Dicer‐Derived MicroRNAs Are Utilized by the Fragile X Mental Retardation Protein for Assembly on Target RNAs

Abstract

In mammalian cells, fragile X mental retardation protein (FMRP) has been reported to be part of a microRNA (miRNA)-containing effector ribonucleoprotien (RNP) complex believed to mediate translational control of specific mRNAs. Here, using recombinant proteins, we demonstrate that human FMRP can act as a miRNA acceptor protein for the ribonuclease Dicer and facilitate the assembly of miRNAs on specific target RNA sequences. The miRNA assembler property of FMRP was abrogated upon deletion of its single-stranded (ss) RNA binding K-homology domains. The requirement of FMRP for efficient RNA interference (RNAi) in vivo was unveiled by reporter gene silencing assays using various small RNA inducers, which also supports its involvement in an ss small interfering RNA (siRNA)-containing RNP (siRNP) effector complex in mammalian cells. Our results define a possible role for FMRP in RNA silencing and may provide further insight into the molecular defects in patients with the fragile X syndrome.

Human Dicer C-terminus functions as a 5-lipoxygenase binding domain

“…Recombinant human Dicer full-length or deletion mutant 1650-1912 proteins were incubated in the absence or presence of proteinase K for 10 min or human 5LO for 30 min prior to addition of a randomly, 32 P-labeled microRNA precursor substrate (pre-let-7a-3) in a reaction buffer containing 20 mM Tris•HCl pH 7.5, 5 mM MgCl 2 , 1 mM dithiothreitol (DTT), 1 mM ATP, 5% Superase•In (Ambion), at 37°C for 1 h. The resulting RNA products were analyzed by denaturing PAGE and autoradiography, as described previously [30,31].…”

MiRNAs-based Gene Therapy on the Horizon: Novel and Effective Therapeutic Advancement

“…Biochemical studies using recombinant FMRP and FXR1P suggest that FMR1P, FXR1P, and FXR2P proteins perform as acceptor molecules for Dicer-processed miRNAs and help direct gathering of miRNA [80]. The dAGO1 is indispensable for miRNA-directed gene silencing in Drosophila, showing a functional interaction with dFMR1, strengthens the dispute that failing in miRNA-mediated translation repression during neural development could result into fragile X syndrome [81,82].…”

Protein Components of the microRNA Pathway and Human Diseases

“…Our group recently showed that human FMRP can act as a miRNA acceptor protein for the RNase III Dicer and facilitate assembly of miRNAs on specific target RNA sequences (95). We have proposed a model in which FMRP could facilitate miRNA assembly on target mRNAs.…”

“…This may be expected given the requirement of FMRP for efficient small RNA-guided gene regulation (95). Elucidation of the exact role and function of FMRP in miRNA-guided gene regulation may hold key to determining the molecular basis of the fragile X syndrome and establishing a causal link between dysfunction of the RNA-silencing machinery and a human genetic disease.…”