Purified recombinant Fmrp exhibits selective RNA binding as an intrinsic property of the fragile X mental retardation protein.
Fragile X syndrome is caused by the transcriptional silencing of the FMR1 gene due to a trinucleotide repeat expansion. The encoded protein, Fmrp, has been found to be a nucleocytoplasmic RNA-binding protein containing both KH domains and RGG boxes that associates with polyribosomes as a ribonucleoprotein particle. RNA binding has previously been demonstrated with in vitro-translated Fmrp; however, it remained uncertain whether the selective RNA binding observed was an intrinsic property of Fmrp or required an associated protein(s). Here, baculovirus-expressed and affinity-purified FLAG-tagged murine Fmrp was shown to bind directly to both ribonucleotide homopolymers and human brain mRNA. FLAG-Fmrp exhibited selectivity for binding poly(G) > poly(U) >> poly(C) or poly(A). Moreover, purified FLAG-Fmrp bound to only a subset of brain mRNA, including the 3' untranslated regions of myelin basic protein message and its own message. Recombinant isoform 4, lacking the RGG boxes but maintaining both KH domains, was also purified and was found to only weakly interact with RNA. FLAG-purified I304N Fmrp, harboring the mutation of severe fragile X syndrome, demonstrated RNA binding, in contrast to previous suggestions. These data demonstrate the intrinsic property of Fmrp to selectively bind RNA and show FLAG-Fmrp as a suitable reagent for structural characterization and identification of cognate RNA ligands.
Brown
V
V
Howard Hughes Medical Institute, Emory University School of Medicine, Atlanta, Georgia 30322, USA.
Small
K
K
Lakkis
L
L
Feng
Y
Y
Gunter
C
C
Wilkinson
K D
KD
Warren
S T
ST
D000818:Animals; D051860:Fragile X Messenger Ribonucleoprotein 1; D005600:Fragile X Syndrome / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D008607:Intellectual Disability / Q000235:genetics* / Q000378:metabolism*; D051379:Mice; D009419:Nerve Tissue Proteins / Q000302:isolation & purification / Q000378:metabolism*; D011068:Poly G / Q000378:metabolism; D011485:Protein Binding; D012313:RNA / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D011817:Rabbits; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D014466:Ultraviolet Rays
D051860:Fragile X Messenger Ribonucleoprotein 1; D009419:Nerve Tissue Proteins; D011068:Poly G; D012313:RNA; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; C497683:FMR1 protein, human; C497684:Fmr1 protein, mouse