.