Regulation and Function of Alternative mRNA Isoform Expression in Mammals

哺乳动物中替代 mRNA 同工型表达的调节和功能

• 批准号: 10540339
• 负责人: CHRISTOPHER B BURGE
• 金额: $ 52.87万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-01-06 至 2024-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10540339
• 关键词: 5' Splice Site; 5' Untranslated Regions; Abbreviations; Affect; Animals; Antisense Oligonucleotides; Bar Codes; Biological Assay; Cells; Complex; Conserved Sequence; DNA; Data; Dependence; Detection; Development; Elements; Enhancers; Exons; Fluorescence; Gene Expression; Gene Expression Regulation; Genes; Genetic Transcription; Genome; Genomics; Human; Human Genome; Introns; Learning; Libraries; Location; Logic; Mammals; Measurement; Mediating; Messenger RNA; Methods; Plants; Property; Protein Isoforms; Proteins; RNA; RNA Decay; RNA Processing; RNA Splicing; RNA-Binding Proteins; Regulation; Reporter; Reporting; Research; Ribonucleoproteins; Structure; System; Technology; Testing; Therapeutic; Thiouridine; Transcript; Transcription Initiation Site; Transcriptional Activation; Translations; Tumor Suppressor Proteins; Untranslated RNA; Variant; Work; design; disease phenotype; fungus; genomic locus; improved; insight; mRNA Precursor; mammalian genome; novel; novel strategies; promoter; recombinase-mediated cassette exchange

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.

总结 大多数人类基因含有内含子，内含子的存在通常会增加宿主基因的表达， 内含子介导的增强（intron mediated enhancement，IME）IME在不同的基因中被观察到， 动物、植物和真菌，并且在内含子之间经常在幅度上变化。然而，人们对如何做到这一点知之甚少。 内含子影响表达或什么样的内含子特征调节IME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激活起始（EMATS）的现象，其中 内部外显子影响所用启动子的谱和基因的表达水平。EMATS在一个 距离可达几kb，可以改变基因表达至少几倍，并且在 某些启动子-特别是固有弱启动子。详细的顺序要求和方式 EMATS的行动尚不清楚。该提案旨在了解管理IME的规则， EMATS，以改善基因表达的预测，并使方法能够调节基因表达， 改变剪接。它是围绕以下目标组织的。 SA 1.确定内含子介导的增强的序列依赖性。 SA 2.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插入内含子的不同随机序列的文库， 双荧光报告系统，染色体整合到人类细胞。该设计将使 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， 这些数据将用于鉴定增强或沉默剪接依赖性表达的基序， 方式从intronic位置。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 启动子最后，我们将使用有关IME和EMATS的信息来改进基因预测。 从一级序列表达。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 因为剪接依赖性表达激活剂或沉默剂可用于改善表达预测 从基因组序列，并可以使检测内含子变异，改变表达。理解 剪接如何影响表达也可能使基因表达调节的新方法成为可能。