The glycine arginine-rich domain of the RNA-binding protein nucleolin regulates its subcellular localization

Abstract

Nucleolin is a multifunctional RNA Binding Protein (RBP) with diverse subcellular localizations, including the nucleolus in all eukaryotic cells, the plasma membrane in tumor cells, and the axon in neurons. Here we show that the glycine arginine rich (GAR) domain of nucleolin drives subcellular localization via protein-protein interactions with a kinesin light chain. In addition, GAR sequences mediate plasma membrane interactions of nucleolin. Both these modalities are in addition to the already reported involvement of the GAR domain in liquid-liquid phase separation in the nucleolus. Nucleolin transport to axons requires the GAR domain, and heterozygous GAR deletion mice reveal reduced axonal localization of nucleolin cargo mRNAs and enhanced sensory neuron growth. Thus, the GAR domain governs axonal transport of a growth controlling RNA-RBP complex in neurons, and is a versatile localization determinant for different subcellular compartments. Localization determination by GAR domains may explain why GAR mutants in diverse RBPs are associated with neurodegenerative disease.

Keywords: axonal transport; cell size regulation; local translation; protein-membrane interaction; subcellular localization.

Figure 1. The nucleolin GAR domain binds DNA aptamer AS1411 and kinesins

1. A

   Domain structure of nucleolin. RRM—RNA recognition motif; GAR—glycine–arginine‐rich domain. Amino acid sequence of the GAR domain is shown below. R3 and R4: peptides derived from the GAR domain C‐terminus, efficiently binding AS1411 (see panel B). A3 designates the R3 peptide with three arginines substituted with alanines, and N4 designates the R4 peptide with four arginines substituted with asparagines.

2. B

   R3 and R4, but not the A3 and N4 peptides efficiently bind AS1411 in an ELISA assay with biotinylated peptides shown in (A) immobilized to streptavidin‐coated plates. n = 4 technical replicates; means ± SEM; ****P < 0.0001 in AS1411 versus control aptamer, 2‐way ANOVA with Sidak’s post‐test. a.u.—arbitrary units.

3. C

   Kinesin protein family members affinity purified by biotinylated R3 peptide from mouse sciatic nerve axoplasm, identified by mass spectrometry (schematics are based on (Hirokawa et al, 2009). Biotinylated A3 peptide served as a control. Mean spectral counts (normalized plus pseudocounts) ± SEM, n = 3 independent biological repeats. All proteins shown were found to significantly purify with R3, but not A3, with probability > 0.95 and a mean fold change > 2 by SAINTexpress analysis (http://crapome.org/).

4. D

   Schematic of kinesin pulldown from mouse sciatic nerve axoplasm, using biotinylated GAR peptides bound to streptavidin beads (STRP).

5. E

   Automated capillary electrophoresis immunoassay traces of Kif5a pulled down by biotinylated R3 and R4 peptides. Biotinylated A3 and N4 peptides, respectively, served as controls. 4.5% of input used for pulldowns was loaded alongside the pulldown samples. a.u.—arbitrary units.

6. F

   Quantification of (E). n = 3 independent biological repeats; means ± SEM; **P < 0.01, paired Student’s t‐test.

See also Fig EV1 and Appendix Fig S1.

Figure EV1. Mass spectrometry of R3‐interacting proteome in sciatic nerve axoplasm (related to Fig 1)

1. A

   Venn diagram showing filtering of R3 peptide‐binding proteins identified by mass spectrometry. A total of 2,367 proteins were identified in the R3 and A3 pulldowns (at least one peptide in one out of the 6 samples). This set was filtered to a subset of 965 with at least one peptide in all three experimental replicates of R3, further narrowed down to 525 proteins with a SAINT probability score > 0.95 (equivalent of alpha < 0.05). Finally, the latter subset was filtered for R3/A3 fold change > 2, yielding 352 proteins.

2. B

   Top 15 canonical pathways represented in the 352 R3‐binding proteins (Ingenuity Pathway Analysis), ranked by –log10(P‐values) using the embedded right‐tailed Fisher’s exact test.

3. C

   Subcellular localization categories as defined by Ingenuity Pathway Analysis (IPA) for the 352 R3‐binding proteins (numbers indicate percentages of total).

Figure 2. Nucleolin–kinesin interaction is directly mediated by the GAR domain

1. A

   GAR domain deletion or mutation perturbs binding to Kif5a. Co‐immunoprecipitation analysis of Kif5a and HA‐Dendra2‐tagged nucleolin (both overexpressed in HEK‐293 cells). IP was performed with HA antibody and probed in Western blot with anti‐Kif5a and anti‐HA antibodies. Ncl FL—HA‐Dendra2‐full‐length nucleolin; Ncl ΔGAR—HA‐Dendra2‐nucleolin with a GAR domain deletion; Ncl GAR(N)—HA‐Dendra2‐nucleolin with all 10 arginines in the GAR domain mutated to asparagines; DEN—Dendra2. Note that HA input blots for HA‐Dendra2 (Dendra) and the other three constructs are from the same membrane, but shown discontinuously owing to the different migration of these proteins in PAGE.

2. B

   Quantification of (A). n = 3 independent biological repeats; means ± SEM; ****P < 0.0001, ANOVA with Tukey’s post‐test.

3. C–E

   R4, but not N4 peptide, reduces Kif5a co‐immunoprecipitation with nucleolin from mouse sciatic nerve axoplasm, assayed by automated capillary electrophoresis immunoassay for Kif5a. Schematic of the assay is shown in (C). Representative traces of the Kif5a immunoreactive peaks are shown in (D), and quantifications are shown in (E). Immunoprecipitated Kif5a levels are normalized with input levels and expressed relative to N4. n = 3 independent biological repeats; means ± SEM; *P < 0.05, paired Student’s t‐test.

4. F

   Surface plasmon resonance analysis of KLC2 binding to biotinylated R4 peptide. Recombinant KLC2 was injected at different concentrations (5‐160 nM) on biotinylated R4 or N4 peptides immobilized to a streptavidin sensor chip. The dissociation steady‐state constant, KD, was determined by fitting the sensogram to a 1:1 model.

5. G

   Kinetic analyses of photoconverted Dendra fusion proteins in cultured DRG neuron axons. Adult mouse DRG neurons were transfected by electroporation with constructs expressing HA‐Dendra‐GAR(WT)—wild‐type nucleolin GAR domain N‐terminally fused with HA‐Dendra, or HA‐Dendra‐GAR(N) with 10 arginines in the GAR domain mutated to asparagines. 48 h after plating and transfection, Dendra fluorescence was photoconverted using a DMD module to restrict conversion within the cell body. Time‐lapse images were collected from the entire field of view, and red signal intensity was analyzed within axons at distances of 60–90 µm from the cell body. Photoconverted signal sampled at 10 s intervals is shown, normalized to signal intensity before photoconversion. n = 3 biological repeats comprising 7 cells each. Means ± SEM; ****P < 0.0001 (ANOVA).

See also Appendix Fig S2. Source data are available online for this figure.

Figure 3. GAR domain interactions with phospholipid membranes

1. A

   Molecular dynamics simulations of native (R4) or variant (K4 and N4) nucleolin GAR‐derived peptides interacting with an idealized phospholipid bilayer (phosphatidylcholine (PC): phosphatidylserine (PS), 4:1) in water. Amino acid sequences of peptides are shown above. Shaded area—standard deviations of three independent runs, each 50 ns long. Mean values are indicated by colored lines, and overlay of these is shown on the right.

2. B

   Flow cytometry histograms of TAMRA‐labeled peptide uptake into HEK‐293 cells after 1‐h incubation at 4°C in growth medium.

3. C

   Quantification of mean fluorescence intensities in (B). n = 6 independent biological repeats; means ± SEM; *P < 0.05, ***P < 0.001, ****P < 0.0001, ANOVA with Tukey’s post‐test.

4. D

   Uptake of TAMRA‐labeled R4 and N4 peptides into cultured DRG neurons. Cells were incubated with peptides dissolved to 5 μM in growth medium for 30 min at 4°C, washed, fixed, and analyzed by confocal microscopy. PC—phase contrast. Scale bar—10 µm.

5. E

   Quantification of (D). n = 3 independent biological repeats; means ± SEM; *P < 0.05, paired Student’s t‐test a. u. – arbitrary units.

6. F

   Analysis of cell surface nucleolin in HEK‐293 cells. Cells were treated with 0.5 mM sulfo‐NHS‐SS‐biotin for 30 min at 4°C, lysed, and subjected to affinity pulldown of biotinylated proteins with streptavidin Dynabeads, eluted by 50 mM DTT, and probed in Western blot with antibodies indicated on the left. Endogenous nucleolin (Ncl) was readily detected in the biotinylated membrane protein pool. Glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) and importin β1 (Impβ1) served as negative controls.

7. G

   HEK‐293 cells were transfected with plasmids expressing HA‐Dendra2‐full‐length nucleolin (Ncl FL), HA‐Dendra2‐nucleolin with a GAR domain deletion (Ncl ΔGAR), or HA‐Dendra2‐nucleolin with all 10 arginines in the GAR domain mutated to asparagines (Ncl GAR(N)) and processed as in (F).

8. H

   Quantification of (G). Levels of nucleolin in pulldowns were normalized to input levels and expressed as % relative to full‐length HA‐Dendra2‐nucleolin. n = 4 independent biological repeats; means ± SEM; ****P < 0.0001, ANOVA with Dunnett’s post‐test.

See also Fig EV2 and Appendix Fig S3. Source data are available online for this figure.

Figure EV2. Molecular simulations of GAR peptide interactions with phospholipid membranes (related to Fig 3)

1. A

   Interaction of three R4 peptides with a POPC:POPS (4:1) membrane in water, 500‐ns simulation time. POPC—1‐palmitoyl‐2‐oleoyl‐sn‐glycero‐3‐phosphocholine; POPS—1‐palmitoyl‐2‐oleoyl‐sn‐glycero‐3‐phosphoserine; 4:1 refers to molar ratio. Arginines are shown in blue, glycines in green and phenylalanines in light gray. Water is indicated in red, hydrophobic phospholipid tails in cyan and POPC/POPS polar groups in gray. Note the deep penetration of phenylalanine side chains into the hydrophobic bilayer core.

2. B

   Distributions of mass densities of molecules in the simulation box shown in (A), with the center of symmetry in the middle of the POPC:POPS membrane. Enlargement of the region bounded by the dashed line is shown at the bottom.

3. C

   Number of contacts between the membrane and individual residues in the peptides, at a distance of < 0.6 nm from the membrane. Average number of contacts from three trials is shown. Error bars—SD.

Figure 4. GAR domain and nucleolar localization of nucleolin

1. A

   GAR deletion in nucleolin reduces partitioning to nucleoli in adult DRG neurons, transduced with the peripheral neuron‐specific AAV‐PHP.S vector expressing full‐length HA‐Dendra2‐nucleolin (Ncl FL) or respective ΔGAR mutant (Ncl ΔGAR) upon seeding, and analyzed by epifluorescence microscopy 9–10 days later. Shown are representative Dendra2 fluorescence images collected from the non‐activated (green) emission line. Hoechst 33342 (10 µM) was used to outline nuclei. Superimposed phase contrast and Dendra2 fluorescence images are shown on the right. Scale bar—25 µm.

2. B

   Quantification of the nucleolar/nuclear intensity ratios in DRG cells shown in (A). n = 38–54 cells per sample in three independent biological repeats; means ± SEM; ****P < 0.0001, Mann–Whitney test.

3. C

   Time‐lapse live imaging of nuclei in DRG neurons transduced with AAV‐PHP.S HA‐Dendra2‐nucleolin (full‐length and ΔGAR mutant), after a hypotonic challenge with double‐distilled water (ddH20). Shown are epifluorescence images (Dendra2 green emission line) taken at indicated times after medium replacement with ddH20. Scale bar—10 µm.

4. D

   Quantification of nucleolar Dendra2 signal in (C) with intensity values immediately before medium replacement set as 100%. n = 17–24 cells per sample in three biological repeats; means ± SEM; two‐way ANOVA with Sidak’s post‐test detected a significant time‐dependent reduction in nucleolar signal (P < 0.0001) and no difference between full‐length and ΔGAR mutant nucleolin (P > 0.05).

See also Appendix Fig A4.

Figure 5. Reduced levels of axonal nucleolin in nucleolin GAR^+/− mice

1. A

   Targeted deletion of nucleolin GAR domain by CRISPR‐Cas9. Schematic shows mouse nucleolin exons targeted by single guide RNAs (sgRNAs) and resulting deletion in the GAR domain amino acid sequence.

2. B

   Western blot analysis of nucleolin in DRG neurons from wild‐type (WT) and GAR^+/− mice cultured in Boyden chambers. Tuj1 was used as a loading control. The higher nucleolin band in GAR^+/− corresponds to WT nucleolin and lower band to nucleolin with a 41 aa deletion (corresponding to about 4 kDa reduction in protein size).

3. C

   Quantification of (B), total nucleolin levels (sum of both bands where applicable) were normalized to Tuj1 and expressed as GAR^+/− / WT ratios. n = 3 independent biological repeats; means ± SEM; *P < 0.05, 1 sample t‐test.

See also Appendix Fig S5. Source data are available online for this figure.

Figure 6. Reduced levels of nucleolin‐mRNA cargos in axons of nucleolin GAR^+/− mice

1. A, B

   Representative maximum projections of exposure‐matched confocal images of immunofluorescence for nucleolin and neurofilament (NF) and the neuronal marker Tuj1 in sciatic nerve axons from WT and GAR^+/− mice shown in (A). Upper panels show total nucleolin stain. Middle panels show merged image of nucleolin (gray), NF and Tuj1 (magenta), and DAPI (blue). Lower panels show nucleolin overlaps with NF and Tuj1 signals as the “axon only” signal. B shows quantification of nucleolin immunofluorescence with approximately a 50% reduction in nucleolin in the axons of GAR^+/− mice in vivo. n = 3 WT, n = 4 GAR^+/−; means ± SEM; *P < 0.05, unpaired Student’s t‐test. Scale bar – 10 µm.

2. C–F

   Representative, exposure‐matched maximum projection confocal images of FISH for Kpnb1 (C) or mTOR (E) mRNA and NF plus Tuj1 immunostaining from sciatic nerve sections from WT (left) or GAR^+/− mice. Upper panels for each show total mRNA signal. Middle panels show mRNA (gray) signals merged with NF plus Tuj1 (magenta) and DAPI (blue). Lower panels show mRNA signal that overlap with NF plus Tuj1 signal (labeled “axon only” signal). Quantification of axonal Kpnb1 (D) and mTOR (F) mRNA signals compared to the negative control, DapB mRNA, show a significant reduction in these axonal mRNAs in the GAR^+/− mice. n = 3 WT, n = 3 GAR^+/−; means ± SEM; **P < 0.01, ***P < 0.001, ****P < 0.0001 unpaired Student’s t‐test. Scale bar – 10 µm.

See also Appendix Fig S6.

Figure 7. Axonal mRNAs associated with the Ncl‐Kif5a complex

1. A

   Workflow for profiling mRNAs bound by the Ncl‐Kif5a complex. Nucleolin (Ncl) and Kif5a‐binding RNAs were isolated from wild‐type (WT) adult mouse sciatic nerve axoplasm by immunoprecipitation; in addition, DRG neurons from adult WT and GAR^+/− mice were cultured in modified Boyden chambers and RNA was isolated from cell body and axonal sides. RNA‐seq analysis from the resulting four datasets yielded 11,771 overlapping transcripts. The latter were further processed into a subset of 488 transcripts enriched in Ncl and Kif5a pulldown and depleted in axons of nucleolin GAR^+/− mice compared with WT (B). Please see Fig EV3 for a detailed workflow.

2. B

   Clustering of 488 Ncl/Kif5a‐enriched transcripts reduced in GAR^+/− versus WT axons. Yellow cluster— transcripts not significantly enriched in the soma of GAR^+/− DRG neurons versus the WT control, Purple cluster—transcripts enriched in the soma of GAR^+/− DRG neurons versus the WT control. Heatmap shows mean log2‐fold changes across four datasets (see also Fig EV3A), from left to right: (i) nucleolin‐binding mRNAs in mouse sciatic nerve axoplasm; (ii) Kif5a‐binding mRNAs in mouse sciatic nerve axoplasm; (iii) mRNA abundance in DRG neuron cell bodies of GAR^+/− mice relative to abundance in WT mice; and (iv) mRNA abundance in DRG neuron axons of GAR^+/− mice relative to abundance in wild‐type mice. All transcripts chosen for this cluster analysis showed significant enrichment in Ncl IP and reduction in GAR^+/− axons versus WT, as determined by rank–rank hypergeometric overlap (RRHO) as well as a fold change in Kif5a IP versus control > 2.

3. C

   Genes comprising the purple cluster—transcripts significantly enriched by both Ncl and Kif5a immunoprecipitation and showing a reduction in GAR^+/− axons concurrent with an enrichment in GAR^+/− soma (compared to the WT control). Inpp5f (highlighted in red) was chosen for follow‐up.

4. D

   Representative images for FISH analysis of Inpp5f in DRG neurons treated for 48 h with 10 µM AS1411 or 10 µM control aptamer, replated, and grown for 18 h. FISH signal is shown in gray; cell somata (left) and axons (right) are visualized by neurofilament immunostaining (magenta). Scr—scrambled FISH probe served as a negative control. Scale bar—10 µm.

5. E

   Quantification of axonal Inpp5f signal in (D) as density of RNA granules along axons. n = 11–19 cells per sample; means ± SEM; *P < 0.05, unpaired Student’s t‐test.

6. F

   RT–qPCR analysis of axonal Inpp5f mRNA levels in DRG neurons grown in Boyden chambers and treated for 48 h with 10 µM AS1411 or 10 µM control aptamer. Axon/cell body ratios of Inpp5f normalized to Gapdh levels are shown. n = 4 independent biological repeats; means ± SEM; *P < 0.05, unpaired Student’s t‐test.

See also Fig EV3.

Figure EV3. RNA‐seq to identify nucleolin interacting mRNAs (related to Fig 7)

1. A

   Heatmaps showing genes depleted or enriched relative to controls in the four RNA‐seq datasets generated in this study. Clusterograms show Z‐scores for individual biological replicates (Rep). Controls used as a reference for finding differentially expressed genes were as follows: nucleolin immunoprecipitation (IP): IP with nucleolin antibody pre‐blocked by its target peptide; Kif5a IP: rabbit IgG; GAR^+/− cell bodies and axons: wild‐type littermates.

2. B

   The number of enriched and depleted genes in the four RNA‐seq datasets shown in (A).

3. C

   Workflow for identifying the 488‐member transcript set shown in the clusterogram in Fig 7B. Rank–rank hypergeometric overlap (RRHO) analysis (Plaisier et al, 2010) was used to identify transcripts that are enriched in the nucleolin IP dataset and depleted from GAR^+/− axons, as compared to WT. The resulting set was further narrowed down by using a cutoff fold enrichment > 2 in both nucleolin and Kif5a IPs. The final set, comprising 488 transcripts, was further clustered by the “hclust” function in R, using the Pearson method.

4. D

   Venn diagram showing the number of transcripts in subsets generated by the filtering workflow described in (B).

5. E

   Ingenuity Pathway Analysis (top 10 canonical pathways) of the 488‐member transcript set shown in Fig 7B, ranked by –log10(P‐values) using the embedded right‐tailed Fisher’s exact test.

Figure 8. Wild‐type, but not ΔGAR mutant nucleolin, rescues defective axonal localization of its cargo mRNAs in DRG neurons treated with nucleolin siRNA

1. A, B

   Representative exposure‐matched FISH/IF images for DRG neurons cotransfected with control versus nucleolin (Ncl) siRNA plus siRNA‐resistant wild‐type (WT; A) or ∆GAR (B) Ncl cDNAs. Axonal Kpnb1 mRNA is decreased with the Ncl knockdown, and this is not rescued by expression of ∆GAR Ncl mutant (scale bars—10 µm).

2. C, D

   Quantification of RNA signals from FISH/IF for Kpnb1, mTOR, and Inpp5f in cell bodies (C) and axons (D) for DRG cultures transfected as in A are shown. Axons of neurons cotransfected with Ncl siRNA plus ∆GAR show significantly lower signals for each mRNA compared with Ncl siRNA plus WT and control siRNA plus WT or ∆GAR; N ≥ 10 for cell bodies, N ≥ 25 for axons over 3 biological repeats; means ± SEM; ****P ≤ 0.001 by one‐way ANOVA with Tukey HSD post hoc.

See also Figs EV4 and EV5, and Appendix Fig S7

Figure EV4. Rescue of axonal mTOR mRNA in DRG neurons treated with nucleolin siRNA (related to Fig 8)

1. A, B

   Representative exposure‐matched FISH/IF images for DRG neurons cotransfected with control versus Ncl siRNA plus siRNA‐resistant wild‐type (WT) or ∆GAR Ncl cDNAs. Axonal mTor mRNA is decreased with the Ncl knockdown, and this is not rescued by expression of ∆GAR Ncl mutant. Scrambled Quasar RNA probe (Scr) (scale bars – 10 µm).

Figure EV5. Rescue of axonal Inpp5f mRNA in DRG neurons treated with nucleolin siRNA (related to Fig 8)

1. A, B

   Representative exposure‐matched FISH/IF images for DRG neurons cotransfected with control versus Ncl siRNA plus siRNA‐resistant wild‐type (WT) or ∆GAR Ncl cDNAs. Axonal Inpp5f mRNA is decreased with the Ncl knockdown, and this is not rescued by expression of ∆GAR Ncl mutant. Scrambled Quasar RNA probe (Scr) (scale bars – 10 µm).

Figure 9. Reduced levels of full‐length axonal nucleolin increase axonal outgrowth in DRG neurons

1. A

   Cultured DRG neurons from wild‐type (WT) Thy1‐YFP mice and Thy1‐YFP / GAR^+/− mice (YFP signal is shown in green). Cells were imaged every hour for a period of 48 h. Scale bar—100 μm.

2. B

   Quantification of the time‐lapse imaging experiment shown in (A). Growth rate of the longest neurite of each cell was calculated from the time point of starting growth. n = 4 independent biological repeats; means ± SEM, ****P < 0.0001, two‐way ANOVA.

3. C

   Fluorescence images of cultured DRG neurons from adult C57BL/6 mice infected with AAV‐PHP.s expressing HA‐Dendra fused with the wild‐type nucleolin GAR domain (GAR WT) or with the GAR domain with all 10 arginines substituted with asparagines, GAR(N). Neurons were replated 9–10 days after AAV infection. 24 h after replating, cells were fixed and stained with anti‐HA (gray) and anti‐NFH (magenta) antibodies. Scale bar—100 μm.

4. D

   Quantification of total axonal outgrowth and mean process length in HA‐positive cells in (C). Outgrowth measurements were based on NFH staining. n = 6 independent biological repeats; means ± SEM; *P < 0.05, ***P < 0.001 (Student’s‐test).

Figure 10. Essential roles for the GAR domain in subcellular localization of nucleolin

1. A, B

   The nucleolin GAR domain binds a kinesin light chain, directly linking nucleolin–mRNA complexes to kinesin motors for axonal transport (A). The GAR domain further mediates membrane association of nucleolin. GAR‐mediated subcellular targeting of nucleolin complexes enables export of key mRNAs to the axon, and the local translation of their encoded proteins for local functions in the axon, or for retrograde transport to the cell body. This reciprocal transport mechanism provides intrinsic regulation of axon length and growth, and indeed, deletion or mutation of the GAR domain (B) perturbs mRNA localization to axons and increases axonal elongation.