Prof. Dr. Ohler
Colaboration with Prof. Dr. Georg Stoecklin
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.
KEY TECHNOLOGIES :
- Algorithms for deep sequence data analysis
- Machine learning approaches to model gene regulatory mechanisms
- Genomics protocols including RNA-seq, metabolic labelling of RNA, CLIP, ribo-seq
Wessels HH, Imami K, Baltz AG, Kolinski M, Beldovskaya A, Selbach M, Small S, Ohler U*, Landthaler M*. The mRNA-bound proteome of the early fly embryo. Genome Res 26:1000-1009, 2016.
Calviello L, Mukherjee N, Wyler E, Zauber H, Hirsekorn A, Selbach M, Landthaler M, Obermayer B, Ohler U. Detecting actively translated open reading frames in ribosome profiling data. Nat Methods 13:165-170, 2016.
Mukherjee N, Jacobs NC, Hafner M, Kennington EA, Nusbaum JD, Tuschl T, Blackshear PJ, Ohler U. Global target mRNA specification and regulation by the RNA-binding protein ZFP36. Genome Biol 15:R12, 2014.
Ascano M Jr, Mukherjee N, Bandaru P, Miller JB, Nusbaum JD, Corcoran DL, Langlois C, Munschauer M, Dewell S, Hafner M, Williams Z, Ohler U*, Tuschl T*. FMRP targets distinct mRNA sequence elements to regulate protein expression. Nature 492:382-386, 2012.
Corcoran DL, Georgiev S, Mukherjee N, Gottwein E, Skalsky RL, Keene JD, Ohler U. PARalyzer: definition of RNA binding sites from PAR-CLIP short-read sequence data. Genome Biol 12:R79, 2011.