Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2009 Sep 9:2:177.
doi: 10.1186/1756-0500-2-177.

Normal RNAi response in human fragile x fibroblasts

Charlotte Madsen  1 , Karen Grønskov, Karen Brøndum-Nielsen, Thomas G Jensen
Affiliations

Normal RNAi response in human fragile x fibroblasts

Charlotte Madsen et al. BMC Res Notes. .

Abstract

Background: Fragile x syndrome is caused by loss of expression of the FMRP protein involved in the control of a large number of mRNA targets. The Drosophila ortholog dFXR interacts with a protein complex that includes Argonaute2, an essential component of the RNA-induced silencing complex (RISC). Furthermore dFXR associates with Dicer, another essential processing enzyme of the RNAi pathway. Both microRNA and microRNA precursors can co-immunoprecipitate with dFXR. Consequently it has been suggested that the Fragile x syndrome may be due to a defect in an RNAi-related apparatus.

Findings: We have investigated the RNAi response in Fragile x patient cells lacking FMRP compared with normal controls. RNAi responses were successfully detected, but no statistically significant difference between the response in normal cells compared to patients cells was found - neither one nor two days after transfection.

Conclusion: Our data show that in human fibroblasts from Fragile x patients lacking FMRP the RNAi response is not significantly impaired.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Relative luciferase expression showing the RNAi response in human fibroblasts. Primary human skin fibroblasts from two fragile × patients (Fra-X1 and 2) and a normal control person with a normal karyotype were transfected with the plasmid pGL3-basic (Promega) encoding luciferase and siRNA against the luciferase (GL3 siRNA, MWG-Biotech). GL3 siRNA targets the luciferase sequence 5'-CUUACGCUGAGUACUUCGATT -3' [11]. As control for the transfection frequency, cells were also transfected with the plasmid pCMVβ (Clontech) encoding beta-galactosidase. Luciferase and beta-galactosidase activities were measured one and two days after transfection using kits from Pierce and Promega, respectively. Transfections were performed in triplicates using X-tremeGeNE siRNA Transfection Reagent as suggested by the manufacturer (Roche). Subconfluent cells in 25 cm2 tissue culture flasks were transfected with a mixture of 100 μl transfection reagent diluted in Opti-MEM together with 6 μg of each of the reporter plasmids and 2 μg of the siRNA oligonucleotides. The primary fibroblasts were obtained from 2 patients with a trinucleotide expansion leading to a full mutation in the FMR1 gene, and, thus, no FMRP protein detectable using western blotting (data not shown). A negative control siRNA with no significant homology to any known gene sequences from mouse, rat, or human was also purchased from MWG-Biotech. Shown on the Y-axes are the luciferase expression divided by the beta-galactosidase expression, setting the ratio to 100 for cells transfected without siRNA. Error bars: Standard deviation. The experiments comply with the National regulations on cells from approved biobanks. The primary human fibroblasts were cultured according to standard procedures and used in early passages.
See this image and copyright information in PMC

Similar articles

See all similar articles

References

    1. Tan H, Li H, Jin P. RNA-mediated pathogenesis in fragile X-associated disorders. Neurosci Lett. 2009 - PMC - PubMed
    1. Caudy AA, Myers M, Hannon GJ, Hammond SM. Fragile X-related protein and VIG associate with the RNA interference machinery. Genes Dev. 2002;16:2491–2496. doi: 10.1101/gad.1025202. - DOI - PMC - PubMed
    1. Jin P, Zarnescu DC, Ceman S, Nakamoto M, Mowrey J, Jongens TA, Nelson DL, Moses K, Warren ST. Biochemical and genetic interaction between the fragile × mental retardation protein and the microRNA pathway. Nat Neurosci. 2004;7:113–117. doi: 10.1038/nn1174. - DOI - PubMed
    1. Plante I, Davidovic L, Ouellet DL, Gobeil LA, Tremblay S, Khandjian EW, Provost P. Dicer-Derived MicroRNAs Are Utilized by the Fragile × Mental Retardation Protein for Assembly on Target RNAs. J Biomed Biotechnol. 2006;2006:64347. - PMC - PubMed
    1. Plante I, Provost P. Hypothesis: A Role for Fragile × Mental Retardation Protein in Mediating and Relieving MicroRNA-Guided Translational Repression? J Biomed Biotechnol. 2006;2006:16806. - PMC - PubMed

LinkOut - more resources