Similar poly(C)-sensitive RNA-binding complexes regulate the stability of the heavy and light neurofilament mRNAs

Abstract

The potential role of RNA processing in regulating neurofilament (NF) subunit expression and in mediating the neuropathic effects of NF transgenes was explored by determining whether similar regulatory elements and cognate binding factors are present in NF mRNAs. Gel-shift studies were used to compare RNA-binding complexes that assemble on the 3′UTR of the heavy (NF-H), mid-sized (NF-M) and light (NF-L) NF mRNAs when radioactive RNA probes are incubated with high-speed supernatants (S100) of rat brain homogenates. RNA-binding complexes were characterized by their rate of migration in non-denaturing gels and by their ability to be competed with specific homoribopolymers. Similar RNA-binding complexes formed on probes to the 3′UTRs of NF-L and NF-H mRNAs. The complexes were competed with poly(C) and are referred to as poly(C)-sensitive complexes. Their binding sites were localized to a 36 nt sequence in the mid-distal region of the NF-H 3′UTR and to a 45 nt sequence at the proximal edge of the 3′UTR of the NF-L transcript. Although the binding sites showed limited sequence homology, the complexes were cross-competed with unlabeled probes and radioactivity in either probe was cross-linked to a 43 kDa protein. The 43 kDa protein also bound directly to NF-L and NF-H probes in Northwestern blots. Functional studies showed that deletion of the binding sites markedly increased expression of a luciferase reporter gene containing the 3′UTR of NF-L or NF-H by stabilizing the fusion transcripts. Point mutations in the NF-H binding site which prevented formation of the poly(C)-sensitive complex also stabilized the fusion mRNA. The findings reveal a common destabilizing element in the 3′UTR of NF-L and NF-H mRNAs that may be important in coordinating NF subunit expression and in mediating the neuropathic effects of the NF-L and NF-H transgenes in transgenic mice.