RNA switch model for localization and translation of the myelin basic protein mRNA

Abstract

Oligodendrocytes myelinate the central nervous system by extending cellular projections that ensheath axons and elongate to form lipid-rich myelin. Classic studies visualizing RNA dynamics showed that myelin basic protein (MBP), one of the most abundant myelin proteins, is locally synthesized at the myelin sheath through the transport and local translation of Mbp mRNA. Mbp transport requires its 1.5-kb 3' untranslated region (3' UTR) and prior work identified candidate sub-sequences that may act as cis-acting transport stimulating RNA elements, including one with putative secondary structure. Here, a high-throughput reporter assay, dimethyl sulfate (DMS)-based RNA structure probing , and microscopy in primary rat oligodendrocytes identify a structured 127-nt region that we name the Mbp localization signal (MLS) as both necessary and sufficient for RNA enrichment to oligodendrocyte projections. Lysate pulldown experiments further identify hnRNP-F - a known constituent of the Mbp RNA granule that can suppress mRNA translation - as associated with the MLS; paradoxically, binding of this protein should compete with the ordered MLS RNA structure. These results suggest a model in which the MLS switches between two RNA conformations with distinct protein partners during the transition from Mbp mRNA transport to Mbp translation at the myelin sheath. Such regulation of RNA behavior by structure switching may generalize to other eukaryotic mRNAs whose behaviors shift across space and time.

Figure 1:

The Mbp localization signal (MLS). Schematics of (A) DMS-MaPseq protocol for mapping the secondary structure of the Mbp 3’ UTR in primary oligodendrocytes and (B) SLAP-seq to test a library of 3’ UTR sequences in their ability to stimulate reporter enrichment to oligodendrocyte processes. (C) RNA Transport Sequence (RTS), RNA Localization Region (RLR) and MLS locations along Mbp 3’ UTR. (D) In-cell DMS reactivity (top) and reactivity-guided secondary structure (bottom). Arcs represent base pairing between distal nucleotides. (E) Secondary structure of the MLS subsequence. (F). SLAP-seq reporter enrichment of windows along the Mbp 3’ UTR in cell bodies vs. projections. (G)Representative micrographs from reporter assay testing MLS role in peripheral RNA enrichment (scale bar = 30 μm), and (H) quantitative analysis supporting MLS necessity and sufficiency.

Figure 2.

MLS is necessary and sufficient for distal localization of an EGFP reporter in primary oligodendrocytes. (A) Representative micrographs from reporter assay testing MLS role in peripheral RNA enrichment (scale bar = 30 μm), and (B) quantitative analysis supporting MLS necessity and sufficiency.

Figure 3.

The MLS is a cis-acting inhibitor of RNA translation that interacts with hnRNP-F. (A) Volcano plot of mass spectrometry results of MLS RNA pulldown vs a scrambled negative control. hnRNP-F is significantly enriched at a threshold of p_adj < 0.01 (above dashed line). TRiC complex proteins (green) are also enriched.

Figure 4.

(A) Representative micrographs of an oligodendrocyte expressing cytoplasmic EGFP and stained with anti-hnRNP-F antibody (scale bar = 50 μm). In contrast to cytoplasmic EGFP, hnRNP-F is highly punctate, localizing both proximally and at the leading edge. (B)Testing MLS necessity and sufficiency in regulating reporter translation. Signal is the ratio of EGFP fluorescence to the number of reporter RNA copies per cell. Addition of the full-length Mbp 3’ UTR or the MLS to the 3’ end of an EGFP reporter dramatically reduces per-RNA translational output. Deletion of the MLS from the full-length Mbp 3’ UTR partially rescues translation, indicating that the MLS is sufficient for translational suppression, but not necessary. (C) Architecture of the hNRNP-F protein, which contains three qRRM RNA-binding domains with sequence specificity of 5’-GGG-3’. Enlarged MLS structure with GGG(A) sub-sequences highlighted. (D) Conservation of GGG(A) motif (purple boxes) in Mbp 3’UTR across mammalian species. (E) RNA switch model: during Mbp mRNA granule transport into oligodendrocyte projections, hnRNP-F binds to GGG(A) sequences in the unstructured Mbp 3’UTR, inhibiting translation. Once the granule localizes to the periphery of the cell, Fyn kinase phosphorylates hnRNP-F, resulting in its release from Mbp RNA and allowing the folding of the mRNA into the predicted translation-ready secondary structure.