mRNA Structure and its Effects on Posttranscriptional Gene Silencing

Abstract

Posttranscriptional gene silencing (PTGS) is a process by which a protein’s synthesis is impaired by targeting its messenger RNA (mRNA). The antisense oligodeoxynucleotide (AON) and RNA interference (RNAi) pathways can both accomplish PTGS by hybridization of a reverse complementary oligonucleotide and subsequent enzymatic degradation of the mRNA by an RNase mediated mechanism. We have investigated the influence of specific mRNA structural elements on short interfering RNA (siRNA) and AON targeting efficiency. Under identical conditions, both PTGS pathways are significantly inhibited by target mRNA secondary structure. Surprisingly, we found that an AON was less stringent in its requirement for mRNA target accessibility than the corresponding siRNA. By determining that the AON and siRNA guide strand have the same apparent K D in the absence of protein, we show that nucleic acid binding affinity does not explain their difference in in vivo silencing activity. Rather, it appears that RISC must increase the binding affinity of the siRNA for the target. Furthermore, RNA binding proteins are also potent inhibitors of AON activity. We conclude that mRNA secondary and quaternary structure play important roles in PTGS by significantly affecting the ability of a siRNA or AON to hybridize with their intended target. Recognition of these effects will facilitate the design of more efficient antisense molecules for therapeutically motivated gene silencing and argue for continued mechanistic studies on AON and siRNA mediated mRNA destruction.