CHARACTERIZATION OF RIBONUCLEOPROTEIN COMPLEXES AND THEIR BINDING-SITES ON THE NEUROFILAMENT LIGHT SUBUNIT MESSENGER-RNA

Levels of neurofilament (NF) gene expression are important determinant s of basic neuronal properties, but overexpression can lead to motoneu ron degeneration in transgenic mice. In a companion study (Canete-Sole r, R., Schwartz, M. L., Hua, Y., and Schlaepfer, W. W. (1998) J. Biol. Chem. 273, 12650-12654), we show that levels of NF expression are reg ulated by altering mRNA stability and that stability determinants are present in the 3'-coding region (3'-CR) and 3'-untranslated region (3' -UTR) of the NF light subunit (NF-L) transcript. This study characteri zes the ribonucleoprotein complexes that bind to the NF-L mRNA when cy toplasmic brain extracts are incubated with radioactive probes. Gel re tardation assays reveal ribonucleoprotein complexes that are selective ly competed with poly(C) or poly(U))/poly(A) homoribopolymers and are referred to as C-binding and U/A-binding complexes, respectively. The C-binding complex forms on the proximal 45 nucleotides of 3'-UTR, but its assembly is markedly enhanced by 23 nucleotides of flanking 3'-CR sequence. U/A-binding complexes form at multiple binding sites in the 3'-CR and 3'-UTR, A pattern of reciprocal binding suggests that the C- binding and U/A-binding complexes interact and may compete for common components or binding sites. Cross-linking studies reveal unique polyp eptides in the C-binding and U/A-binding complexes. The findings provi de the basis for probing mechanisms regulating NF-L mRNA stability and the relationship between NF overexpression and motoneuron degeneratio n in transgenic mice.