Fragile × syndrome is caused by loss of expression of the FMRP protein involved in the control of a large number of mRNA targets [1]. The Drosophila ortholog dFXR interacts with a protein complex that includes Argonaute2, an essential component of the RNA-induced silencing complex (RISC) [2]. Furthermore dFXR associates with Dicer, another essential processing enzyme of the RNAi pathway [3]. Both microRNA and microRNA precursors can co-immunoprecipitate with dFXR [3], and it was shown that FMRP can act as a miRNA acceptor protein for the ribonuclease Dicer and facilitate the assembly of miRNAs on specific target RNA sequences [4, 5].
Consequently, it has been suggested that the Fragile × syndrome may be due to a defect in an RNAi-related apparatus [6] although in Drosophila S2 cells dFXR is not required for an RNAi response [7]. RNAi is a naturally occurring mechanism of gene regulation that induces sequence-specific knock-down of gene expression at the post-transcriptional level [8]. Regulation of gene expression by RNAi utilizes endogenous cellular pathways in which double-stranded RNA molecules produced from endogenous or foreign DNA are processed into short double-stranded RNA molecules of 21-23 nucleotides. These small interfering RNA (siRNA) molecules are incorporated into RISC that facilitates degradation of the target.
Recently the RISC function was analysed in mouse embryo fibroblasts (MEFs) from FMRP wildtype or knockout (FMR1-/-) littermates [9]. Here it was shown that cells lacking FMRP have normal RISC activity, since FMRP and RISC were associated with distinct pools of mRNAs.
We have investigated the RNAi response in Fragile × patient cells lacking FMRP compared to fibroblasts from normal individuals. Primary fibroblasts from patients and controls were co-transfected with a luciferase plasmid and siRNA oligonucleotides against the luciferase gene. As control for the transfection frequency, cells were also transfected with the plasmid lacZ encoding the beta-galactosidase. As seen in Fig 1 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 (two-tailed T-test). However, we cannot rule out that the short term kinetics of the RNAi reponse in normal and patient cells differ.