Myelin is a specialized membrane produced by oligodendrocytes that insulates and supports axons. Oligodendrocytes extend numerous cellular processes, as projections of the plasma membrane, and simultaneously wrap multiple layers of myelin membrane around target axons. Notably, myelin sheaths originating from the same oligodendrocyte are variable in size, suggesting local mechanisms regulate myelin sheath growth. Purified myelin contains ribosomes and hundreds of mRNAs, supporting a model that mRNA localization and local protein synthesis regulate sheath growth and maturation. However, the mechanisms by which mRNAs are selectively enriched in myelin sheaths are unclear. To investigate how mRNAs are targeted to myelin sheaths, we tested the hypothesis that transcripts are selected for myelin enrichment through consensus sequences in the 3′ untranslated region (3′ UTR). Using methods to visualize mRNA in living zebrafish larvae, we identified candidate 3′ UTRs that were sufficient to localize mRNA to sheaths and enriched near growth zones of nascent membrane. We bioinformatically identified motifs common in 3′ UTRs from 3 myelin-enriched transcripts and determined that these motifs are required and sufficient in a context-dependent manner for mRNA transport to myelin sheaths. Finally, we show that 1 motif is highly enriched in the myelin transcriptome, suggesting that this sequence is a global regulator of mRNA localization during developmental myelination.

Myelin, a specialized membrane produced by oligodendrocytes, insulates and supports axons.Oligodendrocytes extend numerous cellular processes to wrap multiple axons, and myelin sheaths differ in length and thickness. Notably, neuronal activity can modify sheath characteristics. How myelin formation is controlled at sites distant from oligodendrocyte cell bodies is not well known. Using zebrafish, we tested the possibility that 3' untranslated regions (UTRs) influence localization of myelin mRNAs, thereby enabling localized gene expression. By visualizing mRNA in living animals, we found that some candidate 3' UTRs were enriched in myelin sheaths and localized near growth zones of nascent myelin membrane. Injecting zebrafish larvae with tetrodotoxin to block action potentials reduced the amounts of mRNAs localized to myelin in a 3' UTR-dependent manner. Our data indicate that 3' UTRs contain information for neuronal activity-regulated localization of mRNAs to myelin, suggesting that changes in sheath characteristics result from mRNA regulation within nascent sheaths.Keywords: myelin, oligodendrocyte, mRNA, zebrafish 2010) and are locally translated to control axon growth and synapse formation (Kang and Schuman, 1996;Huber, Kayser and Bear, 2000;Zhang and Poo, 2002;Leung et al., 2006). Frequently, mRNA localization in neurons is determined by 3' UTRs (Taliaferro et al., 2016). RNA sequencing recently revealed that myelin purified from mouse brain has hundreds of transcripts . Many of these mRNAs encode proteins that function in cellular differentiation, translation regulation and cell-cell signaling, which might be important for sheath formation and myelination. Here we describe experiments to test the hypothesis that neuronal activity promotes myelin sheath localization of mRNAs via their 3' UTRs. RESULTS mbpa mRNA accumulates at distinct sites within nascent myelin sheathsMyelin sheaths have transcripts encoding Myelin Basic Protein (MBP) (Kristensson et al., 1986;Trapp et al., 1987;Ainger et al., 1993) but we lack information about the distribution of MBP mRNA within sheaths in vivo. We therefore began by using techniques to visualize and quantify the subcellular distribution of mRNA encoded by mbpa, a zebrafish ortholog of human and rodent MBP. First, we performed single molecule fluorescent in situ RNA hybridization (smFISH) (Femino et al., 1998;Raj et al., 2008) to detect mbpa transcripts during early stages of larval development. To mark oligodendrocytes and myelinating sheaths we used Tg(mbpa:EGFP-CAAX) larvae, which express a membrane-tethered EGFP under control of mbpa regulatory DNA. In the zebrafish hindbrain at 4 days post-fertilization (dpf), a time of active axon ensheathment and myelination (Czopka, Ffrench-Constant and Lyons, 2013), mbpa mRNA occupied cell bodies and myelin sheaths ( Figure 1A).