RNA-Pt Adducts Following Cisplatin Treatment of <i>Saccharomyces cerevisiae</i>

Abstract

The numerous regulatory roles of cellular RNAs suggest novel potential drug targets, but establishing intracellular drug–RNA interactions is challenging. Cisplatin (<i>cis</i>-diamminedichloridoplatinum­(II)) is a leading anticancer drug that forms exchange-inert complexes with nucleic acids, allowing its distribution on cellular RNAs to be followed <i>ex vivo</i>. Although Pt adduct formation on DNA is well-known, a complete characterization of cellular RNA–Pt adducts has not been performed. In this study, the action of cisplatin on <i>S. cerevisiae</i> in minimal media was established with growth curves, clonogenic assays, and tests for apoptotic markers. Despite high toxicity, cisplatin-induced apoptosis in <i>S. cerevisiae</i> was not observed under these conditions. In-cell Pt concentrations and Pt accumulation on poly­(A)-mRNA, rRNA, total RNA, and DNA quantified <i>via</i> ICP-MS indicate ∼4- to 20-fold more Pt accumulation in total cellular RNA than in DNA. Interestingly, similar Pt accumulation is observed on rRNA and total RNA, corresponding to one Pt per (14,600 ± 1,500) and (5760 ± 580) nucleotides on total RNA following 100 and 200 μM cisplatin treatments, respectively. Specific Pt adducts mapped by primer extension analysis of a solvent-accessible 18S rRNA helix occur at terminal and internal loop regions and appear as soon as 1 h post-treatment. Pt per nucleotide accumulation on poly­(A)-mRNA is 4- to 6-fold lower than on rRNA but could have consequences for low copy-number or highly regulated transcripts. Taken together, these data demonstrate significant accumulation of Pt adducts on cellular RNA species following <i>in cellulo</i> cisplatin treatment. These and other small molecule–RNA interactions could disrupt processes regulated by RNA