Enzymatic processing of platinated RNAs

Abstract

The broadly prescribed antitumor drug cisplatin coordinates to DNA, altering the activity of cellular proteins whose functions rely upon sensing DNA structure. Cisplatin is also known to coordinate to RNA, but the effects of RNA-Pt adducts on the large number of proteins that process the transcriptome are currently unknown. In an effort to address how platination of an RNA alters the function of RNA processing enzymes, we have determined the influence of [Pt(NH(3))(2)](2+)-RNA adducts on the activities of 3'-->5' and 5'-->3' phosphodiesterases, a purine-specific endoribonuclease, and a reverse transcriptase. Single Pt(II) adducts on RNA oligonucleotides of the form (5'-U(6)-XY-U(5)-3': XY = GG, GA, AG, GU) are found to block exonucleolytic digestion. Similar disruption of endonucleolytic cleavage is observed, except for the platinated XY = GA RNA where RNase U2 uniquely tolerates platinum modification. Platinum adducts formed with a more complex RNA prevent reverse transcription, providing evidence that platination is capable of interfering with RNA's role in relaying sequence information. The observed disruptions in enzymatic activity point to the possibility that cellular RNA processing may be similarly affected, which could contribute to the cell-wide effects of platinum antitumor drugs. Additionally, we show that thiourea reverses cisplatin-RNA adducts, providing a chemical tool for use in future studies regarding cisplatin targeting of cellular RNAs.

Figure 1. Platination and VPD Exonuclease Digestion of RNAs

MALDI-MS spectra of a) 5’-(U)₆-AG-(U)₅-3’ RNA b) platinated 5’-(U)₆-AG-(U)₅-3’ RNA c) platinated 5’-(U)₆-AG-(U)₅-3’ RNA digested by VPD (3’→5’) exonuclease d) platinated 5’-(U)₆-GU-(U)₅-3’ RNA digested by VPD (3’→5’) exonuclease.

Figure 2. SPD Digestion of Platinated RNAs

MALDI-MS spectra of a) SPD (5’→3’) digestion of platinated 5’-(U)₆-AG-(U)₅-3’ RNA b) SPD (5’→3’) digestion of platinated 5’-(U)₆-GU-(U)₅-3’ RNA.

Figure 3. U2 Digestion of Platinated RNAs

MALDI-MS spectra obtained following a) RNase U2 digestion of 5’-(U)₆-GU-(U)₅-3’ RNA b) RNase U2 digestion of platinated 5’-(U)₆-GU-(U)₅-3’ RNA c) RNase U2 digestion of 5’-(U)₆-GA-(U)₅-3’ RNA. d) RNase U2 digestion of platinated 5’-(U)₆-GA-(U)₅-3’ RNA.

Figure 4. Reverse Transcription of Platinated PEBBD

a) Sequence of the BBD region of PEBBD. 5’ and 3’ sequences attached for in vitro PCR amplification of coding DNA, transcription, and reverse transcription are represented by blue lines. RT stops are represented by arrows. b) Reverse transcription of platinated PEBBD analyzed by 15% sequencing dPAGE along with a plot of band intensities quantified using ImageQuant software.

Figure 5. Removal of Cisplatin From RNA by Thiourea

MALDI-MS spectra of the reaction of platinated 5’-(U)₆-GG-(U)₅-3’ RNA with thiourea taken over the course of 72 h. Similar results are observed with a platinated 5’-(U)₆-GU-(U)₅-3’ RNA (data not shown).