Mechanisms of dynein-based mRNA transport processes in human cells

Protein function in cells is intricately regulated in both space and time through the positioning of mRNA molecules in the cytoplasm. This process is commonly mediated by molecular motors, which transport mRNAs along the cytoskeleton to specific cellular regions. Despite the widespread occurrence of mRNA transport, the precise mechanisms by which transcripts are recognised and recruited to motor complexes in mammals are not understood. This thesis investigates how the major minus-end-directed microtubule motor, dynein, traffics mRNAs in human cells. A co-immunoprecipitation-based proteomics approach was first employed to identify candidate adaptors between dynein and mRNA in HeLa cells. Analysis of the interactomes of dynein and its co-factor BICD2 revealed that the RNA binding proteins FXR1 and FXR2 associate with the transport machinery independently of RNA, and that they link dynein-BICD2 to components of ribonucleoprotein particles (RNPs), including ribosomal proteins. Ribosomal proteins were shown to interact with dynein and BICD2 in a nascent chain-dependent manner, supporting the existence of a co-translational mRNA transport mechanism. Protein-protein interaction studies with purified factors further showed that FXR1 and FXR2 directly bind to both dynein and BICD2. This result supports a novel mode of cargo recognition by human dynein that involves interactions with multiple components of the transport machinery. Moreover, analysis of protein truncations demonstrated that the interaction of FXR1 and FXR2 with BICD2 occurs through the C-terminal domain of the protein but not through a previously reported cargo binding site within this domain. These findings strongly implicate FXR1 and FXR2 as dynein-mRNA adaptors and challenge the current understanding of modes of cargo linkage to the motor. As FXR1 and FXR2 are highly enriched in stress granules – large RNPs that sequester mRNAs when cells receive an insult – this thesis also explores the role of the dynein-BICD2 complex in the assembly of these structures. For this purpose, biochemical assays, as well as fixed and live cell imaging approaches were employed. The findings provide evidence that dynein, BICD2 and FXR1 and FXR2 all contribute to stress granule formation, thereby providing molecular insights into how microtubule-based transport regulates the assembly of these structures. As stress granules have been implicated in neurodegenerative diseases, cancer and viral infections, further exploration of motor-driven assembly of these structures may open potential therapeutic avenues. Together, this research leads to a working model that describes a co-translational connection between microtubules, dynein and RNPs in human cells, both in basal and stressed conditions.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2023

Author dc:creator

• Andres Jeske, Yaiza

Advisor dc:contributor.advisor

• Bullock, Simon

• Cellular Stress
• Dynein
• Microtubules
• Molecular motors
• mRNA transport
• RNA
• RNA granules
• Stress granules

Licence

• https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/03f698d8-5716-47bc-80e7-97b27002a6a3/download
• https://www.rioxx.net/licenses/all-rights-reserved/

Language dc:language

eng

DOI dc:identifier.doi

https://doi.org/10.17863/CAM.102088

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/358433