Title

Myelin basic protein is a sequence-specific RNA-binding protein

Date of Completion

January 1997

Keywords

Biology, Molecular|Biology, Neuroscience

Degree

Ph.D.

Abstract

Myelin basic protein (MBP) accounts for 30% to 40% of the protein in CNS myelin. MBP has been localized immunocytochemically to the major dense line of myelin, which corresponds to the apposed cytoplasmic leaflets of the myelin membrane. It is believed that MBP play a role in mediating close membrane apposition during myelin compaction.^ In this work we show that: (1) MBP binds RNA both on nitrocellulose and in solution. (2) Recombinant 18.5kD mouse MBP binds RNA with higher affinity than other MBP isoforms. Insertion of exon 2, or deletion of exon 6, in 21.5kD, 17kD and 14kD MBP isoforms reduces the binding affinity. (3) UV crosslinking and tryptic peptide mapping indicates that three tryptic peptides in 18.5kD MBP contact RNA. (4) Systematic Evolution of Ligands by EXponential enrichment (SELEX) analysis indicates that MBP has higher affinity to selected RNA sequences than to a pool of random sequences. Selected sequences have significantly higher GC content than the pool of random sequence (t $<$ 0.0005). Many selected RNA sequences contain one or more copies of the sequence, CAGUGU, or related sequences. The selected RNA sequence with the highest affinity for MBP contains three copies of CAGUGU. The dissociation constant (Kd) of 18.5kD MBP for these sequences in 250mM NaCl, pH7.2 is $10\sp{-7}$ to $10\sp{-8}$M. Homologous sequences are found in connexin 32 (Cx32) and MBP mRNA. (5) Computer modeling, using neural network secondary structure prediction and homology modeling reveal that the 18.5kD MBP could adopt a characteristic alpha-beta sandwich fold, which is found in U1A and other RNA-binding proteins.^ MBP-RNA interactions may be involved in Cx32 and/or MBP gene regulation, or may play a role in transport and/or localization of their mRNAs. ^