The RNA-binding protein SFPQ orchestrates an RNA regulon to promote axon viability


To achieve accurate spatiotemporal patterns of gene expression, RNA-binding proteins (RBPs) guide nuclear processing, intracellular trafficking and local translation of target mRNAs. In neurons, RBPs direct transport of target mRNAs to sites of translation in remote axons and dendrites. However, it is not known whether an individual RBP coordinately regulates multiple mRNAs within these morphologically complex cells. Here we identify SFPQ (splicing factor, poly-glutamine rich) as an RBP that binds and regulates multiple mRNAs in dorsal root ganglion sensory neurons and thereby promotes neurotrophin-dependent axonal viability. SFPQ acts in nuclei, cytoplasm and axons to regulate functionally related mRNAs essential for axon survival. Notably, SFPQ is required for coassembly of LaminB2 (Lmnb2) and Bclw (Bcl2l2) mRNAs in RNA granules and for axonal trafficking of these mRNAs. Together these data demonstrate that SFPQ orchestrates spatial gene expression of a newly identified RNA regulon essential for axonal viability.

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Figure 1: SFPQ binds axonal mRNAs.
Figure 2: SFPQ regulates localization of axonal mRNAs.
Figure 3: SFPQ is required for axonal trafficking of Lmnb2 and Bcl2l2 mRNA.
Figure 4: SFPQ is required for coassembly of Bcl2l2 and Lmnb2 mRNA in RNA transport granules.
Figure 5: SFPQ binds motifs in the 3′ UTRs of Bcl2l2 and Lmnb2.
Figure 6: SFPQ regulates functionally related genes to promote axonal viability.


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We thank members of the Segal laboratory, A. Eisner, M. McClintock and M. Greenberg for discussions, and G. McGregor (University of California, Irvine) for providing Bcl2l2−/− mice. This work was supported by US National Institutes of Health grants R01 NS050674 and R01 MH091662 to R.A.S. and F31 NS077620 to S.J.F.; Harvard/MIT Joint Research Program in Basic Neuroscience Grant to R.A.S. and M. Heiman; fellowships from the Harvard Mahoney, the Harvard NeuroDiscovery Center and the Alice and Joseph E. Brooks fund to K.E.C.; and a fellowship from the Victoria Quan fund to S.J.F.

Author information

K.E.C., S.J.F. and R.A.S. designed experiments and wrote the manuscript. K.E.C., S.J.F. and M.F.P.-M. performed compartmentalized culture experiments. K.E.C. performed formaldehyde cross-linking experiments. M.F.P.-M. performed axonal degeneration and protein transfection experiments. H.L.E. analyzed FISH data. K.E.C. and S.J.F. performed all other experiments.

Correspondence to Rosalind A Segal.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 SFPQ is in axons and binds axonal mRNAs

(a) Previously identified axonal mRNAs analyzed for SFPQ-binding motifs10, 20. (b) qRT-PCR analysis of axonal genes expressed in Trk-PC12 cells and DRG sensory neurons normalized to gapdh mRNA. Data shows mean ± SEM, n = 3 experiments; *P < 0.05 (Unpaired two-tailed t-test; P = 0.3, t(3) = 1.25 for laminb2, P = 0.54, t(3) = 0.68 for bclw, P = 0.028, t(3) = 3.9 for impa1, P = 0.26, t(3) = 1.37 for creb, P = 0.33, t(3) =1.17 for β-actin, P = 0.31, t(3) = 1.22 for cox4, P = 0.01, t(3) = 5.62 for smad, P = 0.06, t(3) = 3.01 for rpl4). (c) qRT-PCR analysis of SFPQ-precipitated mRNAs from Trk-PC12 cells in the absence or presence of NGF stimulation following formaldehyde crosslinking. Data, normalized to no antibody control, shows mean ± SEM, n = 4 experiments; *P < 0.05 (Paired two-tailed t-test; P = 0.05, t(3) = 2.44 for laminb2, P = 0.04, t(3) = 2.48 for bclw, P = 0.01, t(3) = 4.03 for impa1, P = 0.05, t(3) = 2.53 for creb, P = 0.35, t(3) = 0.42 for β-actin, P = 0.08, t(3) = 1.85 for cox4, P = 0.4, t(2) = 0.29 for smad, P = 0.2, t(2) = 1.26 for rpl4, P = 0.3, t(3) = 0.6 for gapdh). (d) SFPQ immunostaining of whole-mount DRG at P0 shows SFPQ within neuronal nuclei (white arrow heads). Scale bar, 100 µm. (e) Western blot of sensory neurons cultured in compartmented cultures and lysates from cell bodies (CB) and distal axons (DA) probed for SFPQ, GAPDH and Histone with SFPQ protein quantification; data shows mean ± SEM, n = 3 experiments. (f) SFPQ immunostaining of cultured sensory neurons expressing control or SFPQ shRNA. Scale bar, 10 µm. (g) SFPQ immunostaining of cultured sensory neuron axons expressing control or SFPQ shRNA. Scale bar, 5 µm.

Supplementary Figure 2 Validation of single-molecule FISH probes

(a) qRT-PCR analysis of sfpq mRNA, normalized to gapdh, in cell bodies of sensory neurons grown in compartmented cultures expressing control or SFPQ shRNA. Data, normalized to control, shows mean ± SEM, n = 8; *P = 0.0003, t(7) = 6.74 (Unpaired two-tailed t-test). (b) Single-molecule FISH for laminb2 mRNA in cells expressing control or laminb2 shRNA (max projection). Quantification of laminb2 mRNA spots per cell of a single image stack; data shows mean ± SEM, n = 458 for shCntrl and n = 638 for shLmnB2 cells from 3 experiments; *P < 0.0001, t(20) = 5.19 (Unpaired two-tailed t-test). Scale bar, 10 μm. (c) Single-molecule FISH for bclw mRNA on frozen sections of DRG (L4) from bclw+/+ and bclw−/− mice (6 months), stained for Tuj1 and DAPI. Scale bar, 100 μm. (d) Number of single-molecule FISH mRNA puncta per μm2 in DA following 2h neurotrophin stimulation of DA. Data shows mean ± SEM, n = 3 individual microfluidic cultures; *P = 0.11, t(79) = 1.58 (Unpaired two-tailed t-test between Cn and NT). (e) Number of single-molecule FISH mRNA puncta per μm2 in DA of sensory neurons expressing SFPQ shRNA. Data shows mean ± SEM, n = 3 individual microfluidic cultures; P = 0.422, t(68) = 0.82 (Unpaired two-tailed t-test between shCntrl and shSFPQ).

Supplementary Figure 3 Reverse colocalization analysis

(a,b) Reverse colocalization analysis from Fig. 4c,d in control (a) and shSFPQ (b) conditions. Normalized density is frequency of adjacent mRNAs at each distance on x-axis normalized to the alternate frequency expected by chance (reverse randomization).

Supplementary Figure 4 The Bcl2l2 3′ UTR is critical for neurotrophin-dependent axonal localization

(a) qRT-PCR analysis of egfp mRNA levels in sensory neurons grown in compartmented cultures expressing eGFP or eGFP-Bclw-3’UTR following 2h neurotrophin stimulation of distal axons (DA); data shows mean ± SEM, n = 6 experiments; P = 0.05, t(10) = 2.18 (Unpaired two-tailed t-test). In DA, neurotrophin-dependent regulation of egfp mRNA occurs only in the presence of the bclw 3’UTR.

Supplementary Figure 5 SFPQ and cell body apoptosis

(a) DAPI staining of cell bodies (CB) of sensory neurons grown in compartmented cultures expressing control or SFPQ shRNA and quantification of percentage condensed nuclei (arrow heads). Scale bar, 40 μm. n = 4746 cells for shCntrl and n = 3817 cells for shSFPQ from 3 experiments; n.s., not significant (two-way ANOVA with Bonferroni correction; P = 0.49, F(1,80) = 0.48). (b) Western blot of LaminB2 in CB of sensory neurons grown in compartmented cultures expressing control or laminb2 shRNA. Quantification of protein levels normalized to GAPDH, data shows mean ± SEM, n = 3 experiments; *P < 0.05 (Unpaired two-tailed t-test; P = 0.003, t(4) = 6.36). (c) Western blot for His after selective introduction of His-tagged Bclw protein into distal axons (DA) of sensory neurons grown in compartmented cultures.

Supplementary Figure 6 Model for SFPQ and the axonal mRNA regulon

SFPQ binds both bclw mRNA and laminb2 mRNA enabling neurotrophin-dependent localization to distal axons. Following neurotrophin stimulation of distal axons, SFPQ binds bclw mRNA enabling nuclear export. In the cytoplasm, SFPQ and bclw join with SFPQ-bound laminb2 and/or other axonally targeted mRNAs to form an RNA transport granule that traffics to axons. There, mRNAs are locally translated at mitochondria to generate Bclw and LaminB2 protein that promote axon survival.

Supplementary Figure 7 Full-length blots and gels

(a) Figure 2e. (b) Figure 3a. (c) Figure 5a. (d) Figure 5b. (e) Figure 5c. (f) Figure 5d. (g) Figure 6a. (h) Supp. Figure 1e. (i) Supp. Figure 5b. (j) Supp. Figure 5c.

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Supplementary Figures 1–7 and Supplementary Table 1 (PDF 1883 kb)

Supplementary Methods Checklist (PDF 429 kb)

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Cosker, K., Fenstermacher, S., Pazyra-Murphy, M. et al. The RNA-binding protein SFPQ orchestrates an RNA regulon to promote axon viability. Nat Neurosci 19, 690–696 (2016).

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