Project Summary — Translational regulation has emerged as a key process in the evolution of hepatocellular
carcinoma (HCC)1,11. Support of rapid proliferation in cancer requires enhanced protein production as well as
gene-specific translational changes that facilitate reprogrammed cellular activities. Recent research has revealed
that ribosome composition, including rRNA modification stoichiometry, can affect translational function in cells
and bias translation of oncogenic transcripts, altering cell state6,17–20. Although 2% of all rRNA bases are
modified, only two of them are N6-methyladenosine (m6A) — 28S m6A4220 and 18S m6A1832. Our lab previously
characterized the 28S m6A4220 methyltransferase (ZCCHC4)21, and we have now biochemically characterized
METTL5 (M5) as the 18S m6A1832 methyltransferase, stabilized by the cofactor TRMT112. The function of this
site is still unknown, but structural analysis suggests roles in translation initiation and re-initiation events29,31, as
it is near the mRNA channel and at the binding site of ribosome recycling factors. Furthermore, our M5 knockout
HeLa cells display a markedly hypoproliferative phenotype while overexpression of M5 has been associated with
hyperproliferation, including in hepatocellular carcinoma (HCC)8,9,22. We have also shown by imaging and
biochemical fractionation that M5 is localized in both the nucleolus and cytosol, suggesting that the 18S m6A site
may be dynamically methylated in the cytosol, even after ribosome biogenesis, to regulate and/or fine tune
translational processes. The goal of this proposal is to define the functional effects of M5 on translation and
investigate the mechanism by which it supports cell proliferation and cancer development. We hypothesize that
M5 dynamically methylates m6A1832 in response to oncogenic cell stress with functional consequences in
ribosome composition, translational function, and ribosome recycling activities that support proliferation and
tumorigenesis. To investigate this hypothesis, we will first thoroughly define the impact of M5 on ribosome
composition and function as follows: quantifying M5 and m6A1832 under normal and stress conditions by LC-
MS/MS and HPLC; characterizing the effect of M5 on translation through nascent protein synthesis assays,
ribosome profiling, and translation reporter assays; and monitoring M5-related changes in stoichiometry of
ribosomal proteins and translation-related factors. Then, we will investigate the role and mechanism of M5 in
HCC proliferation and tumorigenesis by examining the effects of M5 on ribosome binding and translational
activities of ribosome recycling factors, and by evaluating the role of M5 in proliferation of HCC cell lines and
tumorigenesis in HCC xenograft mouse models with respect to ribosome recycling processes. Successful
completion of this proposal will unveil the function of 18S m6A1832 in translation, clarify the link between M5 and
HCC prognosis, potentially guide new translation-based therapy development for HCC, and contribute to our
understanding of how dynamic regulation of rRNA modifications can affect the proteome and cellular state in
support of cancer development.