RNA interference is not involved in natural antisense mediated regulation of gene expression in mammals
© Faghihi and Wahlestedt; licensee BioMed Central Ltd. 2006
Received: 3 October 2005
Accepted: 13 April 2006
Published: 9 May 2006
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.
We have examined the mechanism of S-AS RNA base pairing, using thymidylate synthase and hypoxia inducible factor-1α 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.
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.
Naturally occurring antisense transcripts (NAT) have been reported for 20% of the human genome [1–3]. Recent reports indicate the existence of NAT for at least 72% of mouse transcripts [4, 5]. Most NAT are cis-encoded antisense RNA [6, 7]. By definition, cis-NAT are complementary mRNA with an overlapping transcription unit at the same chromosomal locus. Trans-NAT are complementary RNA transcribed from different chromosomal locations . Chimeric transcripts are mRNA with identity to more than one region of the genome and might be an artifact of cDNA library production . Over 70% of cis-NAT have a tail-to-tail format with a 3' overlap, while 15% have a head-to-head format with a 5' overlapping region. The remaining molecules have intronic or coding sequence overlaps . Many NAT show no open reading frame and are, therefore, classified as non-coding RNA [11–13].
The interaction between antisense and corresponding sense transcript partners does not follow a unified and predictable pattern . Here, we investigated the interactions between two well-validated NAT targeting the human genes encoding hypoxia inducible factor-1α (HIF) and thymidylate synthase (TS). The antisense transcript for HIF (aHIF) is a non-coding RNA that may alter HIF splicing and also the ratio between the two splice forms of HIF [14–16]. Specifically, it has been hypothesized that the antisense molecule may destabilize one splice variant of HIF mRNA and shift the balance in favor of the other variant [17, 18]. Editing is another proposed function of NAT through transformation of the adenosine to inosine nucleotide in pre-mRNA. The antisense sequence for TS (rTSα) induces editing of the sense RNA molecule, and thereby drives TS mRNA down-regulation [19, 20]. Importantly, the NAT for TS is protein coding, whereas there are no predicted open reading frames for aHIF. Thus, we chose to study these two known candidates from coding and non-coding subgroups of NAT, which could potentially modulate sense mRNA through two distinct modes of action.
One of the most exciting findings in genome biology in recent years has been the discovery of RNA interference (RNAi), which has been proposed as a possible mechanism by which NAT may regulate gene expression [9, 21]. RNAi is an innate cellular process activated when a double-stranded RNA (dsRNA) enters the cell. Originally discovered in Caenorhabditis elegans, RNAi is an evolutionarily conserved, post-transcriptional gene silencing mechanism. The dsRNA is processed by the RNase III enzyme called Dicer into small duplex RNA molecules of approximately 21 to 22 nucleotides, termed small interfering RNA (siRNA). The siRNA molecules then interact with a multi-protein complex, termed RNA-induced silencing complex (RISC), resulting in sequence specific association of the activated RISC complex with the cognate RNA transcript. This interaction leads to sequence-specific cleavage of the target transcript . It has been suggested that dsRNA derived from endogenous sense-antisense (S-AS) duplexes may act through the RNAi pathway by serving as a substrate for Dicer, and the subsequent generation of siRNA. The siRNA would then regulate one or both of the S-AS transcripts [9, 23].
In summary, NAT have been proposed to regulate gene transcription, RNA splicing, polyadenylation, editing, stability, transport, and translation . The aim of this study was to explore the mechanism of NAT action. Shared complementary regions in exons of NAT imply the probability of cytoplasmic duplex formation, and intronic overlap sequences suggest that they form nuclear dsRNA duplexes. In theory, all proposed regulatory mechanisms would require RNA duplex formation in the cytoplasm or nucleus; therefore, cellular evidence for RNA duplexes, using HIF and TS as model genes, were the main focus of this work.
Taken together, the present results suggest that NAT do not form cytoplasmic RNA duplexes that activate RNAi mechanisms. Overlapping transcripts in an antisense orientation, be they protein coding or non-coding, have the potential to form dsRNA, a substrate for a number of different RNA-modification pathways. One prominent route for dsRNA is its breakdown by Dicer enzyme complexes into small RNA. We used several experimental approaches to identify the presence of RNA duplexes in the cytoplasm of cells, and to detect Dicer products involved in processing of dsRNA. Our results, using synthetic S-AS constructs as well as endogenous NAT, did not support the presence of cytoplasmic RNA duplexes or engagement of the RNAi mechanism.
The concomitant presence of both sense and antisense mRNA is one requirement for NAT regulation and many in silico predicted NAT candidates can be ruled out on this criterion alone. Expression levels of S-AS pairs are also important, as these could predict the mode of regulation. High levels of S-AS pairs in a single cell, as suggested from our experimental model, argue against RNAi involvement. However, another explanation for this phenomenon is a translation block or other kind of RNA mediated regulation of gene expression, without alteration of mRNA levels. Expression assessment and evaluation of mRNA levels would be recommended as a first step in studying other predicted S-AS candidates.
Alterations in antisense transcript levels can affect the sense mRNA level; however, S-AS changes are not necessarily reciprocal. Recently, we showed that antisense transcript knock down elevated sense transcript levels but the reverse interaction was not observed . This observation suggests antisense mRNA is involved in sense transcript regulation, but sense mRNA does not appear to control antisense expression. If endogenous RNAi were involved in mammalian S-AS phenomena, then it may be expected that both transcripts exhibit similar expression profiles in knockdown experiments.
Overall, the above observations are consistent with the conclusion that RNAi mechanisms are not engaged by S-AS gene regulation. Indeed, further support is derived from two other observations. First, small RNA molecules were not detected even for highly expressed S-AS pairs, implying Dicer-independent RNA processing. Second, the IFN cascade was not activated by NAT. Indeed, it may have been expected that, if at least 70% of mammalian genes have NAT and the mechanism is through RNA duplex formation, there would be a cumulative IFN response. Our studies show a dramatic IFN-β and OAS2 mRNA induction with dsRNA transfection, but not in cells overexpressing S-AS pairs, indicating the absence of duplexes of NAT.
To date, there are no reports of endogenous mammalian siRNA derived from NAT in the literature . It is possible, however, that endogenous siRNA could be programmed into RISC and that this effect would be long term and lead to down-regulation of target RNA. In theory, a 500 bp dsRNA would produce a library of siRNA. This siRNA collection could impair protein production at two levels, either by degrading many 'off targeted' mRNAs or by blocking translation. The extent of this non-specific effect would be much greater when considering the large number of genes known to have antisense sequences. It is worth noting that many research groups have identified and cloned all known small regulatory RNAs, such as miRNA and repeat associated siRNA [26, 27]. An interesting observation is that no perfect match RNA oligonucleotides have been reported.
Consistent with data in the present investigation, Jen et al.  pursued a meta-analysis of NAT expression and suggested that RNA degradation by dsRNA formation is not a predominant route of gene regulation in Arabidopsis thaliana. Additionally, endogenous siRNA has been defined for plants; however, only 11 pairs of NAT had siRNA sequences mapped uniquely to the overlapping region of NAT, which substantiates the notion that RNAi is not involved in the processing of S-AS pairs . In other words, although the presence of endogenous miRNA has been reported, no endogenous mammalian siRNA originating from NAT has been described so far. This observation also argues against processing of endogenous RNA duplexes in a Dicer-dependent pathway and further substantiates our findings.
Our data suggest that antisense expression is not linked to transcript degradation pathways. However, our methods do not completely exclude the formation of RNA duplexes in the cell nucleus, or any proposed functions for NAT regulation of gene expression, such as editing, nuclear retention, splicing or transport. Although many different functions and mechanisms have been suggested for NAT, no systematic approaches for the classification or prediction of these mechanisms have been suggested to date. Our study could be a start for a functional approach to NAT studies that could lead to a categorization of NAT based on their unique bioinformatic features. Our methodology could also be expanded to provide a systematic approach to natural antisense mediated regulation of gene expression.
Materials and methods
HeLa cells were grown on the surface of silane-coated slides overnight and fixed with 4% paraformaldehyde (pH 7.4) for 4 minutes. After air drying of the slides, a chamber was utilized for easy treatment of the attached cells with DNase at 37°C for 16 hours. DNase Master Mix contained 10× TurboDNase Buffer (Ambion Europe, Cambridgeshire, UK), 100 units DNase1, 100 units of TurboDNase, and 100 units of Suprasin in a final volume of 200 μl. The cells were then washed with 1× phosphate-buffered saline (PBS) and subsequently incubated at 95°C for 5 minutes. First strand cDNA was synthesized with an RT-Master Mix of 10× RT buffer (Applied Biosystems, Foster City, CA, USA), 2.5 mM MgCl2, 10 mM dNTP mixture, 10 pM random hexamers, 100 units RNase inhibitor, and 500 units of reverse transcriptase in a final volume of 200 μl. The reverse transcription (RT) reactions were completed using the following conditions: 30 minutes at room temperature, 3 hours at 42°C, and 5 minutes at 95°C. For in situ hybridization, the cells were incubated at 65°C for one hour in blocking buffer (10 mM Tris-HCl, 50 mM KCl, 1.5 mM MgCl2, 1% Triton-X, 20 μM random DNA in a final volume of 200 μl). After blocking, the cells were hybridized at 70°C for one hour with 10 μM of specific intron spanning probes (the sequences are given in Additional data file 1). The slides were then washed two times with pre-warmed PBS. Hybridization of the probe directly to the RNA was done under the same conditions without RT.
Dilutional single cell real-time PCR
The HeLa cultures were diluted to a few cells in each bright field. RNA was extracted from 15 individual cells that were picked under the guide of a confocal microscope. First strand cDNA synthesis was made from the RNA by using SMART and CDS III 3' oligonucleotides and Powerscript reverse transcriptase from Clontech (Mountain View, CA, USA) according to the manufacturer's instructions. The first strand cDNA was then used for PCR amplification using the LD primer, DSIII PCR primer, and Advantage2 Polymerase mix from the Clontech cDNA library kit.
Preparation and fractionation of cell extracts
Cytoplasmic extracts were prepared from HeLa cells transfected with different vectors. Cells were harvested after 24 hour transfections and centrifuged at 1,000 g for 5 minutes at 4°C. Cell pellets were washed three times with ice-cold PBS, pH 7.2, and lysed for 10 minutes on ice in three packed cell volumes of lysis buffer (20 mM Tris-HCl, pH 7.4, 200 mM NaCl, 14 mM MgCl2, 20 units of suprasin, 100 units of protease inhibitor; 100 μg/ml cyclohexamide, 0.1% (v/v) Triton X-100). Nuclei were isolated by centrifugation at 5,000 g for 10 minutes at 4°C. The supernatant contained the cytoplasmic extract and was immediately used for RNA extraction with Trizol (Invitrogen, Carlsbad, CA, USA) Nuclear extracts were prepared by washing the pellet once in lysis buffer and twice in 1× PBS, pH 7.2. Nuclear RNA was then collected using Trizol reagent. Purity (>98%) and integrity of nuclei were determined microscopically.
Ribonuclease protection assay (RPA)
Using the Direct Protect Lysate RPA kit from Ambion, cytoplasmic lysate was treated with RNase cocktail buffer and incubated with RNase A and T cocktail at 37°C for 30 minutes. Nucleases were removed by incubation with sodium sacrosyl and proteinase at 37°C for 30 minutes. RNA was precipitated using 99% ethanol and glycogen blue and subsequently DNase treated with TurboDNase (Ambion) prior to separation on a 5% denaturing PAGE/8 M urea. RNAse negative samples were treated exactly the same, except for addition of RNAse A and T to assess the specificity of the probe and efficacy of RNAse treatment.
Northern blot for the Dicer products
Total RNA was collected using Trizol (Invitrogen) and precipitated with 99% ethanol. Total RNA (30 μg) was loaded per lane and separated out on a 10% PAGE/urea gel. The RNA was then transferred onto a nylon membrane (Amersham, Little Chalfont, UK) and blocked with salmon sperm DNA for six hours. The blocked membrane was hybridized overnight with radiolabeled S-AS probes spanning the overlap region of the TS and rTSα genes. The probe was made by random priming of overlap DNA using 32P-labeled nucleotide and the Amersham random priming kit. All membranes were washed one time with low stringency and two times with high stringency buffer, each for 1 hour, and signal was detected with a Typhoon (Amersham) phosphor-imaging instrument.
Cell culture and transfection
HeLa cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. The cells in logarithmic growth were transfected with plasmids containing the luciferase gene with either the sense or antisense overlap region or both. At 24 hours post-transfection, cells were used for further applications. The pGL3 control vector (Promega, Madison, WI, USA) was used for making all S-AS constructs. We engineered Pst1 and EcoR1 restriction sites downstream of the firefly luciferase for cloning. A BamH1 sequence was used to form a hairpin between overlap regions and to construct a vector with a consecutive S-AS sequence (primers and probe sequences are listed in Additional data file 1). The same vector was used as a template for IVT of S-AS overlap mRNA, using a MEGAscript transcription kit (Ambion). For IVT of HIF, transcript primers with a flanking T7 promoter sequence were designed and the PCR product then used for synthesizing duplex RNA.
Real-time PCR was carried out with the GeneAmp 7000 machine (Applied Biosystems). The PCR reactions contained 20 ng cDNA, Sybrgreen or Universal Mastermix (Applied Biosystems), 300 nM of forward and reverse primers, and 200 nM of probe in a final reaction volume of 50 μl (primers and probe sequences are listed in Additional data file 1). The primers and probe were designed using PrimerExpress software (Applied Biosystems). They were strand specific for each S-AS pair and the probe covered exon boundaries to eliminate the chance of genomic DNA amplification. The PCR conditions for all genes were as follows: 50°C for 2 minutes and 95°C for 10 minutes, 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. The results are based on the cycle threshold (Ct) values. Differences between the Ct values for the experimental genes and the reference gene (either β2 M or glyceraldehyde 3-phosphate dehydrogenase) were calculated as ΔΔCt.
Additional data files
The following additional data are available with the online version of this paper. Additional data file 1 is a table containing sequence information for all the primers and probes used in this study. Primers included were used for real-time PCR, in situ hybridization, cloning of Ts and rTSα and for in vitro transcription of HIF.
We thank Omid Faridani and Dr Hakan Thonberg for their help discussions on this topic and technical assistance. We thank Dr Jannet Kocerha, Dr Paul Kenny and Dr Patricia McDonald for careful reading and making corrective comments on this manuscript.
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