Control of mRNA fate by mRNP acetylation

通过 mRNA 乙酰化控制 mRNA 的命运

• 批准号: 313024148
• 负责人: Professor Dr.-Ing. Uwe Ohler
• 金额: --
• 依托单位: Institut für Biologie
• 依托单位国家: 德国
• 项目类别: Priority Programmes
• 财政年份: 2016
• 资助国家: 德国
• 起止时间: 2015-12-31 至 2021-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/313024148?language=en
• 关键词: Control; mRNA; fate; mRNP; acetylation

Acetylation of histones and transcription-associated proteins is known to exert a pervasive effect on epigenetic and transcriptional regulation of gene expression. Within the first SPP period, we discovered that the histone acetyltransferases p300/CBP and histone deacetylases HDAC1&2 also have profound effects at the posttranscriptional level by exerting widespread control over poly(A) RNA stability. The regulatory switch is based on acetylation of the CCR4-CAF1-NOT deadenlyase complex, and it appears to promote a dynamic mode of gene expression where active transcription is coupled to rapid mRNA degradation. In the following, we identified RNA-binding proteins (RBPs) that show altered association with poly(A) RNA upon HDAC inhibition, and pursued CPEB4 as candidate RBP that we found to be regulated by acetylation. In parallel, we also advanced computational methods for the identification RBP target sites from crosslinking and immunoprecipitation (CLIP) data, and we developed tools for accurate assignment of open reading frames based on ribosome footprinting (Ribo-Seq).Within the second funding period of the SPP 1935, we want to extend the successful collaboration between the Ohler and Stoecklin labs, which allows us to combine our expertise in RNA biochemistry, genomics and computational biology. Our goal is to obtain a molecular understanding of mechanisms underlying acetylation-induced mRNA turnover and changes in RBP function, concurrent with next generation algorithms for accurate quantification of alterations in mRNA translation. To this end, we have generated essential tools such as HeLa cell lines with endogenously tagged NOT1 and CPEB4, and demonstrated successful CLIP-seq of CPEB4. We want to pursue three specific aims: 1) To functionally characterize novel components of the CCR4-CAF1-NOT complex, based on our results on quantitative purification of the complex through endogenously tagged NOT1. 2) To elucidate CPEB4 binding specificity and regulation by acetylation, based on our poly(A) RNA capture analysis that allowed us to identify RBPs controlled by acetylation. 3) To develop computational approaches for the quantification of translation changes, which will be essential for characterizing acetylation-induced alterations in protein synthesis and CPEB4 function. Taken together, the proposed research will advance our understanding of the mechanistic principles by which acetylation controls gene expression at the posttranscriptional level, and provide the community with improved computational tools for translatome analysis.

已知组蛋白和转录相关蛋白的乙酰化对基因表达的表观遗传和转录调控具有普遍影响。在第一个SPP期间，我们发现组蛋白乙酰转移酶p300/CBP和组蛋白去乙酰化酶hdac1和2也通过广泛控制poly(A) RNA的稳定性在转录后水平上产生深远的影响。调节开关是基于CCR4-CAF1-NOT deadenlyase复合物的乙酰化，它似乎促进了基因表达的动态模式，其中活性转录与mRNA的快速降解相结合。在接下来的研究中，我们确定了RNA结合蛋白（RBP）在HDAC抑制下与poly(A) RNA的关联发生改变，并将CPEB4作为候选RBP，我们发现CPEB4受乙酰化调节。同时，我们还开发了从交联和免疫沉淀（CLIP）数据中识别RBP靶点的计算方法，并开发了基于核糖体足迹（Ribo-Seq）准确分配开放阅读框的工具。在SPP 1935的第二个资助期内，我们希望扩展Ohler和Stoecklin实验室之间的成功合作，这使我们能够结合我们在RNA生物化学，基因组学和计算生物学方面的专业知识。我们的目标是获得对乙酰化诱导的mRNA转换和RBP功能变化的分子机制的理解，同时使用下一代算法精确量化mRNA翻译的变化。为此，我们制作了必要的工具，如内源性标记NOT1和CPEB4的HeLa细胞系，并成功展示了CPEB4的CLIP-seq。我们希望实现三个具体目标：1)基于我们通过内源性标记的NOT1对CCR4-CAF1-NOT复合物进行定量纯化的结果，对CCR4-CAF1-NOT复合物的新组分进行功能表征。2)为了阐明CPEB4的结合特异性和乙酰化调控，基于我们的poly(A) RNA捕获分析，使我们能够识别由乙酰化控制的rbp。3)开发量化翻译变化的计算方法，这对于表征乙酰化诱导的蛋白质合成和CPEB4功能的改变至关重要。综上所述，该研究将促进我们对乙酰化在转录后水平控制基因表达的机制原理的理解，并为翻译组分析提供改进的计算工具。