title: Characterization of ribonucleoprotein complexes and their binding sites on the neurofilament light subunit mRNA.
abstract: Levels of neurofilament (NF) gene expression are important determinants of basic neuronal properties, but overexpression can lead to motoneuron degeneration in transgenic mice. In a companion study (Cañete-Soler, 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 regulated 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 characterizes the ribonucleoprotein complexes that bind to the NF-L mRNA when cytoplasmic brain extracts are incubated with radioactive probes. Gel retardation assays reveal ribonucleoprotein complexes that are selectively 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 polypeptides in the C-binding and U/A-binding complexes. The findings provide the basis for probing mechanisms regulating NF-L mRNA stability and the relationship between NF overexpression and motoneuron degeneration in transgenic mice.
authors.affiliation: Division of Neuropathology, Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA. racansol@mail.med.upenn.edu
authors.forename: R
authors.initials: R
authors.lastname: Cañete-Soler
authors.forename: M L
authors.initials: ML
authors.lastname: Schwartz
authors.forename: Y
authors.initials: Y
authors.lastname: Hua
authors.forename: W W
authors.initials: WW
authors.lastname: Schlaepfer
mesh_terms: D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D001704:Biopolymers; D051379:Mice; D008969:Molecular Sequence Data; D016900:Neurofilament Proteins / Q000235:genetics* / Q000378:metabolism; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D012261:Ribonucleoproteins / Q000378:metabolism*; D012689:Sequence Homology, Nucleic Acid
chemical_list: D001704:Biopolymers; D016900:Neurofilament Proteins; D012333:RNA, Messenger; D012261:Ribonucleoproteins; C044882:neurofilament protein L