Receptor-specific interactome as a hub for rapid cue-induced selective translation in axons
- University of Cambridge, United Kingdom
- Harvard Medical School, United States
Figures
Figure 1 with 1 supplement
Multiple guidance cue receptors interact with ribosomes.
(A) Volcano plots showing statistically enriched proteins in DCC-IP and Nrp1-IP samples identified by permutation-based FDR-corrected t-test based on three biological replicates. The LFQ intensity …
Figure 1—figure supplement 1
Multiple guidance cue receptors interact with ribosomes in SH-SY5Y cells.
(A–C) Western blot validation of RP co-immunoprecipitation with DCC, Nrp1 and Robo2 in SH-SY5Y cells. Western blots were repeated 2 to 4 times, Rps4X Western blots are from one experiment, …
Figure 2 with 1 supplement
Receptor-ribosome coupling is mRNA dependent and DCC and Nrp1 bind to specific RBPs and mRNAs.
(A) Relative 18S and 28S ribosomal RNA abundance after control (IgG) pulldown or receptors pulldowns with or without EDTA or RNase A/T1 treatments (two-way ANOVA with Bonferroni’s multiple …
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Figure 2—source data 1
Spreadsheet containing all Manders Overlap Coefficient values for each axonal growth cone in Figure 2E and F.
- https://cdn.elifesciences.org/articles/48718/elife-48718-fig2-data1-v2.xlsx
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Figure 2—source data 2
Spreadsheet containing RNA-sequencing analysis of DCC and Nrp1 bound mRNAs and GO analysis of high abundant (FPKM >100) detected mRNAs for DCC and Nrp1.
- https://cdn.elifesciences.org/articles/48718/elife-48718-fig2-data2-v2.xlsx
Figure 2—figure supplement 1
Polysome fractionation analysis, RNase sensitivity of Nrp1-Staufen1 interaction and additional RNA-seq analyses.
(A) Control and (B) EDTA treated polysome fractions and Western blot showing the distribution of DCC and Nrp1 across fractions. (C) Relative quantification of DCC and Nrp1 protein levels in …
Figure 3 with 1 supplement
DCC and Nrp1 are in close proximity to ribosomes in axonal growth cones in a cue-dependent manner.
(A) Expansion imaging shows partial co-localization of DCC and (B) Nrp1 with ribosomal proteins (Scale bars, 5 μm). (C) Representative proximity ligation assay signal in axonal growth cones between …
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Figure 3—source data 1
Spreadsheet containing PLA counts and relative comparisons from each axonal growth cone in Figure 3E, all inter-ribosome distances and distribution shown in Figure 3G, and all normalized PLA count values for each axonal growth cone in Figure 3H–L.
- https://cdn.elifesciences.org/articles/48718/elife-48718-fig3-data1-v2.xlsx
Figure 3—figure supplement 1
DCC and Nrp1 are in close proximity to ribosomes in axonal growth cones in a cue-dependent manner.
(A) Pearson’s Correlation coefficients of DCC-RPL5/uL18 and Nrp1-RPS3A/eS1 from expanded axonal growth cones (data obtained from four biological replicates, bars indicate mean, error bars indicate …
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Figure 3—figure supplement 1—source data 1
Spreadsheet containing all Pearson’s correlation values for each expanded growth cone in Figure 3—figure supplement 1A, all normalized PLA count values for each axonal growth cone in Figure 3—figure supplement 1F and H, and all normalized puromycin intensity values for each axonal growth cone in Figure 3—figure supplement 1G.
- https://cdn.elifesciences.org/articles/48718/elife-48718-fig3-figsupp1-data1-v2.xlsx
Figure 4 with 1 supplement
EphrinA1 co-stimulation blocks Netrin-1 induced receptor-ribosome dissociation and selective translation.
(A) Relative PLA quantification of DCC and RPL5/uL18 compared to control after Netrin-1, EphrinA1, or co-stimulation (one-way ANOVA with Bonferroni’s multiple comparisons test; bars indicate mean, …
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Figure 4—source data 1
Spreadsheet containing all normalized PLA count values for each axonal growth cone in Figure 4A, all normalized puromycin intensity values for each axonal growth cone in Figure 4C, all normalized ß-Catenin intensity values for each axonal growth cone in Figure 4F and all normalized hnRNPH1 intensity values for each axonal growth cone in Figure 4H.
- https://cdn.elifesciences.org/articles/48718/elife-48718-fig4-data1-v2.xlsx
Figure 4—figure supplement 1
EphrinA1 co-stimulation blocks Netrin-1 induced receptor-ribosome dissociation and selective translation of rps14.
(A) Relative PLA quantification of Nrp1 and RPS23/uS12 compared to control after Sema3A, EphrinA1, or co-stimulation with Sema3A and EphrinA1 (one-way ANOVA with Bonferroni’s multiple comparisons …
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Figure 4—figure supplement 1—source data 1
Spreadsheet containing all normalized PLA count values for each axonal growth cone in Figure 4—figure supplement 1A, all normalized pERK1/2 intensity values for each axonal growth cone in Figure 4—figure supplement 1B and all normalized RPS14 intensity values for each axonal growth cone in Figure 4—figure supplement 1D and E.
- https://cdn.elifesciences.org/articles/48718/elife-48718-fig4-figsupp1-data1-v2.xlsx
Figure 5
Model diagram depicting the proposed interactions between receptors, RBPs, mRNAs and ribosomes under basal and cue stimulation conditions.
Author response image 1
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Biological sample (Xenopus laevis) | Xenopus laevis | NASCO | Cat# LM00715 (male); RRID:XEP_Xla100; Cat# LM00535 (female); RRID:XEP_Xla | |
| Cell line (Homo-sapiens) | SH-SY5Y | ATCC | Cat# CRL-2266; RRID:CVCL_0019 | |
| Antibody | anti-RPS3A (Rabbit polyclonal) | Abcam | Cat# ab194670; RRID:AB_2756396 | ICC/PLA (1:100) WB (1:1000) |
| Antibody | Anti-Neuropilin-1 (Rabbit monoclonal) | Abcam | Cat# ab81321; RRID:AB_1640739 | ICC/PLA (1:100) WB (1:2000) IP (5 µg) |
| Antibody | Anti-Neuropilin-1 (Mouse monoclonal) | Proteintech | Cat# 60067–1-Ig; RRID:AB_2150840 | ICC (1:100) |
| Antibody | Anti-DCC (mouse monoclonal | BD Biosciences | Cat# 554223; RRID:AB_395314 | ICC/PLA (1:100) WB (1:1000) IP (5 µg) |
| Antibody | Anti-RPL5 (rabbit polyclonal | Proteintech | Cat# 15430–1-AP; RRID:AB_2238681 | ICC/PLA (1:100) |
| Antibody | Anti-RPS4X (Rabbit polyclonal) | Proteintech | Cat# 14799–1-AP; RRID:AB_2238567 | PLA (1:100) WB (1:1000) |
| Antibody | Anti-RPL10A (Rabbit polyclonal) | Proteintech | Cat# 16681–1-AP; RRID:AB_2181281 | PLA (1:100) WB (1:500) |
| Antibody | Anti-RPS23 (mouse monoclonal) | Abcam | Cat#: ab57644; RRID:AB_945314 | PLA (1:100) WB (1:1000) |
| Antibody | Anti-RPS26 (Rabbit polyclonal | Proteintech | Cat# 14909–1-AP; RRID:AB_2180361 | WB (1:500) |
| Antibody | Anti-Robo2 (goat polyclonal) | R and D Systems | Cat# AF3147; RRID:AB_2181857 | WB (1:250) |
| Antibody | Anti-EphB2 (mouse monoclonal) | Santa Cruz | Cat# sc130068; RRID:AB_2099958 | WB (1:100) IP (5 µg) |
| Antibody | Anti-EphB2 (mouse monoclonal) | Thermo Fisher Scientific | Cat# 37–1700; RRID:AB_2533302 | PLA (1:100) |
| Antibody | Anti-Staufen1 (Rabbit polyclonal) | Abcam | Cat# ab73478; RRID:AB_1641030 | ICC (1:100) WB (1:500) |
| Antibody | Anti-hnRNPA2B1 (Rabbit polyclonal) | Abcam | Cat# ab31645; RRID:AB_732978 | ICC/PLA (1:100) |
| Antibody | Anti-RPS14 (Rabbit polyclonal) | Abcam | Cat# ab174661 | ICC (1:100) |
| Antibody | Anti-ß-Catenin (Rabbit polyclonal) | Sigma-Aldrich | Cat# C2206; RRID:AB_476831 | ICC (1:500) |
| Antibody | Anti-hnRNPH1 | Abcam | Cat# ab154894 | ICC (1:500) |
| Antibody | Anti-IgG (Rabbit) | Abcam | Cat# ab37415; RRID:AB_2631996 | PLA (1:100) IP (5 µg) |
| Antibody | Anti-IgG1 (Mouse) | R and D Systems | Cat# MAB002; RRID:AB_357344 | PLA (1:100) IP (5 µg) |
| Antibody | Anti-IgG2b (Mouse) | R and D Systems | Cat# MAB004; RRID:AB_357346 | IP (5 µg) |
| Antibody | Anti-IgG (Goat) | R and D Systems | Cat# AB-108-C; RRID:AB_354267 | IP (5 µg) |
| Antibody | Anti-Puromycin-Alexa Fluor 488 conjugate (mouse monoclonal) | Millipore | Cat# MABE343-AF488; RRID:AB_2736875 | ICC (1:200) |
| Antibody | Anti-RPL19 (mouse monoclonal) | Abcam | Cat#ab58328; RRID:AB_945305 | WB (1:1000) |
| Antibody | Anti-FxR | Gift from Dr. Edward Khandjan, University of Quebec | N/A | WB (1:1000) |
| Antibody | Anti-pERK1/2 | Cell Signaling | Cat# 9101; RRID:AB_331646 | ICC (1:250) |
| Antibody | Goat-anti-rabbit Alexa Fluor 568 | Abcam | Cat# ab150077; RRID:AB_2630356 | ICC (1:1000) |
| Antibody | Goat-anti-mouse Alexa Fluor 568 | Abcam | Cat# ab150117; RRID:AB_2688012 | ICC (1:1000) |
| Antibody | Goat-anti-mouse-HRP | Abcam | Cat# ab6789; RRID:AB_955439 | WB (1:15000) |
| Antibody | Goat-anti-rabbit-HRP | Abcam | Cat#: ab97080; RRID:AB_10679808 | WB (1:15000) |
| Commercial assay or kit | RNeasy mini kit | Qiagen | Cat# 74104 | |
| Commercial assay or kit | SuperScript III First-strand Synthesis kit | Thermo Fisher Scientific | Cat# 18080051 | |
| Commercial assay or kit | Quantitect SYBR green PCR kit | Qiagen | Cat# 204143 | |
| Commercial assay or kit | KAPA HyperPrep kit | Roche | Cat# KK8503 | |
| Commercial assay or kit | NextSeq 500/550 high output v2 kit (150 cycles) | Illumina | Cat# FC-404–2002 | |
| Commercial assay or kit | Duolink In situ PLA Detection reagents green | Sigma-Aldrich | Cat# DUO92014 | |
| Commercial assay or kit | Duolink In situ PLA Detection reagents red | Sigma-Aldrich | Cat# DUO92008 | |
| Commercial assay or kit | Duolink In situ PLA probe Anti-Rabbit PLUS | Sigma-Aldrich | Cat# DUO92002 | |
| Commercial assay or kit | Duolink In situ PLA probe Anti-Mouse MINUS | Sigma-Aldrich | Cat# DUO92004 | |
| Chemical compound, drug, reagent | Cycloheximide | Sigma Aldrich | Cat# C4859 | |
| Chemical compound, drug, reagent | RNase A | Ambion | Cat# EN0531 | |
| Chemical compound, drug, reagent | RNase T1 | Ambion | Cat# EN0541 | |
| Chemical compound, drug, reagent | Puromycin | Sigma-Aldrich | Cat# P8833 | |
| Chemical compound, drug, reagent | Recombinant mouse Netrin-1 | R and D systems | Cat# 1109-N1 | |
| Chemical compound, drug, reagent | Recombinant human Sema3A | R and D systems | Cat# 1250-S3 | |
| Chemical compound, drug, reagent | Dynasore | Sigma-Aldrich | Cat# D7693 | |
| Chemical compound, drug, reagent | SUPERase In RNAse inhibitor | Ambion | Cat# AM2696 | |
| Software, algorithm | Volocity | PerkinElmer | Version 6.0.1; RRID:SCR_002668 | |
| Software, algorithm | GraphPad Prism | GraphPad | v.5; RRID:SCR_002798 | |
| Software, algorithm | R | Other | v.3.2.2; RRID:SCR_001905 | https://www.r- project.org |
| Software, algorithm | MATLAB | Mathworks | v.R2016b; RRID:SCR_001622 | |
| Software, algorithm | HISAT2 | Other | v.2.1.0; RRID:SCR_015530 | https://ccb.jhu.edu/software/hisat2/index.shtml |
| Software, algorithm | Cufflinks | Other | v.2.2.1; RRID:SCR014597 | http://cole-trapnell-lab.github.io/cufflinks/ |
