Control of mRNA fate by mRNP acetylation

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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.