p190RhoGEF Binds to a destabilizing element in the 3' untranslated region of light neurofilament subunit mRNA and alters the stability of the transcript.
Stabilization of neurofilament (NF) mRNAs plays a major role in regulating levels of NF expression and in establishing axonal size and rate of axonal conduction. Previous studies have identified a 68-nucleotide destabilizing element at the junction of the coding region and 3' untranslated region of the light NF subunit (NF-L) mRNA. The present study has used the destabilizing element (probe A) to screen a rat brain cDNA library for interactive proteins. A cDNA clone encoding 1068 nucleotides in the C-terminal domain of p190RhoGEF (clone 39) was found to bind strongly and specifically to the RNA probe. The interaction was confirmed using a glutathione S-transferase/clone 39 fusion protein in Northwestern, gel-shift, and cross-linkage studies. The glutathione S-transferase/clone 39 fusion protein also enhanced the cross-linkage of a major 43-kDa protein in brain extract to the destabilizing element. Functional studies on stably transfected neuronal cells showed that p190RhoGEF expression increased the half-life of a wild-type NF-L mRNA but did not alter the half-life of a mutant NF-L mRNA lacking the destabilizing element. The findings reveal a novel interactive feature of p190RhoGEF that links the exchange factor with NF mRNA stability and regulation of the axonal cytoskeleton.
Cañete-Soler
R
R
Department of Pathology and Laboratory Medicine, University of Pennsylvania Medical School, Philadelphia, Pennsylvania 19104, USA. racansol@mail.med.upenn.edu
Wu
J
J
Zhai
J
J
Shamim
M
M
Schlaepfer
W W
WW
D020413:3' Untranslated Regions; D000595:Amino Acid Sequence; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D020662:Guanine Nucleotide Exchange Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016900:Neurofilament Proteins / Q000235:genetics*; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D015347:RNA Probes; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D012097:Repressor Proteins; D017386:Sequence Homology, Amino Acid
D020413:3' Untranslated Regions; C513040:Arhgap35 protein, rat; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D020662:Guanine Nucleotide Exchange Factors; D016900:Neurofilament Proteins; D009687:Nuclear Proteins; D015347:RNA Probes; D012333:RNA, Messenger; D012097:Repressor Proteins