Abstract:The targeting of mRNAs to specific subcellular locations is believed to facilitate the rapid and selective incorporation of their protein products into complexes that may include membrane organelles. In oligodendrocytes, mRNAs that encode myelin basic protein (MBP) and select myelin-associated oligodendrocytic basic proteins (MOBPs) locate in myelin sheath assembly sites (MSAS). To identify additional mRNAs located in MSAS, we used a combination of subcellular fractionation and suppression subtractive hybridization. More than 50% of the 1,080 cDNAs that were analyzed were derived from MBP or MOBP mRNAs, confirming that the method selected mRNAs enriched in MSAS. Of 90 other cDNAs identified, most represent one or more mRNAs enriched in rat brain myelin. Five cDNAs, which encode known proteins, were characterized for mRNA size(s), enrichment in myelin, and tissue and developmental expression patterns. Two of these, peptidylarginine deiminase and ferritin heavy chain, have recognized roles in myelination. The corresponding mRNAs were of different sizes than the previously identified mRNA, and they had tissue and development expression patterns that were indistinguishable from those of MBP mRNA. Three other cDNAs recognize mRNAs whose proteins (SH3p13, KIF1A, and dynein light intermediate chain) are involved in membrane biogenesis. Although enriched in myelin, the tissue and developmental distribution patterns of these mRNAs differed from those of MBP mRNA. Six other cDNAs, which did not share significant sequence homology to known mRNAs, were also examined. The corresponding mRNAs were highly enriched in myelin, and four had tissue and developmental distribution patterns indistinguishable from those of MBP mRNA. These studies demonstrate that MSAS contain a diverse population of mRNAs, whose locally synthesized proteins are placed to contribute to myelin sheath assembly and maintenance. Characterization of these mRNAs and proteins will help provide a comprehensive picture of myelin sheath assembly.

The nervous systems of all jawed animals are fully myelinated. During development, the largest caliber axons are separated from smaller neighbors by oligodendrocyte (OL) processes that envelop them with compacted multilayered myelin sheaths. Usually a single OL myelinates many axon segments. Proteins used in the sheaths are synthesized at two sites, OL soma and OL processes. Myelin basic protein (MBP) and isoforms of a second small, highly basic protein, myelin-associated oligodendrocytic basic protein (MOBP) are the only proteins known to be synthesized in OL processes. The synthesis of these two proteins is ideally situated to coordinate the compaction of the cytoplasmic leaflets into the major dense line. As a prelude to their synthesis in OL processes, the mRNAs encoding MBP and MOBP must be transported to each of the sites where myelin sheaths are formed. Morphologically, these sites are thin cytoplasmic fingers, called outer tongue processes, that overlie the compacted myelin. When one homogenizes nervous tissue, these cytoplasmic fingers become entrapped in vesicles that form from compacted myelin lamellae. The resulting myelin vesicles are readily purified by subcellular fractionation. Because they trap cytoplasm derived from OL processes, they have high levels of MBP mRNA (1). In contrast, they contain relatively little of the mRNAs that originate in other neural cell compartments, including OL soma, astrocytes, neuronal soma, and their dendritic processes (2, 3). We used mRNAs purified from a low-speed supematant (S) of homogenized rat brain and mRNAs purified from the myelin fraction (M)-material that accumulates at a 0.25 M sucrose/O.85 M sucrose interface-as starting materials for suppression-subtractive hybridization (4). Briefly, the "S" mRNA is used to make double-stranded cDNA "driver," and "M" mRNA is used to make "M" cDNA tester. Both cDNAs are digested with RsaI to make small pieces. Different adaptors are ligated to each of two separate batches of digested "M" cDNAs. One round of hybridization is performed in which each batch of "M" cDNA is melted and annealed with excess "S" cDNA. A second hybridization is performed with the individual hybridization reactions combined. Then, the double-stranded cDNAs, which are derived from the separate testers (i.e., they have different adaptors at each end), are selectively amplified by nested PCR. All of the above protocols follow the Clontech kit manual. The PCR product, which represents mRNAs enriched in myelin, is incorporated into vector, transformed into bacteria, and colonies that represent individual cDNAs are screened by southern blot hybridization with full