title: Studies on the role of enhancer RNAs in the release of RNA polymerase II from promoter-proximal pausing
Creators: name: Gorbovytska, Vladyslava
Publisher: name: University of Bayreuth
Year: 2021
Subjects: subject: Enhancer RNAs
subject: eRNAs
subject: promoter-proximal pausing
subject: Pol II
subject: gene regulation
subject: non-coding RNAs
subject: 500

description: Enhancers are cis-regulatory elements on genomic DNA that bind activator proteins and facilitate transcription of target genes. About ten years ago, active enhancers were found to be transcribed, giving rise to long non-coding RNAs, termed enhancer RNAs (eRNAs). Enhancer transcription correlates with enhancer activation, hence further elevating the transcription rates of target genes. This finding revolutionized our view of enhancers, which now are seen to exploit several mechanisms to promote target gene transcription. The eRNA transcripts themselves were thereby reported to employ various mechanisms to activate target gene transcription, e.g., in neurons eRNAs were suggested to facilitate the release of the RNA polymerase II (Pol II) from the promoter-proximally paused state. To that end, eRNAs are believed to compete off negative elongation factor (NELF) from paused Pol II at target genes, thereby facilitating its transition into productive elongation. As the molecular mechanism of eRNA function was unknown to date, this study was aimed at investigating, whether eRNAs share common secondary structures that enable them to perform their activatory function. Moreover, this work was focused on developing a biochemical in vitro framework to systematically test the impact of eRNAs on paused Pol II, and to thereby gain insights into their underlying mechanism of action. Within the scope of this thesis, I determined the precise 5'-ends of mouse neuronal eRNAs using the Exo-seq protocol and I performed chemical structure probing for a set of 39 eRNA 5'-end fragments (first 200 nucleotides), employing the SHAPE-MaP protocol. The data I obtained revealed that eRNAs adopt a broad spectrum of structures without sharing specific structural motifs. However, with the aid of biochemical in vitro assays, we were able to demonstrate that, indeed, eRNAs can trigger the dissociation of NELF from the paused Pol II complex and that they thereby facilitate transcription. Furthermore, we found that the efficiency of an eRNA to detach NELF from paused Pol II correlates with its length and with the presence of unpaired guanosines only. The overall structure of an eRNA seems to only play a minor role. Last, by using a combination of biochemical assays, RNA-protein crosslinking coupled to mass spectrometry, and experiments with NELF mutants, we rationalized the strict length-dependence of eRNA-driven NELF dissociation: eRNAs make allosteric contacts with multiple, distant regions on the NELF complex, namely the RRM domain of NELF-E and positively charged patches on the NELF-AC lobe. These multivalent interactions then likely trigger the dissociation of NELF from paused Pol II. Taken together, by revealing the molecular determinants for eRNA function, this study mechanistically links eRNAs to Pol II pause release and to activity-induced transcription in neurons. Moreover, this study sets the stage for a further detailed investigation of the interaction of eRNAs with the paused elongation complex by providing an established set of biochemical assays, that can be developed further to better represent transcription conditions in vivo.