Molecular basis of neurodegeneration in a mouse model of Polr3-related disease
- Department of Biochemistry, Albert Einstein College of Medicine, United States
- Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, United States
- Department of Systems and Computational Biology, Albert Einstein College of Medicine, United States
Figures
Figure 1 with 2 supplements
Body weight, length, and histological characteristics of Polr3a-tamKI mice.
(A) Schematic of the modified Polr3a locus showing floxed WT sequences encoding exons 15–31 with adjacent sequences for termination and polyadenylation (pA) and Polr3a exon15 containing the …
Figure 1—figure supplement 1
Analysis of recombination frequency in Polr3a-tamKI mice.
(A) Flow cytometry analysis of a cerebral homogenate from tamoxifen-treated Polr3a-tamKI mice carrying a dual tdTomato-EGFP recombination reporter (Muzumdar et al., 2007). Five tamoxifen injections …
Figure 1—figure supplement 2
Cerebellar histology and glucose homeostasis of Polr3a-tamKI mice.
(A) LFB- and Nissl-stained sagittal sections of cerebellum from P90 mice. No differences in staining intensity were noted. (B) Purkinje cell density was quantified in Nissl-stained sections for …
Figure 2 with 1 supplement
Behavioral studies of WT and Polr3a-tamKI mice.
(A–C) Locomotor activity is reported as the total distance traveled (Track length) and the total time during which the mice are running (Run sum) or standing still (Still sum). (D) Risk assessment …
Figure 2—figure supplement 1
Behavioral spectrometer analysis of WT and Polr3a-tamKI mice.
(A) Center visit counts. (B). Center visit duration. (C). Center track length. Anxiety-like behaviors in panels (A–C) were scored in a 15 cm2 area in the center of the 40 cm2 arena. (D). Orient …
Figure 3 with 2 supplements
Transcriptome analysis of Polr3a-tamKI cerebra at P75.
(A) Northern blot of Pol III transcripts. Precursor tRNAs (Ile-TAT, Leu-CAA), mature tRNAs (Leu-CAA, iMet, Ser-CGA, His), RPPH1, U6 and 7SL RNAs and the loading control U3 snRNA were detected by …
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Figure 3—source data 1
PDF file containing original northern blots for Figure 3A, indicating the relevant bands and conditions.
- https://cdn.elifesciences.org/articles/95314/elife-95314-fig3-data1-v1.pdf
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Figure 3—source data 2
Original files for northern analysis displayed in Figure 3A.
- https://cdn.elifesciences.org/articles/95314/elife-95314-fig3-data2-v1.zip
Figure 3—figure supplement 1
Gene expression in WT and Polr3a-tamKI (KI) cerebra at P75.
(A) Heatmap represents Z scores of DESeq2 normalized read counts for ATF4-dependent ISR genes activated in cerebra at P75 (p-adj <0.05). (B) RT-qPCR analysis of XBP1 splicing. The relative …
Figure 3—figure supplement 2
Pol III and Pol II transcript levels in various tissues from WT and Polr3a-tamKI (KI) mice at P75.
(A) The relative abundance of precursor and mature tRNAs in total RNA from cerebella (Cb), heart, kidney, and liver. Cerebral (Ca) data (from Figure 3B and C) are included for ease of comparison. …
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Figure 3—figure supplement 2—source data 1
PDF file containing original northern blots for Figure 3—figure supplement 2A.
- https://cdn.elifesciences.org/articles/95314/elife-95314-fig3-figsupp2-data1-v1.pdf
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Figure 3—figure supplement 2—source data 2
Original files for northern analysis displayed in Figure 3—figure supplement 2A.
- https://cdn.elifesciences.org/articles/95314/elife-95314-fig3-figsupp2-data2-v1.zip
Figure 4
Cell-type-specific changes in Polr3a-tamKI cerebra at P75.
(A–C) Oligodendrocyte (A), neuron (B), and microglia (C) cell type-specific DE genes. Heatmaps show Z-scores of DE genes identified in the top 500 mouse cell-type-specific genes defined in McKenzie …
Figure 5 with 1 supplement
Transcriptome analysis of Polr3a-tamKI cerebra at P42.
(A) Northern blots of Pol III transcripts. Precursor tRNAs (Ile-TAT, Ser-CGA), mature tRNAs (Leu-CAA, iMet, Ser-CGA, His) and U3 snRNA were detected as in Figure 3A. (B) Quantitation of Pol III …
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Figure 5—source data 1
PDF file containing original northern blots for Figure 5A, indicating the relevant bands and conditions.
- https://cdn.elifesciences.org/articles/95314/elife-95314-fig5-data1-v1.pdf
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Figure 5—source data 2
Original files for northern analysis displayed in Figure 5A.
- https://cdn.elifesciences.org/articles/95314/elife-95314-fig5-data2-v1.zip
Figure 5—figure supplement 1
Gene expression and Iba1 staining in adolescent WT and Polr3a-tamKI (KI) cerebra.
(A) RT-qPCR analysis of a set of ATF4-regulated ISR genes in WT and KI cerebra at P42. Data were calculated, normalized and plotted as in Figure 3D, n=3–5 biological replicates. ns, not significant; …
Figure 6 with 1 supplement
Cytosolic tRNA abundance in Polr3a-tamKI cerebra at P42.
(A) Total cytoplasmic tRNA reads in WT and KI cerebra, normalized to the sum of spike-in and mitochondrially-encoded tRNA reads are expressed relative to the mean WT value. Mean ± SD, KI n=4, WT n=6 …
Figure 6—figure supplement 1
Gene expression in WT and Polr3a-tamKI (KI) cerebella at P42.
(A–B) The relative abundance of Pol III transcripts in total RNA from WT and KI cerebella (Cb) at P42 determined by northern analysis (A) and RT-qPCR (B). (A) Precursor (pre-tRNAIle-TAT) and mature …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Genetic reagent (Mus musculus) | C57BL/6J-Polr3atm1Iwil | Merheb et al., 2021 | PMCID:PMC8501794 | |
| Genetic reagent (M. musculus) | B6.Cg-Tg(CAG-cre/Esr1*5Amc/J) | Jackson Laboratory | #004682, RRID:IMSR_JAX:004682 | |
| Genetic reagent (M. musculus) | B6.129(Cg)-Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo/J | Jackson Laboratory | #007676, RRID:IMSR_JAX:007676 | |
| Antibody | Anti-MBP, rat monoclonal | Abcam | #ab7349, RRID:AB_305869 | 1:100 |
| Antibody | Anti-APC (CC1), mouse monoclonal | Millipore | #OP80, RRID:AB_2057371 | 1:20 |
| Antibody | Anti-NeuN, mouse monoclonal | Abcam | #ab104224, RRID:AB_10711040 | 1:100 |
| Antibody | Anti-Iba1, rabbit polyclonal | FUJIFILM Wako | #019–19741, RRID:AB_839504 | 1:250 |
| Antibody | Anti-mouse IgG, Alexa Fluor 488 goat polyclonal | Invitrogen | #A28175, RRID:AB_2536161 | 1:1000 |
| Antibody | Anti-mouse IgG2b Alexa-Fluor 568 goat polyclonal | Invitrogen | #A21144, RRID: AB_2535780 | 1:1000 |
| Antibody | Anti-rat IgG Alexa Fluor 633 goat polyclonal | Invitrogen | #A21094, RRID: AB_2535749 | 1:1000 |
| Antibody | Anti-rabbit IgG Alexa Fluor 488 goat polyclonal | Invitrogen | #A11008 RRID: AB_143165 | 1:1000 |
| Commercial assay or kit | Adult brain dissociation kit, mouse | Miltenyi | #130-107-677 | |
| Commercial assay or kit | Lightcycler 480 SYBR Green I Master mix | Roche LifeScience | #04707516001 | |
| Chemical compound, drug | ProLong diamond antifade with DAPI | Invitrogen | #36962 | |
| Chemical compound, drug | Paraformaldehyde 32% | Electron Microscopy Sciences | #15,714 S | |
| Chemical compound, drug | Tamoxifen | Millipore Sigma | #T5648 | |
| Chemical compound, drug | Corn oil | Millipore Sigma | #C8267 | |
| Chemical compound, drug | SuperScript III | Thermo Fisher | #18080051 | |
| Chemical compound, drug | SuperScript IV | Thermo Fisher | #18091050 | |
| Chemical compound, drug | TRIzol Reagent | Thermo Fisher | #15596018 | |
| Chemical compound, drug | RNaseOUT | Invitrogen | #10777019 | |
| Chemical compound, drug | T4 polynucleotide kinase | New England Biolabs | #M0201 | |
| Software, algorithm | CaseViewer v2.4 software | 3DHistech | RRID:SCR_017654 | |
| Software, algorithm | Viewer software | Biobserve | RRID:SCR_014337 | |
| Software, algorithm | Volocity v5.3 | Perkin Elmer | RRID:SCR_002668 | |
| Software, algorithm | Prism v9.0 | GraphPad Software | RRID:SCR_002798 | |
| Software, algorithm | ImageQuant v5.2 | GE Healthcare | RRID:SCR_014246 | |
| Software, algorithm | ImageJ v1.53 | Github | RRID:SCR_003070 | |
| Software, algorithm | R studio v1.3.10.93 | Posit | RRID:SCR_000432 | |
| Software, algorithm | DEseq2 | Bioconductor | RRID:SCR_015687 |
Additional files
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MDAR checklist
- https://cdn.elifesciences.org/articles/95314/elife-95314-mdarchecklist1-v1.docx
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Supplementary file 1
RNAseq analysis of WT and Polr3a-tamKI cerebra at P75.
- https://cdn.elifesciences.org/articles/95314/elife-95314-supp1-v1.xlsx
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Supplementary file 2
Mouse cell type-specific DE genes in Polr3a-tamKI cerebra at P75.
- https://cdn.elifesciences.org/articles/95314/elife-95314-supp2-v1.xlsx
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Supplementary file 3
RNAseq analysis of WT and Polr3a-tamKI cerebra at P42.
- https://cdn.elifesciences.org/articles/95314/elife-95314-supp3-v1.xlsx
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Supplementary file 4
RNAseq of WT and Polr3a-tamKI cerebella at P42.
- https://cdn.elifesciences.org/articles/95314/elife-95314-supp4-v1.xlsx
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Supplementary file 5
tRNAseq P42_Ca. tRNAseq results and analysis of P42 cerebra.
- https://cdn.elifesciences.org/articles/95314/elife-95314-supp5-v1.xlsx
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Supplementary file 6
tRNAseq analysis of WT and Polr3a-tamKI cerebella at P42.
- https://cdn.elifesciences.org/articles/95314/elife-95314-supp6-v1.xlsx
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Supplementary file 7
Oligonucleotide sequences.
- https://cdn.elifesciences.org/articles/95314/elife-95314-supp7-v1.xlsx
