Insights into the mechanisms of translation surveillance using constitutive RNA binding ASC-1 complex mutants

Upon encountering stretches of problematic or damaged mRNA, ribosomes can slow, stall, and collide. Although ribosome stalling and collisions can be caused by numerous translation stresses, from mRNA damage to amino acid starvation, collided ribosomes exist abundantly even in the absence of overt translation stress. Consequently, co-translational quality control mechanisms, such as ribosome-associated quality control (RQC), have evolved to limit the pro- teostatic damage caused by truncated nascent polypeptides associated with stalled ribosomes. During RQC, the stalled ribosome is removed from its substrate mRNA through the splitting of its component subunits by the ASC-1 complex (ASCC), enabling the ubiquitylation, extraction and degradation of the potentially toxic incomplete polypeptide. The core member of the ASCC, ASCC3, is responsible for subunit splitting through its 3’-5’ RNA helicase activity. However, the mechanistic details of ASCC3’s ribosome splitting activity and its broader role in RQC are unclear. Moreover, the functional and structural roles of other ASC-1 proteins, including ASCC1, are poorly understood. In this thesis, I have developed an inducible cell line that expresses an ASCC3 product- release trap mutant, capable of interacting with its RNA partners in the absence of exogenous translational stress. Using this cell line, I have investigated the role of ASCC3 in transla- tion surveillance under steady-state cellular conditions, including the identification of the RNA substrates of ASCC3, the functional impact of preventing the ASCC-dependent resolution of endogenously colliding ribosomes, and determination of changes in the ASCC3 protein binding interactome following ASCC3 RNA binding. Additionally, a cell line expressing ASCC1 with mutations in its putative phosphodiesterase domains has been generated, to probe the functional role of ASCC1 in ASC-1 complex ribosome splitting.

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

• Cole, Cameron

Advisors dc:contributor.advisor

• Willis, Anne
• Stoneley, Mark

• Molecular Biology
• Ribosome associated quality control
• Ribosome stalling
• Translation

Licence

• https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/f10bec3e-d8c0-4032-ad94-3cd243dba061/download
• https://creativecommons.org/licenses/by/4.0/

Language dc:language

eng

DOI dc:identifier.doi

https://doi.org/10.17863/CAM.115839

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/379868