Investigations into the role of RNA modification in the function of the c-Myc IRES

During patho-physiological conditions, which lead to cell stress, there is a rapid and large reduction in protein synthesis rates. This is mediated, in part, by a decrease in cap-dependent initiation. In several cases, it has been shown that cap independent pathways are used for the translation of mRNAs required as part of the cell stress response/permit cell survival, and internal ribosome entry segments (IRES) have been shown to recruit the mRNAs to ribosomes directly in these conditions. It is known that the majority of cellular IRESs, unlike viral IRES, require a “nuclear event” before they are active the cytoplasm and recent data have shown that m6A methylation alone, which occurs in the nucleus, is sufficient to promote internal ribosome entry. Therefore, a testable hypothesised is that the “nuclear” event required for IRES function is RNA modification. Prior data from a yeast 3 Hybrid screen identified a novel S-adenosyl-L-methionine dependent methyltransferase Fibrillarin-Like-1 (FBLL1) as binding to the IRES of c-Myc. This protein, largely unstudied, shares homology with Fibrillarin; a well characterised 2’O-methyltransferase ubiquitously expressed and located in the nucleolus, consistent with its role in ribosomal RNA modification and, ribosomal biogenesis. Fibrillarin adds a methyl group to the 2' position of the ribose sugar of an RNA molecule. This modification can affect RNA stability, function and interactions with proteins and small molecules. Such interactions can either stabilize or destabilize the RNA. The c-Myc IRES contains a highly structured RNA sequence which is essential for its role in cap-independent translation. Therefore, this novel 2’O-methylatransferase, FBLL1, may be essential for regulating its complex structure. This thesis investigates the role of RNA modification, by FBLL1 and additional methyltransferases, on the activation of the c-Myc IRES. Recombinant FBLL1 was produced to investigate the direct effect of FBLL1 on the c-Myc IRES in vitro. In addition, proteins that "read" and "erase" such RNA modifications were also assessed for their impact on the expression of c-Myc protein, cell growth and global protein synthesis rates. Bicistronic reporter vectors were also used to specifically observe the effects of these altered methylation effects on a range of IRESs function including c-Myc, Bag1, MNT, Apaf-1, HCV and EMCV.

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

• Goodacre, Alexander

Advisor dc:contributor.advisor

• Willis, Anne

• 2'O methylation
• c-Myc
• FBLL1
• IRES
• m6A methylation
• Methylation
• Nm methylation

Licence

• https://www.repository.cam.ac.uk/bitstreams/bb5b55d2-8a23-4464-a66b-db25df56ac79/download
• https://www.rioxx.net/licenses/all-rights-reserved/

Language dc:language

eng

DOI dc:identifier.doi

https://doi.org/10.17863/CAM.107483

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/366626