Abstract
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.
Highlights
Neurofilaments (NFs)1 are a major component of the neuronal cytoskeleton and principal determinant of axonal size [1]
C-terminal Domain of p190RhoGEF (Clone 39) Binds an RNA Probe to the Destabilizing Element of the NF light subunit (NF-L) mRNA—To identify RNA-binding proteins that participate in regulating the stability of NF-L mRNA, a rat brain cDNA library was probed with the 68-nt sequence to the stability determinant of NF-L mRNA
A functional role of p190RhoGEF in altering NF-L mRNA stability was tested by examining the effects of p190RhoGEF expression on levels of wild-type NF-L mRNA or levels of mutant NF-L mRNA lacking the destabilizing element in transcript [11]
Summary
Neurofilaments (NFs)1 are a major component of the neuronal cytoskeleton and principal determinant of axonal size [1]. The present study has used the destabilizing element (probe A) to screen a rat brain cDNA library for interactive proteins. The glutathione S-transferase/clone 39 fusion protein enhanced the cross-linkage of a major 43-kDa protein in brain extract to the destabilizing element.
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