SiRNA Has Greatly Elevated Mismatch Tolerance at 3′-UTR Sites

Na Wei,Lei Zhang,Jie Zheng,Quan Du,Xiao Zhou,Fan Yi,Zicai Liang,Yue Chen,Huang Huang,Alfred Lewin

doi:10.1371/journal.pone.0049309

Abstract

It has been noted that target sites located in the coding region or the 3′-untranslated region (3′-UTR) can be silenced to significantly different levels by the same siRNA, but little is known about at what specificity the silencing was achieved. In an exploration of positional effects on siRNA specificity by luciferase reporter system, we surprisingly discovered that siRNA had greatly elevated tolerance towards mismatches in target sites in the 3′-UTR of the mRNA compared with the same target sites cloned in the coding region. Assessment of changes in protein and mRNA levels suggested that the differential mismatch tolerance might have resulted from location-specific translational repression in the 3′-UTR. Ablation of argonaute proteins by AGO-specific siRNAs revealed that the AGO2 had major impact on siRNA silencing activity against sites in both coding region and 3′-UTR, while the silencing of nonnucleolytic AGO proteins (AGO1, AGO3 and AGO4) did not significantly affect silencing of sites in either region. This paper revealed the discovery that the specificity of an siRNA can be affected by the location of its target site.

Highlights

RNA interference (RNAi) is the process of post-transcriptional gene silencing mediated by double-stranded RNA [1,2,3]
The absolute activity of the two sets of constructs bearing the target sites in the coding region or the 39-untranslated region (39-UTR) was equal in the absence of an Small interfering RNAs (siRNA), and mFold analysis was shown that there were no significant differences in accessibility to the RNAi machinery between the 39-UTR site and the site located in the coding region of a transcript (Supplementary Figure S1A and B)
Some SNPs located in the 39-UTR of genes were found to affect mRNA stability or be associated with susceptibility to diseases [31,32,33] there is no evidence that allele-specific siRNAs can efficiently discriminate SNPs in 39-UTR

Summary

Introduction

RNA interference (RNAi) is the process of post-transcriptional gene silencing mediated by double-stranded RNA [1,2,3]. Small interfering RNAs (siRNA), a class of 19–21-bp double-stranded RNAs can be incorporated into an RNA-induced silencing complex (RISC) [4] to regulate the expression of target genes by either cleavage of target mRNAs or repressing translation and/or promoting mRNA decay based on the complementarity between the siRNA and its target [5,6,7]. Structural and biochemical studies indicated that all human AGO proteins are capable of associating with both siRNAs and miRNAs indiscriminately of their sequences and structures [9,10,11,12]. This is somewhat different from sorting of small RNAs in flies and worms [13,14,15]. All AGOs were proposed to exert translational repression when tethered to an mRNA [18,19]

Methods

Results

Conclusion