Transcriptional silencing by single-stranded RNAs targeting a noncoding RNA that overlaps a gene promoter

Abstract

RNAi using single-strand RNA would provide new options for therapeutic development and for investigating critical questions of mechanism. Using chemically modified single-strands, we test the hypothesis that single-stranded RNAs can engage the RNAi pathway and silence gene transcription. We find that a chemically modified single-stranded silencing RNA (ss-siRNA) designed to be complementary to a long noncoding RNA (lncRNA) requires argonaute protein, functions through the RNAi pathway, and inhibits gene transcription. These data expand the use of single-stranded RNA to cell nuclei.

Figure 1. Inhibition of PR gene expression by ss-siRNAs targeting the PR gene promoter

a) Sequences of chemically modified single-stranded RNAs (ss-siRNAs) used in this study (left) and chemical structures of modified nucleotides (right). Oligonucleotides are phosphorylated at the 5' ends. Bases mismatched relative to ssPR9 are underlined. b) Scheme of the PR gene promoter showing the target site for duplex or single-stranded PR9 within the antisense transcript (AT2). c) Western blot analysis of unmodified or chemically modified duplex or single-stranded RNAs for PR inhibition. Hybrid dsPR9 consists of ssPR9 (sense strand) and a complementary unmodified RNA antisense strand. The western blot is representative of three independent experiments. d) qPCR analysis after transfection of unmodified dsPR9, modified ssPR9, or modified control oligomers (ssMM1, ssMM2). n=3. e) Chromatin immunoprecipitation (ChIP) for RNA polymerase II after treatment with unmodified dsPR9, modified ssPR9, or modified ssMM1. n=3. Error shown is SD. **, P < 0.01; ***, P < 0.001 (t-test).

Figure 2. Potency and duration of effect for dsPR9 and ssPR9

a–c) Western blots and their quantitation showing dose response profiles for unmodified dsPR9 (a; n=4), modified ssPR9 (b; n=4), and modified mismatch control ssMM1 (c; n=3). Dose response data were fit to the following model equation: y = 100(1−x^m/(n^m+x^m)), where y is percent expression of protein and x is concentration of oligomers. m and n are fitting parameters, where n is taken as the IC₅₀ values. n.d. = not determined. d,e) Western blots showing time-course profiles for unmodified dsPR9 (d; n=2) and modified ssPR9 (e, n=2). The western blots are representative of independent replicates. Each oligomer was transfected into T47D cells at 50 nM using Lipofectamine RNAiMAX. Error shown is SD.

Figure 3. Requirement for AGO proteins during inhibition of PR expression by ssPR9

a) ssMM1, siAGO1, siAGO2, siAGO3, or siAGO4 was transfected into T47D cells at 25 nM on day 0 (TF1). Two days later, second transfection (TF2) was performed using 50 nM mismatch ssMM1 or ssPR9. Cells were harvested for western blot on day 6. The western blots are representative of three independent experiments. Potency and selectivity of siAGOs were confirmed before this experiment (Supplementary Figure 1). b) Schematic illustration of AGO2-mediated transcriptional silencing of PR by ssPR9.