A STRUCTURAL AND FUNCTIONAL ATLAS OF THE HUMAN RIXOSOME COMPLEX

The rixosome complex is a conserved nuclear protein complex responsible for many essential enzymatic functions during 60S ribosomal subunit synthesis. This complex contains seven protein members including the non-enzymatic PELP1, WDR18, TEX10 proteins along with enzymatic members including LAS1L (endoribonuclease), NOL9 (RNA kinase), SENP3 (SUMO protease), and MDN1 (AAA+-ATPase motor). The rixosome also has conserved RNA processing functions at heterochromatin, effectively triggering polycomb gene silencing. It has become increasingly clear that the seven protein members of the rixosome stably associate with one another as a large >1 mDa mega-complex. The molecular details of how the rixosomal protein factors assemble in the cell is unknown. It is also not clear how the enzymes retained within this complex function. I hypothesize that the structural assembly of the rixosome plays key roles in regulating how the associated enzymes function, both in ribosome biogenesis and heterochromatin maintenance. I also suspect a degree of enzymatic coordination to exist within this complex, especially as its functions relate to the multi-step process of ribosome synthesis. I hypothesize that the molecular architecture of the rixosome holds the key to this coordination. In this study, I aim to elucidate the structural architecture of the human rixosome complex and investigate how this architecture influences rixosomal enzyme function in the cell. A primary focus will be given to the rixosome’s conserved role in 60S subunit synthesis. I have employed a combination of techniques in structural biology, biochemistry, and molecular biology to pursue this aim. Three formal results chapters will present data on the human rixosome formation and function including, 1) the cryo-EM structure of the rixosome scaffolding core and insights on PELP1’s nuclear functions; 2) the reconstitution and structural mapping of the entire rixosome complex architecture with novel functional data on the rixosomal deSUMOylation enzyme SENP3; 3) the cryo-EM structure of the rixosomal RNA processing module RNase PNK and the characterization of a rixosome binding domain that mediates the module’s integration. In addition to the data in these formal results chapters, select data from additional experiments are presented as appendices and discussed in the final discussion chapter. I intend for this work to represent a visualized structural atlas of the human rixosome complex that allows for a better understanding of this important molecular machine. Importantly, I also present how this work reveals many new questions for future study on the rixosome and its functions in the cell.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2024

Author dc:creator

• Gordon, Jacob

Advisor dc:contributor.advisor

• Warren, Alan

• Ribosome assembly
• RNA processing
• Rixosome complex
• SUMOylation
• SENP3 SUMO protease
• PELP1
• RNase PNK (LAS1L-NOL9)
• Structural Biology
• Cryo-electron microscopy
• Biochemistry

Licence

• https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/c4ec8e46-5c04-439c-9a05-620b08411fb4/download
• https://www.rioxx.net/licenses/all-rights-reserved/

Language dc:language

eng

DOI dc:identifier.doi

https://doi.org/10.17863/CAM.113884

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/376803