Control of mRNA fate by mRNP acetylation

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产生普遍影响。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HDAC 1和2也通过广泛控制poly（A）RNA的稳定性在转录后水平上具有深远的影响。调控开关是基于CCR 4-CAF 1-NOT deadenlyase复合物的乙酰化，并且它似乎促进基因表达的动态模式，其中活性转录与快速mRNA降解偶联。在下文中，我们鉴定了在HDAC抑制后显示与多聚（A）RNA的改变的缔合的RNA结合蛋白（RBP），并将CPEB 4作为我们发现通过乙酰化调节的候选RBP。与此同时，我们还提出了从交联和免疫沉淀（CLIP）数据中识别RBP靶位点的计算方法，并开发了基于核糖体足迹的开放阅读框架的精确分配工具在SPP 1935的第二个资助期内，我们希望扩大Ohler和Stoecklin实验室之间的成功合作，这使我们能够联合收割机结合我们在RNA生物化学，基因组学和计算生物学方面的专业知识。我们的目标是获得乙酰化诱导的mRNA周转和RBP功能变化的机制的分子理解，同时与下一代算法准确定量mRNA翻译的改变。为此，我们已经产生了必要的工具，例如具有内源性标记的NOT 1和CPEB 4的HeLa细胞系，并证明了CPEB 4的成功CLIP-seq。我们想要追求三个具体目标：1）基于我们通过内源性标记的NOT 1定量纯化复合物的结果，功能性地表征CCR 4-CAF 1-NOT复合物的新组分。2)为了阐明CPEB 4结合特异性和乙酰化的调节，基于我们的poly（A）RNA捕获分析，该分析使我们能够鉴定由乙酰化控制的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CPEB 4功能改变至关重要。两者合计，拟议的研究将推进我们的机械原理，乙酰化控制基因表达在转录后水平的理解，并提供社会与改进的计算工具translatome分析。

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.