Prof. Dr. Stoecklin
Colaboration with Prof. Dr. Uwe Ohler
PROJECT INFORMATION :
Acetylation of histones and transcription-associated proteins is known to exert a pervasive effect on epigenetic and transcriptional regulation of gene expression. We discovered that histone acetyltransferases (HATs) and histone deacetylases (HDACs) also regulate gene expression at the posttranscriptional level by controlling poly-A RNA stability and translation. Our data show that inhibition of HDAC1 and 2 induces widespread degradation of poly-A RNA in mammalian cells. Moreover, we observe a strong repression of translation by HDAC inhibitors. Acetylation-induced RNA decay depends on the HATs p300 and CBP, which acetylate the exoribonuclease CAF1a, a catalytic subunit of the CCR4-CAF1-NOT deadenlyase complex, and thereby accelerate poly-A RNA degradation. Taking adipocyte differentiation as a model, we observe global stabilization of poly-A RNA during differentiation, concomitant with loss of CBP/p300 expression. Our results demonstrate that reversible acetylation serves as a fundamental switch that controls the overall turnover of poly-A RNA. In parallel, we have also developed methods and computational tools for the transcriptome-wide identification of RNA-binding protein (RBP) target sites based on crosslinking and immunoprecipitation (CLIP), for transcriptomic analysis using nascent RNA labeling, and for the analysis of translation by ribosome footprinting.
Within SPP 1935, we now want to combine our expertise in RNA-biochemistry, molecular biology, transcriptome analysis and computational approaches to address two key questions: What is the influence of acetylation on the composition and function of messenger ribonucleoprotein (mRNP) complexes? And, does acetylation regulate specific subsets of genes/transcripts in a coordinated fashion across different steps of gene expression by simultaneously controlling transcription, processing, translation and/or mRNA decay? Specifically, we want to pursue three Aims: 1) To characterize changes in mRNP composition induced by acetylation through the analysis of candidate RBPs identified by poly-A capture, including an assessment of altered RBP target specificity by CLIP. 2) To determine acetylation-mediated alterations in mRNA turnover using nascent RNA labeling, which will also allow us to derive transcription as well as processing rates. Moreover, this Aim includes the comparison of mRNPs that are stabilized and destabilized by HDAC inhibition. 3) To explore acetylation-dependent regulation of translation on a transcriptome-wide scale using ribosome footprinting and nascent polypeptide quantification. To achieve these goals, we will also advance the computational methodology for the assignment of RBP binding sites based on CLIP as well as the analysis of ribosome footprinting data. The proposed project will allow us to obtain a comprehensive understanding of acetylation-induced alteration of mRNP composition, mRNA stability and translation.
Focus of the group
Regulation of translation, Regulation of mRNA turnover, Proteomics of mRNPs, Posttranslational modifications of RNA-binding proteins
KEY TECHNOLOGIES :
- RNA affinity purification
- Polysome profiling
- RNA decay assays
- Poly-A capture analysis
Sharma S, Poetz F, Bruer M, Ly-Hartig TBN, Schott J, Séraphin B, Stoecklin G. (2016) Acetylation-Dependent Control of Global Poly(A) RNA Degradation by CBP/p300 and HDAC1/2. Mol Cell 63:927-38.
Schott J, Reitter S, Philipp J, Haneke K, Schäfer H, Stoecklin G. (2014) Translational regulation of specific mRNAs controls feedback inhibition and survival during macrophage activation. PLoS Genet 10:e1004368.
Leppek K, Stoecklin G. (2014) An optimized streptavidin-binding RNA aptamer for purification of ribonucleoprotein complexes identifies novel ARE-binding proteins. Nucleic Acids Res 42:e13.
Leppek K, Schott J, Reitter S, Poetz F, Hammond MC, Stoecklin G. (2013) Roquin promotes constitutive mRNA decay via a conserved class of stem-loop recognition motifs. Cell 153:869-81.
Spasic M, Friedel CC, Schott J, Kreth J, Leppek K, Hofmann S, Ozgur S, Stoecklin G. (2012) Genome-wie assessment of AU-rich elements by the AREScore algorithm. PLoS Genet 8:e1002433.
INFO EXTRA :
PhD student funded by SPP 1935: