Structural and biochemical studies on co-transcriptional endonuclease reactions
by Alexander Helmut Rotsch
Date of Examination:2024-08-26
Date of issue:2025-03-25
Advisor:Prof. Dr. Patrick Cramer
Referee:Prof. Dr. Patrick Cramer
Referee:Prof. Dr. Hauke Hillen
Persistent Address:
http://dx.doi.org/10.53846/goediss-11073
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Abstract
English
Influenza, an acute respiratory disease, causes 290,000 to 650,000 human deaths annually (Iuliano et al., 2018; WHO, 2023). During an influenza infection, the virus ensures proper handling of viral mRNA, including nuclear export and translation, by mimicking host mRNAs (Ramanathan et al., 2016). However, the influenza virus genome does not encode for a capping enzyme (Plotch et al., 1978). Instead, the virus uses its dedicated RNA-dependent RNA polymerase (FluPol) to acquire 5'-capped RNA sequences from host mRNAs through a process called cap-snatching (Krischuns et al., 2021; Te Velthuis and Fodor, 2016). FluPol requires actively transcribing host RNA polymerase II (Pol II) as the source of the co-transcriptionally synthesized 5'-cap structure and directly interacts with Pol II during cap-snatching (Engelhardt et al., 2005; Garg et al., 2023; Lukarska et al., 2017; Mahy et al., 1972). Despite its central importance for viral transcription, the molecular mechanism of cap-snatching remains elusive. However, human cells can protect themselves against viruses using the micro-RNA system (miRNA), which targets and degrades viral RNA. Furthermore, the cells can also use this miRNA system to regulate the protein production of a gene after transcription (O’Brien et al., 2018). Alterations in miRNA expression profiles have been found in several pathologies, including cancer, HIV, and neuropsychiatric disorders (Geaghan and Cairns, 2015; Maffioletti et al., 2014; Palmero et al., 2011; Swaminathan et al., 2014), suggesting that dysregulation of miRNA synthesis may play a role in these diseases. Therefore, understanding the mechanism behind miRNA synthesis regulation has great potential for therapeutic approaches. The microprocessor complex (MPC), containing the endonuclease DROSHA, is the first processing enzyme within the miRNA biogenesis pathway (Kwon et al., 2016; Lee et al., 2003; Partin et al., 2020). Multiple studies have shown that the MPC cleaves the RNA co-transcriptional (Ballarino et al., 2009; Morlando et al., 2008; Nojima et al., 2015), which could regulate the MPC. However, the molecular mechanism remains to be determined. To elucidate the process of co-transcriptional cap-snatching, I established a set of in vitro experiments to study recruitment of FluPol to Pol II and to investigate FluPol’s endonuclease activity. I could demonstrate that FluPol recognizes the elongating Pol II as a substrate for cap-snatching. I demonstrated that the phosphorylation of the C-terminal domain (CTD) of Pol II largest subunit, RPB1, recruits FluPol to the nascent transcript. However, this phosphorylated CTD recruitment is insufficient for endonuclease cleavage activation. Only when I add the elongation factor DRB-sensitivity inducing factor (DSIF) to the elongation complex is the endonuclease of FluPol stimulated. Based on this biochemistry, I could solve two cryogenic electron microscopy (cryo-EM) structures of FluPol in a complex with the elongating Pol II, containing a phosphorylated CTD and the elongation factor DSIF. The structures visualize FluPol bound to elongating Pol II before and after RNA cleavage. I observed direct interactions between FluPol, Pol II, and DSIF. Furthermore, we showed that these interactions are important for viral transcription in cells. Based on the obtained structures, the target for cap-snatching is either an early elongation or a paused Pol II. Furthermore, we could show that this mechanism of substrate recognition is conserved between influenza A and B viruses. This study unravels new potential targets for future antiviral therapeutic approaches. Furthermore, I provide biochemical evidence that the activated elongation complex is the preferred substrate for the MPC. Using single-particle cryo-EM, I could structurally characterize the microprocessor complex engaged with the activated elongation complex. This structure revealed that SPT6 and LEO1, components of the activated elongation complex, may serve as potential interfaces between the MPC and the activated elongation complex. These experiments provide evidence for the first time that transcription elongation factors stimulate RNA processing in vitro. Furthermore, the presence of the transcription-stimulating factor RTF1 appears to be mutually exclusive with the MPC on the same Pol II molecule, suggesting a potential regulatory mechanism. A structural overlay with the co-transcriptional splicing complex comprising the U1 snRNP hints toward sequential miRNA biogenesis and splicing process. In summary, these results greatly enhance our understanding of RNA processing. Substrate specificity for RNA processing is not only provided by RNA features but additionally by the transcription machinery. By studying processes from human to viral origin, I provided evidence that this mechanism of substrate recognition, where the transcription machinery contributes to the specificity of RNA processing, is strongly conserved throughout evolution. Furthermore, this conceptual advancement could serve as a foundation for future studies on RNA processing and the development of novel therapeutic strategies targeting co-transcriptional RNA processing in various diseases, such as cancer and viral infections.
Keywords: Influenza virus; Cap-snatching; Cryo-electron microscopy; Transcription; miRNA; DROSHA
