Identification of 3’ UTR motifs required for mRNA localization to myelin sheaths in vivo

Abstract

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).

The Akt-mTOR Pathway Drives Myelin Sheath Growth by Regulating Cap-Dependent Translation

“…The localization of mRNAs (Ainger et al, 1993;Thakurela et al, 2016;Yergert et al, 2021), ribosomes (Lunn et al, 1997), and RNA binding proteins (Doll et al, 2020) in distal myelin sheaths support a model where local protein translation promotes myelination. We therefore investigated the possibility that translation factors that are regulated by mTOR signaling are localized to nascent myelin sheaths.…”

“…In support of this possibility, free ribosomes have been observed by electron microscopy in the distal ends of cultured oligodendrocytes (Lunn et al, 1997) and recently hundreds of other myelin resident mRNAs have been identified (Thakurela et al, 2016) and a subset validated in vivo (Yergert et al, 2021).…”

The RNA Binding Protein FMRP Promotes Myelin Sheath Growth

“…Embryos were raised at 28.5°C in embryo medium and staged as hours or days post fertilization (hpf/dpf) according to morphological criteria [53]. Zebrafish lines used in this study included fmr1 hu2787 [34], Tg(sox10:mRFP) vu234 [32], Tg(sox10:tagRFP) co26 [38], Tg(mbp:EGFP-CAAX) co58 [54], and Tg(olig2:EGFP) vu12 [55]. All other constructs were delivered by transient transgenesis to facilitate sparse labeling and single cell analysis.…”

Molecular characterization of myelin basic protein a (mbpa) gene from red-bellied pacu (Piaractus brachypomus)

“…Oligodendrocytes of the central nervous system (CNS) and Schwann cells in the peripheral nervous system (PNS) generate the myelin sheath, covering axons to a proper propagation of neural signal at a high speed with a diminution of ionic leakiness, as well as preserving structures from degeneration [ 1 – 3 ]. In fish, myelination is conserved across species at a molecular and cellular level and also showed high conserved mechanisms with mammals [ 4 ].…”