RNAa is conserved in mammalian cells - PubMed (original) (raw)
RNAa is conserved in mammalian cells
Vera Huang et al. PLoS One. 2010.
Abstract
Background: RNA activation (RNAa) is a newly discovered mechanism of gene activation triggered by small double-stranded RNAs termed 'small activating RNAs' (saRNAs). Thus far, RNAa has only been demonstrated in human cells and is unclear whether it is conserved in other mammals.
Methodology/principal findings: In the present study, we evaluated RNAa in cells derived from four mammalian species including nonhuman primates (African green monkey and chimpanzee), mouse, and rat. Previously, we identified saRNAs leading to the activation of E-cadherin, p21, and VEGF in human cells. As the targeted sequences are highly conserved in primates, transfection of each human saRNA into African green monkey (COS1) and chimpanzee (WES) cells also resulted in induction of the intended gene. Additional saRNAs targeting clinically relevant genes including p53, PAR4, WT1, RB1, p27, NKX3-1, VDR, IL2, and pS2 were also designed and transfected into COS1 and WES cells. Of the nine genes, p53, PAR4, WT1, and NKX3-1 were induced by their corresponding saRNAs. We further extended our analysis of RNAa into rodent cell types. We identified two saRNAs that induced the expression of mouse Cyclin B1 in NIH/3T3 and TRAMP C1 cells, which led to increased phosphorylation of histone H3, a downstream marker for chromosome condensation and entry into mitosis. We also identified two saRNAs that activated the expression of CXCR4 in primary rat adipose-derived stem cells.
Conclusions/significance: This study demonstrates that RNAa exists in mammalian species other than human. Our findings also suggest that nonhuman primate disease models may have clinical applicability for validating RNAa-based drugs.
Conflict of interest statement
Competing Interests: The authors have declared that no competing interests exist.
Figures
Figure 1. Primate saRNA sequences and target alignments.
Genomic DNA was isolated from WES and COS1 cells. Gene promoter regions surrounding saRNA target sites were sequenced using promoter-specific primers. Indicated are the saRNA sequences aligned to human, Pan troglodytes (PT; chimpanzee), and Cercopithecus aethiops (CA; African green monkey) promoter sequences. Target sites are highlighted in yellow, while non-target sequence is highlighted in blue. Genomic sequences divergent from human are not highlighted. The numerical designation within the name of each saRNA denotes the target location on each gene promoter relative to the transcription start site of the human gene.
Figure 2. RNA activation of different genes in non-human primate cells.
A–G. COS1 and WES cells were transfected with 25 nM of the indicated saRNAs for 3–5 days. Mock samples were transfected in the absence of saRNA. mRNA expression levels were analyzed by real-time PCR and normalized to β-actin. Expression levels were measured as fold induction relative to mock transfections. The results are represented as mean ± SEM of three independent experiments. Species-specific saRNAs were designed to perfectly complement their respective chimpanzee (dsNKX3-1-360-PT) and AGM (dsNKX3-1-360-CA) targeted sequences in WES and COS1 cells, respectively. Statistical significance is indicated (* p<0.05, ** p<0.01) as compared to mock treatments.
Figure 3. Activation of p53 by saRNA causes cell cycle arrest and induction of p21 in WES cells.
A. WES cells were transfected with 25 nM of the indicated saRNAs for 5 days. Protein levels of p53, PARP and p21 were detected by immunoblot analysis. β-actin served as a loading control. B. WES cells were transfected as in A. Cells were collected, stained with propidium iodide, and processed for analysis by flow cytometry to measure DNA content. Shown are examples of resulting FL2A histograms following analysis with FlowJo software. Cell populations distributed in G0/G1, S, and G2/M phases of the cell cycle are indicated. C. WES cells were transfected with 25 nM of the indicated saRNAs for 72 hrs. p21 mRNA expression was analyzed by real-time PCR. The results represent mean ± SEM of two independent experiments and are plotted as fold induction relative to mock transfections.
Figure 4. Ccnb1 activation in mouse cells by promoter-targeting saRNAs.
A. A schematic representation of the mouse Ccnb1 promoter. Indicated is the transcription start site (+1) and locations of the target sites for each putative saRNA. B. saRNAs were transfected into mouse NIH/3T3 and TRAMP C1 cells at 50 nM concentrations for 5 days. Ccnb1 mRNA levels were determined by real-time RT-PCR and normalized to β-actin levels. Results are mean ± SEM of 3 independent experiments. Statistical significance is indicated (* p<0.05, ** p<0.01) as compared to mock treatments. C. NIH/3T3 and TRAMP C1 cells were transfected as in B. Levels of Ccnb1 protein and phospho-histone H3 at serine 10 (p-H3S10) were determined by immunoblot analysis. Representative immunoblots are shown from 3 independent experiments.
Figure 5. RNAa-mediated induction of CXCR4 in primary rat stem cells.
A. A schematic representation of the rat CXCR4 promoter. Indicated are the CpG islands, transcription start site (+1), and locations of the target sites corresponding to each of the five putative saRNAs. B. Each saRNA was transfected at 50 nM concentrations into rADSCs for 4 days. CXCR4 and β-actin mRNA expression levels were assessed by standard RT-PCR. β-actin served as a loading control. C. Expression of CXCR4 was quantified by densitometry and adjusted to β-actin levels. Results are mean ± SEM of 3 independent experiments.
References
- Janowski BA, Younger ST, Hardy DB, Ram R, Huffman KE, et al. Activating gene expression in mammalian cells with promoter-targeted duplex RNAs. Nature Chem Biol. 2007;3:166–173. - PubMed
- Kuwabara T, Hsieh J, Nakashima K, Taira K, Gage FH. A small modulatory dsRNA specifies the fate of adult neural stem cells. Cell. 2004;116:779–793. - PubMed
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