RNA-Pt adducts following cisplatin treatment of Saccharomyces cerevisiae

Abstract

The numerous regulatory roles of cellular RNAs suggest novel potential drug targets, but establishing intracellular drug-RNA interactions is challenging. Cisplatin (cis-diamminedichloridoplatinum(II)) is a leading anticancer drug that forms exchange-inert complexes with nucleic acids, allowing its distribution on cellular RNAs to be followed ex vivo. 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 S. cerevisiae in minimal media was established with growth curves, clonogenic assays, and tests for apoptotic markers. Despite high toxicity, cisplatin-induced apoptosis in S. cerevisiae was not observed under these conditions. In-cell Pt concentrations and Pt accumulation on poly(A)-mRNA, rRNA, total RNA, and DNA quantified via 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 in cellulo cisplatin treatment. These and other small molecule-RNA interactions could disrupt processes regulated by RNA.

Figure 1

Cisplatin inhibits yeast growth and viability. (a) Exponential growth curves of BY4741 S. cerevisiae continuously treated with 0, 100, and 200 µM cisplatin in SD media. (b) Viability of cisplatin-treated yeast plated onto drug-free media,. Results presented as the means ± standard deviation from four (a) and three (b) independent experiments.

Figure 2

Tests for apoptotic markers. (a) DAPI staining of yeast treated with 0 and 200 µM cisplatin for 6 h. (b) TUNEL assay of yeast treated with 0 and 200 µM cisplatin for 6 h, +/− 5 U DNase I. (c) Viability of BY4741, ΔAif1 and ΔYca1 treated with 200 µM cisplatin for 6 h, Inset: average cfu for control and cisplatin-treated cultures. Results presented as means ± standard deviation from three independent experiments.

Figure 3

Estimated cell volumes and in-cell Pt concentrations. (a) Average estimated cell volumes following treatment with 100 µM and 200 µM cisplatin (see Methods). (b) Calculated in-cell Pt concentrations based on Pt/cell ICP-MS measurements and the average estimated cell volumes. Results averaged from at least three independent experiments presented as means ± standard deviation.

Figure 4

Pt accumulation per nucleotide in total RNA, DNA, rRNA, and mRNA. (a) Total RNA from yeast treated with cisplatin. (b) Total RNA and genomic DNA at 12 h treatment. (c) mRNA, total RNA, and rRNA at 6 h. Data values provided in Supplementary Table S1, and presented as means ± standard deviation for at least three independent experiments.

Figure 5

Primer extension analysis of S. cerevisiae small ribosomal subunit helix 18. Primer extension analysis of RNA isolated from 6 h cisplatin-treated BY4741 (left) shows a dosage-dependant increase in termination intensity at the starred sites, indicating major (***) and minor (*) Pt binding sites. Dideoxy sequencing ladders denoted by U, A, G, and C. Experimental results are summarized on secondary structure of the S. cerevisiae helix 18 (right, lower panel). Results from Rijal and Chow (56) are summarized on the E. coli secondary structure (right, lower panel). E. coli numbering used for comparison. Major platinum binding sites and a potential crosslink between G797 and G786 are depicted on a helix 18 crystal structure (right, upper panel, PDB 3O30).