Rapid Cross-Linking of an RNA Internal Loop by the Anticancer Drug Cisplatin

Abstract

Cisplatin is the most prominent member of a series of platinum(II) antitumor drugs that demonstrate activity based on binding to adjacent purines on genomic DNA. The interactions between cisplatin and alternate biomolecules, including chemically similar RNA, are less understood than are those for DNA. In order to investigate potential implications of platinum(II) drug binding to a structurally complex RNA, we have characterized the reaction between cisplatin and the internal loop of a 41-nucleotide subdomain derived from the U2:U6 spliceosomal RNAs. This "BBD" RNA subdomain consists of a hairpin structure containing a purine-rich asymmetric internal loop. Aquated cisplatin is found to cross-link G nucleobases on opposing sides of the internal loop, forming an intramolecular internal loop cross-link in BBD and an analogous intermolecular cross-link in a two-piece construct containing the same internal loop sequence. The two opposing guanine residues involved in the crosslink were identified via limited alkaline hydrolysis. The kinetics of aquated cisplatin binding to the BBD RNA, a related RNA hairpin, and its DNA hairpin analogue were investigated in an ionic background of 0.1 M NaNO 3 and 1 mM Mg(NO 3 ) 2 . Both BBD and the RNA hairpin react 5-6-fold faster than the DNA hairpin, with calculated second-order rate constants of 2.0(2), 1.7(3), and 0.33 (3) M −1 s −1 , respectively, at 37 °C, pH 7.8. MALDI-MS data corroborate the biochemical studies and support a model in which kinetically preferred platinum binding sites compete with less reactive sites in these oligonucleotides. Taken together, these data indicate that cisplatin treatment has potential to create internal loop and other unusual cross-links in structurally complex RNAs, on a time scale that is relevant for RNA-dependent biological processes.

Molecular Pathways: The Immunogenic Effects of Platinum-Based Chemotherapeutics

“…addition, cisplatin causes atypical cross-links in structurally complex RNAs, thereby preventing reverse transcription, indicating that the RNA binding may interfere with communicating sequence information (18,19).…”

Covalent and non-covalent binding of metal complexes to RNA

“…In the sections below the most recent advances in the elucidation of platinum-RNA interactions are summarized, mainly focusing on examples subsequent to the above-mentioned review [39]. In particular, the following aspects are covered: (i) RNA platination kinetics [45,70,[77][78][79] (Sections 3.1 and 3.2); (ii) some examples of platinum binding to structured RNAs [80], focusing on latest insights into platinum binding to ribosomal RNA [44,76,81] (Section 3.3); (iii) the effect of platination on RNA interference [82][83][84] (Section 3.4); (iv) the use of Pt-RNA adducts to study RNA structure and dynamics [85] (Section 3.5) and (v) recent methods to detect RNA-platinum adducts in cells [47,86,87] (Section 3.6).…”

“…This finding suggests that short RNAs may also represent good targets for cisplatin [77,78], and prompted further studies to better elucidate the basis of platinum-RNA interaction. Interestingly, in vitro studies performed using mono-aquated platinum complexes have shown that RNA platination kinetically competes or is even preferred over DNA platination [80,93]. Elmroth and co-workers investigated the interaction of cisplatin with the full length tRNA Ala and a short hairpin model (Mh Ala ) of its GC rich acceptor stem region ( Fig.…”

“…These include, for example, early X-ray investigations of platinated yeast tRNA Phe [95,96] and a spectroscopic study on cisplatin and a platinum(II)-estradiol adduct with yeast tRNA [97]. DeRose and coworkers recently explored the platination profile of a 41-nucleotidelong RNA construct containing a large asymmetric internal loop (Fig. 5a), as model system for the catalytic core of the U2:U6 spliceosomal RNA [80]. The spliceosome is a complex molecular machine responsible for splicing and maturation of mRNA in eukaryotes [98], and earlier studies performed in cell extracts showed that spliceosome activity is inhibited upon addition of platinum(II) compounds [71].…”

“…5a), as confirmed by partial alkaline hydrolysis mapping. This rapid crosslink formation in an internal loop of a structurally complex RNA may in principle affect RNA dynamics and function [80]. However, the effect of RNA-platinum binding on cell functions is still largely unknown [39,75].…”