The divergent PolII complex of Paramecium: transcription initiation, pausing and elongation without Rpb1 heptad repeats and NELF

• Applicants: Professor Dr. Axel Imhof; Professor Dr. Martin Simon
• Subject Area: General Genetics and Functional Genome Biology
• Term: since 2025
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 564092193

Transcription of translatable mRNA is a highly regulated process in which the enzymatic activity of PolII is precisely linked to the transcription unit and also to the biochemical modifications of the mRNA. Transcription factors and many different complexes coordinate the hierarchical modification of heptad repeats in the C-terminal domain of Rpb1 to allow for regulation of the transcriptional cycle consisting of initiation, promoter- proximal pausing and elongation. In addition, chromatin modifications along the transcription unit are involved in this process, on the one hand controlling PolII modification and on the other hand being under the control of PolII activity. This system has been described for a wide range of eukaryotes, from yeast to humans. Here we are analysing a very divergent transcriptional system. Our previous work on the Paramecium macronuclear epigenome revealed many chromatin features that differ from other epigenomes. These include the distribution of nucleosomes, histone modifications and PolII occupancy along genes. These features of the epigenome are accompanied by differences in the transcriptional machinery. Many PolII associated complexes lack canonical components, and Rpb1 lacks heptad repeats in the C-terminal domain, making a hierarchical phosphorylation pattern impossible. Our plan is to unravel the control of mRNA transcription in this highly divergent system and to clarify whether differential phosphorylation of this divergent Rpb1 occurs and how this relates to transcriptional activity. We will analyse the modification/phosphorylation of the serine-rich Rpb1 C-terminus by mass spectrometry and we will characterise PolII-associated complexes by Affinity purifications (AP-MS). Differential modification of Rpb1 can be analysed in knock-down lines for different components required for the transition from the pre-initiation complex to promoter-proximal pausing (TFIIH, Mediator), release from pausing (DSIF), and elongation (Spt6, Paf1). The knockdown lines will be further analysed by Pro-seq and ChIP-seq for their genomic distribution, transcriptional activity of individual genes, promoter-proximal pausing and changes in the chromatin landscape. Our molecular characterisation of this transcriptional machinery, which diverges from all studied systems, will not only contribute to our understanding of the evolution of the Rpb1-CTD, but is also likely to provide explanations for non-canonical transcription in multicellular organisms, where the described pattern of Rpb1 phosphorylation and chromatin marks does not follow the dogmatic rules.

• DFG Programme: Research Grants