Characterization of complexes required for mammalian transcriptional and post transcriptional gene regulation
EMBARGOED UNTIL 2027-05-17
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Date
2024
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Doctoral Thesis
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EMBARGOED UNTIL 2027-05-17
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Abstract
Integrator (INT) is a large protein complex in metazoans, containing at least 16 subunits with a combined molecular weight of more than 1.5 MDa. This modular complex harbors dual enzymatic functionality with a cleavage module that has RNA endonuclease activity, and a phosphatase module with phosphatase activity. INT was initially identified as the 3’-end processing machinery for uridine-rich small nuclear RNAs (UsnRNAs). Later, it was found to be involved in the biogenesis of other noncoding RNAs, such as enhancer RNAs, long non-coding RNAs, and the precursor of micro RNAs etc. More recent studies show that INT plays a role in the premature termination of messenger RNA transcription at promoter-proximal pausing sites. INT recognizes the paused RNA polymerase II (RNAPII) that is bound by negative elongation factors NELF and DSIF, which stall RNAPII, then INT cleaves the nascent RNA and its phosphatase module counteracts RNAPII elongation. INT’s function is crucial in all tissue types and developmental stages in metazoans. Disruption of INT subunits or mutations will lead to various genetic diseases with related neuropathological phenotypes. Until now, many studies were focused on INT’s function and mechanism. In this thesis, we present our new understanding of the INT cleavage module’s biogenesis. In addition, we also explore the reconstitution of INT with paused RNAPII. Furthermore, we present our approach to identify additional binding proteins during UsnRNA 3‘-end processing.
Apart from INT biology, this thesis also presents our studies of three newly identified factors, FOCAD, AVEN, and HBS1LV3, which are interactors of the RNA exosome associated superkiller (SKI)-complex. The exosome and its cofactors play important roles in co-translational mRNA surveillance pathways during which defective transcripts are degraded to safeguard cells from aberrant transcripts. The three factors, FOCAD, AVEN, and HBS1LV3 were found to bind the SKI complex directly. In this thesis, we present our studies in understanding the architecture and functions of FOCAD, AVEN, HBS1LV3, and their influence on the SKI complex.
Chapters 3, 5, and 6 focus on INT biology. BRAT1 and WDR73 have been identified as interactors of INTS9 and INTS11 subunits of INT. Mutations and depletion of BRAT1 and WDR73 cause misregulation of INT’s function. In Chapter 3, I resolved the cryo-electron microscopy (cryo-EM) structures of INTS9-INTS11-BRAT1-WDR73, INTS9-11-BRAT1, and INTS4-9-11-BRAT1, elucidating how BRAT1 and WDR73 associate with INTS9-11 in the cytoplasm, how INTS4 associates with INT9-11- BRAT1 in the nucleus as an intermediate of cleavage module maturation and how WDR73 and BRAT1 play a role in controlling the endonuclease activity of INTS11. Furthermore, we elucidated the stepwise assembly mechanism of the catalytic module and determined why inositol hexaphosphate is crucial for module assembly by using biochemical approaches. Chapter 5 shows our approach to obtain purified RNAPII to characterize the structure of INT with paused RNAPII. I optimized the purification of RNAPII and carried out parts of the EM data analysis. Chapter 6 shows our approach to find 3’-box binding factors that might assist INT in sequence specific UsnRNA processing. I identified 17 potential proteins and further analyzed their knockdown effect on 3’-end processing.
Chapter 4 focuses on the structural analysis of the SKI-complex cofactors FOCAD, AVEN, HBS1LV3. I resolved the cryo-EM structures of FOCAD, the FOCAD-AVEN complex and the HBS1LV3-SKI complex. These structures show, how HBS1LV3 associates with the SKIC3 subunits of the mammalian SKI complex, that FOCAD forms a dimer and how AVEN binds FOCAD. Furthermore, I analyzed the existence of FOCAD and AVEN in other species based on a sequence and predicted structural similarities.
In summary, the findings in this thesis contribute to the understanding of the INT biogenesis, the mechanism of INT during the 3’-end processing of UsnRNAs, and the molecular basis of SKI complex function.
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Examiner : Jonas, Stefanie
Examiner : Ban, Nenad
Examiner : Jinek, Martin
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ETH Zurich
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09609 - Jonas, Stefanie / Jonas, Stefanie