RNA cargoes associating with FMRP reveal deficits in cellular functioning in Fmr1 null mice.
The Fragile X mental retardation-1 (Fmr1) gene encodes a multifunctional protein, FMRP, with intrinsic RNA binding activity. We have developed an approach, antibody-positioned RNA amplification (APRA), to identify the RNA cargoes associated with the in vivo configured FMRP messenger ribonucleoprotein (mRNP) complex. Using APRA as a primary screen, putative FMRP RNA cargoes were assayed for their ability to bind directly to FMRP using traditional methods of assessing RNA-protein interactions, including UV-crosslinking and filter binding assays. Approximately 60% of the APRA-defined mRNAs directly associate with FMRP. By examining a subset of these mRNAs and their encoded proteins in brain tissue from Fmr1 knockout mice, we have observed that some of these cargoes as well as the proteins they encode show discrete changes in abundance and/or differential subcellular distribution. These data are consistent with spatially selective regulation of multiple biological pathways by FMRP.
Miyashiro
Kevin Y
KY
Department of Pharmacology, Department of Psychiatry, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA.
Beckel-Mitchener
Andrea
A
Purk
T Patrick
TP
Becker
Kevin G
KG
Barret
Tanya
T
Liu
Lei
L
Carbonetto
Salvatore
S
Weiler
Ivan Jeanne
IJ
Greenough
William T
WT
Eberwine
James
J
D000818:Animals; D000911:Antibodies, Monoclonal; D015342:DNA Probes / Q000276:immunology; D051860:Fragile X Messenger Ribonucleoprotein 1; D005600:Fragile X Syndrome / Q000235:genetics / Q000378:metabolism*; D066298:In Vitro Techniques; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D009419:Nerve Tissue Proteins / Q000235:genetics* / Q000276:immunology / Q000378:metabolism*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D011687:Purines / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism*; D016601:RNA-Binding Proteins; D013347:Subcellular Fractions
D000911:Antibodies, Monoclonal; D015342:DNA Probes; D051860:Fragile X Messenger Ribonucleoprotein 1; D009419:Nerve Tissue Proteins; D011687:Purines; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; C497684:Fmr1 protein, mouse