RNA interference is not involved in natural antisense mediated regulation of gene expression in mammals

Abstract

Background: Antisense transcription, yielding both coding and non-coding RNA, is a widespread phenomenon in mammals. The mechanism by which natural antisense transcripts (NAT) may regulate gene expression are largely unknown. The aim of the present study was to explore the mechanism of reciprocal sense-antisense (S-AS) regulation by studying the effects of a coding and non-coding NAT on corresponding gene expression, and to investigate the possible involvement of endogenous RNA interference (RNAi) in S-AS interactions.

Results: We have examined the mechanism of S-AS RNA base pairing, using thymidylate synthase and hypoxia inducible factor-1alpha as primary examples of endogenous genes with coding and non-coding NAT partners, respectively. Here we provide direct evidence against S-AS RNA duplex formation in the cytoplasm of human cells and subsequent activation of RNAi.

Conclusion: Collectively, our data demonstrate that NAT regulation of gene expression occurs through a pathway independent of Dicer associated RNAi. Moreover, we introduce an experimental strategy with utility for the functional examination of other S-AS pair interactions.

Figure 1

Thymidylate synthase genomic location. (a) Schematic presentation of TS sense and rTSα antisense mRNA. Exons are presented as boxes and the location of probes used for in situ hybridization (ISH) as well as the 3' overlap region of both sense and antisense mRNA are also indicated. The entire overlap region of both sense and antisense mRNA (red hash shaded region) were cloned into the vector described in (b). (b) Conformation of vectors used for transfection and S-AS RNA production. The sense vector makes luciferase RNA with a 3' sense overlap sequence, the antisense vector makes an analogous RNA with a 3' antisense overlap region, and the S-AS vector makes RNA with a consecutive sense-antisense sequence with a hairpin sequence between them.

Figure 2

Single cell RNA expression of TS transcripts. (a) Antisense probe; (b) sense probe; (c) both sense and antisense probes bound to the fixed and reverse transcribed TS RNA in HeLa cells. Probes were designed to cover exon boundaries and a part of the overlap region in a strand specific manner. (d) Signals from the actin probe show that the method was working optimally. All the probes were intron spanning to avoid background signal from contaminating DNA.

Figure 3

Endogenous single cell expression of TS sense (TS) and its antisense (rTSα) mRNA, as well as HIF sense (HIF) and its antisense (aHIF) RNA. Real-time PCR primers were designed to span between the overlapping and non-overlapping regions. Expression of the low abundant TATA box binding protein (TBP) was also quantified to determine the sensitivity of the assay. All samples were normalized to β2M and the average results from 15 individual cells are plotted.

Figure 4

Cellular localization of TS sense (TS) and its antisense (rTSα) RNA and HIF sense (HIF) and its antisense (aHIF) RNA in three cell lines (HeLa, S-KN-MC and HEPG2). The cytoplasmic and nuclear RNA were normalized to total RNA and graphed as the average for three cell lines.

Figure 5

Duplex RNAs were not detected in HeLa cells using RPA. (a) Ribonuclease protection assay (RPA). of cytoplasmic RNA. Lane 1, HeLa lysate -RNAse; lane 2, HeLa lysate +RNAse; lane 3, HeLa overexpressing sense (S) -RNAse; lane 4, HeLa overexpressing sense (S) +RNAse; lane 5, HeLa overexpressing antisense (AS) -RNAse; lane 6, HeLa overexpressing antisense (AS) +RNAse; lane 7, HeLa overexpressing hairpin vector (S-AS) -RNAse; lane 8, HeLa overexpressing hairpin vector (SAS) +RNAse; lane 9, HeLa transfected with in vitro transcribed S-AS RNA duplex -RNAse; lane 10, HeLa transfected with in vitro transcribed S-AS RNA duplex +RNAse. All of the +RNAse samples treated with RNAse A+T, along with -RNase samples, were separated on denaturing PAGE and probed for the overlap region of TS mRNA. The predicted positive bands (rTSα, 1,800 bp endogenous antisense mRNA; TS 1,600 bp endogenous sense mRNA and 1,100 bp vector based S-AS mRNA) were detected in RNAse negative samples and revealed efficacy of RNAse treatment as well as specificity of the probe. Additionally, signals corresponding to a 200 bp product (protected overlap region) were seen only in the last four lanes, which had synthetically endogenous or exogenous RNA duplex. (b) Additional controls for RPA of cytoplasmic RNA. Lane 1, cytoplasmic lysate of HeLa cells; lane 2, cytoplasmic lysate of HeLa cells overexpressed with sense and antisense vector; lane 3, lysate from HeLa cells transfected with in vitro transcribed S-AS RNA duplex; and lane 4, total RNA from HeLa cells overexpressing sense and antisense vector. All samples were treated with RNAse A+T, separated on denaturing PAGE and probed for overlapping region of TS mRNA. The expected 200 bp product (protected overlapping region) was seen only in lane 3, which included exogenous synthetic RNA duplex.

Figure 6

Northern blot for Dicer products. Total RNA from: lane 1, HeLa cells; lane 2, HeLa cells overexpressed with S-AS mRNA; lane 3, HeLa cells transfected with IVT-overlap dsRNA; lane 4, HeLa cells overexpressing hairpin S-AS RNA; lane 5, marker. The vector based RNA (1,100 bp) band in lanes 2 and 4 represent mRNA originating from the vector. The overlap region (200 bp) band in lane 3 is the transfected overlap RNA, and the same band in lane 4 could represent an intermediate product from siRNA production or a byproduct of the cell interferon response. The Dicer product (approximately 20 bp) band represents 21 nucleotide RNA sequences, characteristic of RNAse III enzyme products.

Figure 7

IFNβ and OAS2 mRNA expression. The interferon response was quantified by qRT-PCR in: HeLa cells transfected with IVT-dsRNA of HIF (1) or IVT-dsRNA of TS (2); HeLa cells overexpressing S-AS of TS (3); or reference HeLa cells (4). The mRNA levels were normalized to glyceraldehydes 3-phosphate dehydrogenase. In cells transfected with duplex RNA originated from TS or HIF, there was a remarkable increase in IFN-β and OAS2 mRNA.