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Characterisation of RNA modifications in human cancer cells



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Wing, Daniel 


Chemical modifications have been studied as key modulators of RNA biology for over 50 years. There are now 171 RNA modifications that have been discovered throughout the three kingdoms of life1. These chemical changes overlay the standard RNA information and have been linked to a wide range of biological processes and human diseases, such as cancer. However, the vast majority of these modifications are uncharacterised, defined on limited substrates and are subject to enzymatic pathways. This shortfall is partly due to lack of reagents and technologies to study these modifications. In an effort to create better tools and reagents, I performed an in-depth characterisation of a group of new anti-RNA modification antibodies generated in collaboration with Abcam plc. Using this platform, I validated a new anti-m1G antibody and utilised this novel reagent to help uncover new roles for m1G and the enzymes that catalyse its deposition. Using pancreatic ductal adenocarcinoma cancer (PDAC) cells as a model system, I discovered that m1G methylation is present on mRNA where it is catalysed by TRMT5, an enzyme previously identified as a tRNA methyltransferase (TRMT). Utilising ribosome profiling, I found that loss of TRMT5 greatly affects global protein translation. Moreover, depletion of TRMT5 from the PDAC cell line, PANC-1, inhibited proliferation and migration during in vitro assays. In contrast, TRMT5 depletion from a non-cancerous pancreatic cell line, hTERTHPNE cells, had no effect on proliferation. By carrying out immunohistochemistry on human PDAC tissue, I identified high TRMT5 expression in cancerous tissue, but not in surrounding normal fibroblasts. Importantly, inducible knockdown of TRMT5 in PANC-1 cells orthotopically injected into mice, significantly interfered with their ability to form metastatic tumours. As a separate project I investigated the role of another TRMT RNA guanosine methyltransferase, TRMT1, in a human ovarian cancer cell line JHOC-5. TRMT1 is predicted to be an m2,2G and m2G methyltransferase in human cells. I found that its loss drastically diminished the level of m2,2G in small (<200nt) and large (>200nt) RNAs, but surprisingly did not affect m2G levels. Furthermore, silencing of TRMT1 by CRISPR knockout technology showed a significant negative impact on migration of JHOC- 5 cells, implicating this enzyme in ovarian cancer biology. In summary, I characterised a new set of RNA modification antibodies using a screening system that exceeded conventional assessment of modification versus non-modification binding. An antibody from this screen was instrumental in (i) characterising m1G as a new mRNA modification, (ii) showing that TRMTs exhibit target promiscuity beyond their canonical tRNA substrates and (iii) linking the m1G RNA modification pathway to the growth of pancreatic cancer cells. My work also identified human TRMT1 as a likely m2,2G RNA methyltransferase whose activity is required for the migration of ovarian cancer cells. Overall, my findings demonstrate the importance of these two RNA modification pathways for cancer cell growth and migration, thereby highlighting TRMT5 and TRMT1, and perhaps TRMTs in general, as potential therapeutic targets in cancer.





Kouzarides, tony


RNA modifications, m1G, methylguanosine, TRMT5, TRMT1, pancreatic cancer, epitranscriptomics


Doctor of Philosophy (PhD)

Awarding Institution

University of Cambridge
Abcam Plc