Platinum Interference with siRNA Non-seed Regions Fine-Tunes Silencing Capacity

Abstract

Knowledge concerning the molecular mechanisms governing the influence of non-coding RNAs on protein production has emerged rapidly during the past decade. Today, two main research areas can be identified, one oriented toward the use of artificially introduced siRNAs for manipulation of gene expression, and the other one focused on the function of endogenous miRNAs. In both cases, the active molecule consists of a ∼20-nucleotide-long RNA duplex. In the siRNA case, improved systemic stability is of central interest for its further development toward clinical applications. With respect to miRNA processing and function, understanding its influence on mRNA targeting and the silencing ability of individual miRNAs, e.g., under pathological conditions, remains a scientific challenge. In the present study, a model system is presented where the influence of the two clinically used anticancer drugs, cisplatin and oxaliplatin, on siRNA's silencing capacity has been evaluated. More specifically, siRNAs targeting the 3' UTR region of Wnt-5a mRNA (NM_003352) were constructed, and the biologically active antisense RNA strand was pre-platinated. Platinum adducts were detected and characterized by a combination of gel electrophoresis and MALDI-MS techniques, and the silencing capacity was evaluated in cellular luciferase-expressing systems using HB2 cells. Data show that platination of the antisense strand of the siRNAs results in adducts with protection against hydrolytic cleavage in the proximity of the platination sites, i.e., with altered degradation patterns compared to native RNAs. The MALDI-MS method was successfully used to further identify and characterize platinated RNA, with the naturally occurring platinum isotopic patterns serving as sensitive fingerprints for metalated sites. Expression assays all confirm biological activity of antisense-platinated siRNAs, here with platination sites located outside of the seed region. A significant reduction of silencing capacity was observed as a general trend, however. Of the two complexes studied, oxaliplatin exhibits the larger influence, thus indicating subtle differences between the abilities of cis- and oxaliplatin to interfere with si- and miRNA processing.

Covalent and non-covalent binding of metal complexes to RNA

“…Several examples of covalent binding of platinum complexes to RNA have been herein commented [44,45,47,70,[76][77][78][79][80][81][82][83][84][85][86][87]. Even if the existence of platinum-RNA adducts is undeniable, further biochemical and structural studies are needed to better understand the nature of platinum-RNA adducts as well as their impact on RNA structure and on RNA biological functions.…”

“…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).…”

“…In both cases, the silencing mechanism relies on the incorporation of one of the two strands, the antisense strand, into a ribonucleoprotein complex, the RNA-induced silencing complex (RISC). This is followed by recognition of the target mRNA which leads to either translation repression or mRNA degradation [84,101,103].…”

“…MiRNAs have strong potential as biomarkers, since their dysregulation has been associated with the occurrence of several pathologies, including cancer [27,102,104], whereas synthetic analogues of siRNAs are currently being developed as biological tools to supress protein production [82][83][84]102,105].…”

“…These results suggest that platinum drugs may interfere with siRNA processing, with subtle differences between the different drugs used. Moreover, the authors speculate that the differences between cisplatin and oxaliplatin could be even more pronounced for endogenous miRNA silencing, and that the different activity and toxicity of the two drugs might be somehow partly related to their ability to interfere with miRNA activity [84].…”