Extracellular N⁶-isopentenyladenosine (i⁶A) addition induces cotranscriptional i⁶A incorporation into ribosomal RNAs

Abstract

N⁶-isopentenyladenosine (i⁶A), a modified adenosine monomer, is known to induce cell death upon its addition to the culture medium. However, the molecular fate of extracellularly added i⁶A has yet to be identified. Here we show that i⁶A addition to cell culture medium results in i⁶A incorporation into cellular RNA in several cell lines, including the 5-fluorouracil (5-FU)-resistant human oral squamous cell carcinoma cell line FR2-SAS and its parental 5-FU-sensitive cell line SAS. i⁶A was predominantly incorporated into 18S and 28S rRNAs, and i⁶A incorporation into total RNA was mostly suppressed by treating these cell lines with an RNA polymerase I (Pol I) inhibitor. i⁶A was incorporated into RNA even upon inactivation of TRIT1, the only cellular i⁶A-modifying enzyme. These results indicate that upon cellular uptake of i⁶A, it is anabolized to be used for Pol I transcription. Interestingly, at lower i⁶A concentrations, the cytotoxic effect of i⁶A was substantially more pronounced in FR2-SAS cells than in SAS cells. Moreover, in FR2-SAS cells, i⁶A treatment decreased the rate of cellular protein synthesis and increased intracellular protein aggregation, and these effects were more pronounced than in SAS cells. Our work provides insights into the molecular fate of extracellularly applied i⁶A in the context of intracellular nucleic acid anabolism and suggests investigation of i⁶A as a candidate for a chemotherapy agent against 5-FU-resistant cancer cells.