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.

概括 大多数人类基因都包含内含子，并且内含子的存在通常会增加宿主基因的表达，A 现象称为内含子介导的增强（IME）。在潜水基因中观察到IME 动物，植物和真菌，在介绍中通常会有所不同。但是，关于如何 内含子影响表达或哪些内含子具有调节IME活性。最近，我们描述了一本小说 我们称为外显子介导的转录激活的现象（emats），其中剪接的剪接 内外外显子会影响所用启动子的光谱和基因的表达水平。 emats在一个 多达几kb的距离可以将基因表达至少几倍改变，并且在 某些启动子 - 尤其是本质上弱的启动子。详细的序列要求和模式 emats的作用尚不清楚。该提议旨在了解管理IME的规则和 emats，改善基因表达的预测并启用方法来调节基因表达 更改剪接。它是围绕以下目标组织的。 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AIM 1中，我们将生成一个库的库，这些库插入了一个内含子 染色体整合到人类细胞中的双荧光报告系统。这个设计将启用 每个内含子对新生RNA，成熟RNA和蛋白质水平的影响的高通量测量， 这些数据将用于识别在剪接依赖性中增强或沉默表达的基序 来自内含子位置的方式。在AIM 2中，我们将系统地得出并测试规则 调节取决于内部外显子的位置和序列以及所涉及的特性 发起人。最后，我们将使用有关IME和EMAT的信息来改善基因的预测 从一级序列表达。这些目标中描述的研究将共同​​建立规则 管理剪接如何影响哺乳动物基因组中的基因表达。识别该功能的主题 因为依赖于剪接的激活剂或表达硅可以用来改善表达的预测 从基因组序列，可以检测改变表达的内含子变体。理解 剪接如何影响表达也可能使基因表达调制的新方法。