Loss of Tsc1 in cerebellar Purkinje cells induces transcriptional and translation changes in FMRP target transcripts
- Department of Neurology, Rosamund Stone Zander Translational Neuroscience Center, Kirby Neurobiology Center, Boston Children’s Hospital, United States
- Department of Cardiology, Boston Children’s Hospital, United States
- Harvard Medical School, United States
- Roche Pharma Research and Early Development, Neuroscience and Rare Diseases, Roche Innovation Center Basel, Switzerland
Figures
Figure 1 with 1 supplement
Labeling and sorting Tsc1fl/fl PCs show predominance of downregulation gene expression.
(a) Immunocytochemical characterization of wild-type control L7-GFP+ mouse PCs on P21 mouse cerebellum. Cell nuclei were stained with Hoechst. GFP+ PCs had dim expression of phosphorylated S6 (red). …
Figure 1—figure supplement 1
Gating strategy for FACS isolation of GFP labeled PCs.
The gating for isolation of GFP-positive PCs is shown for (a) Tsc1+/+ L7GFP and (b) Tsc1fl/fl L7Cre;L7GFP animals. Cells were identified from debris based on the forward and side scatter (P1). …
Figure 2 with 1 supplement
Downregulated transcripts in Tsc1-deficient PCs display enhanced reduction in the 3’UTR and are enriched in synaptic genes.
(a) This plot shows the median coverage of reads across all downregulated transcripts. Each transcript was divided into 100 equally sized intervals and coverage was calculated across each of these …
Figure 2—figure supplement 1
Characterization of transcripts with reduced 3’UTR expression.
(a) Barplot of observed/expected ratios for transcripts with reduced 3’UTR expression in the following sets of genes associated with neurological disorders: (1) ASD – genes from SFARI (https://gene.s…
Figure 3
Expression of GFP-tagged ribosomes and ribosomal binding of transcripts in PCs.
(a) Schematic of TRAP protocol for identifying ribosomally bound transcripts specifically within PCs. (b) Anti-GFP immunofluorescence of in Rosa26fsTRAP L7Cre animals shows labeling of the PCs of …
Figure 4
Alterations in TE in Tsc1fl/fl PCs.
(a) This histogram shows the ΔTE between mutant and control PCs, and the vertical red line in the middle identifies the point where ΔTE = 0. A majority of the genes are situated to the right of the …
Figure 5
Validation of downregulation of transcripts and maintained ribosomal loading in Tsc1-deficient PCs.
(a) This cumulative distribution function shows the distribution of total mRNA expression from sorted PCs for 150 FMRP target genes, previously found to be downregulated in mutant PCs, between …
Figure 6 with 1 supplement
Reduced expression of FMRP targets in mutant cerebellum.
Protein expression of (a) DLG3 and (b) KIF3C assessed by western blot of whole cerebellum lysates from control (Tsc1fl/fl) and mutant (Tsc1fl/fl L7Cre) animals (N = 6). Bands were quantified using …
Figure 6—figure supplement 1
Western blot controls and 5’ TOP genes.
(a) Western blot of GAPDH and total protein showing that GAPDH is unchanged between mutant and control cerebellum, verifying its use as a loading control. (b) Western blot of β-actin in mutant and …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Mus musculus) | Tsc1fl/fl | Jackson Labs | (Stock# 005680) | |
| Strain, strain background (Mus musculus) | Rosa26fsTRAP | Jackson Labs | (Stock# 022367) | |
| Strain, strain background (Mus musculus) | L7Cre | Jackson Labs | (Stock# 010536) | |
| Strain, strain background (Mus musculus) | L7GFP | Jackson Labs | (Stock# 004690) | |
| Antibody | Anti-GFP (Mouse monoclonal) | Memorial Sloan Kettering Centre | HtzGFP-19F7 and HtzGFP-19C8 | 50 µg per IP |
| Antibody | Anti-DLG3 (PSD93) (Mouse monoclonal) | Biolegend | 818302 | (1:1000) |
| Antibody | Anti-KIF3C (Rabbit polyclonal) | Proteintech | 144333–1-AP | (1:800) |
| Antibody | Anti-CDK16 (PCTAIRE) (Rabbit polyclonal) | Proteintech | 10102–1-AP | (1:750) |
| Antibody | Anti-Rpl28 (Rabbit polyclonal) | Proteintech | 16649–1-AP | (1:1000) |
| Antibody | Anti-Gapdh (Mouse monoclonal) | ThermoFisher | AM4300 | (1:10,000) |
| Antibody | Anti-B Actin (Mouse Monoclonal) | Cell Signaling | 3700 | (1:5000) |
| Antibody | Anti-GFP (Chicken polyclonal) | ThermoFisher | A10262 | (1:1000) |
| Antibody | Anti-Shank2 (Rabbit polyclonal) | ThermoFisher | PA5-78652 | (1:500) |
| Peptide, recombinant protein | Purified Recombinant Biotinylated Protein L | Pierce | 29997 | |
| Chemical compound, drug | Streptavidin MyOne T1 Dynabeads | ThermoFisher | 65601 | |
| Chemical compound, drug | Cycloheximide | Sigma Aldrich | 01810–1G | |
| Chemical compound, drug | RNasin | Promega | N2515 | |
| Chemical compound, drug | Superasin | ThermoFisher | AM2694 | |
| Chemical compound, drug | DHPC | Avanti Polar Lipids | 850306 | |
| Chemical compound, drug | TRIzol | ThermoFisher | 15596026 | |
| Chemical compound, drug | Papain | Worthington | LS003126 | |
| Chemical compound, drug | DNase I | Worthington | LK003172 | |
| Commercial assay or kit | RNAeasy Minelute kit | Qiagen | 74204 | |
| Software, algorithm | R v4.0.2 | https://cran.r-project.org/ | ||
| Software, algorithm | Prism v9 | GraphPad | ||
| Software, algorithm | Python v2.7 | https://www.python.org/ |
Additional files
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Source data 1
Raw and annotated western blot images.
- https://cdn.elifesciences.org/articles/67399/elife-67399-data1-v1.zip
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Supplementary file 1
Supplemental table 1.
- https://cdn.elifesciences.org/articles/67399/elife-67399-supp1-v1.csv
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Supplementary file 2
Supplemental table 2.
- https://cdn.elifesciences.org/articles/67399/elife-67399-supp2-v1.csv
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Supplementary file 3
Supplemental table 3.
- https://cdn.elifesciences.org/articles/67399/elife-67399-supp3-v1.csv
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Supplementary file 4
Supplemental table 4.
- https://cdn.elifesciences.org/articles/67399/elife-67399-supp4-v1.csv
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Supplementary file 5
Supplemental table 5.
- https://cdn.elifesciences.org/articles/67399/elife-67399-supp5-v1.csv
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Transparent reporting form
- https://cdn.elifesciences.org/articles/67399/elife-67399-transrepform-v1.pdf
