Analysis of putative miRNA binding sites and mRNA 3' ends as targets for siRNA-mediated gene knockdown.
Tremendous efforts have been made to develop short-interfering RNA (siRNA) design algorithms that generate highly functional siRNAs for gene knockdown. Nevertheless, "difficult-to-silence" target messenger RNAs (mRNAs) still exist for which no functionally validated siRNAs are available. MicroRNA (miRNA) sites in the mRNA 3'UTR, which interact with miRNA-loaded RNA-induced silencing complex (miRISC) for posttranscriptional gene regulation, provide alternative potentially accessible sites for siRNA. To investigate this, we designed siRNAs directed against single putative miRNA sites (misiRNAs) as predicted by miRNA target databases as well as siRNAs against other regions within the 3'UTR of "difficult-to-silence" targets in this proof-of-principle study. Although their design was not fully optimized, the misiRNAs generally caused higher knockdown than previously designed siRNAs for these targets. In general, knockdown by misiRNAs targeting the miRNA seed region was specific for the target mRNA, and misiRNAs targeting 1 nt upstream of miRNA seed region were similarly potent. We also systematically screened the 3'UTR of two mRNA targets using siRNA-tiling experiments. 5' and 3' regions of the p21-activated kinase 6 (PAK6) 3'UTR were found accessible, whereas the middle portion was largely inaccessible for siRNA knockdown. In ribosomal protein S6 kinase (RPS6KB1) 3'UTR, however, only the 5' region was accessible for siRNA knockdown. Detailed analysis of 10 further "difficult-to-silence" targets revealed that siRNA accessibility at the mRNA 3' end is not a general phenomenon, at least in "difficult-to-silence" targets, as we could not detect significant knockdown by siRNAs directed against this region.
Bergauer
Tobias
T
QIAGEN GmbH, Hilden, Germany.
Krueger
Ute
U
Lader
Eric
E
Pilk
Sabrina
S
Wolter
Irene
I
Bielke
Wolfgang
W
D020413:3' Untranslated Regions; D001665:Binding Sites; D055785:Gene Knockdown Techniques; D006367:HeLa Cells; D006801:Humans; D035683:MicroRNAs / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction
D020413:3' Untranslated Regions; D035683:MicroRNAs; D034741:RNA, Small Interfering