Dynamic composition of myelin basic protein mRNA-containing ribonucleoprotein complexes

Abstract

Myelin Basic Protein (MBP) is a major component of the myelin sheath orchestrating the assembly of compact myelin in the central nervous system and thus ensuring saltatory signal propagation and maintenance of the neuronal network. MBP expression is precisely regulated at the posttranscriptional level and depends on the assembly of MBP mRNA-containing granules, which mediate transport and localized translation at the axoglial contact site. This study focused on the identification of the dynamic composition of these RNP complexes and a detailed functional analysis of the previously identified potential MBP mRNA-associating proteins DDX5 and FUS. DDX5 was shown to associate with MBP mRNA in subpopulations of cytoplasmic RNP complexes. In oligodendroglial cells DDX5 functioned as an inhibitor of MBP protein synthesis, acting at the posttranscriptional level and depending on the DDX5 helicase activity. Consequently, knockdown of DDX5 in primary OL correlates with the elevation of MBP protein levels and an imbalance of the known MBP-associating RNA-binding proteins hnRNP A2 and hnRNP F. In addition, DDX5-knockdown selectively increased the expression of the exon 2-containing MBP isoforms 17.22-kDa and 21.5-kDa, possibly by affecting alternative splicing of the pre-mRNA. Alteration of FUS levels did not show a major impact on the myelin protein expression, although MBP mRNA levels were slightly reduced in line with changes in the expression of MBP mRNA-associated proteins hnRNP A2 and DDX5. During oxidative stress, FUS localized to oligodendroglial stress granules and FUS levels inversely correlated with MBP RNA stability upon increasing concentrations of sodium arsenite. To further examine the dynamic composition of MBP mRNA complexes in a more RNA-centric approach, the MS2-RNA-labeling system was adapted to MBP mRNA. The introduction of MS2 hairpin loops into the MBP transcript allowed its visualization and the purification of associated MBP mRNP complexes. Oligodendroglial cell lines stably expressing moderate levels of MS2-labeled MBP14-MS2 mRNA were generated and single molecule FISH confirmed localization and the physiological behavior of the transcript, which reacted to cellular cues such as oxidative stress. The following affinity purification of MBP14-MS2 mRNP complexes under oxidative stress conditions resulted in the identification of numerous candidates potentially playing a role in stress-dependent RNA granule formation and the regulation of MBP. This list includes several proteins connected to neurodegenerative or psychiatric diseases and may thus aid in shedding light on mechanisms regulating MBP expression during OL maturation and myelination in health and disease.