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 leukodystrophy mutations.

(B) Tamoxifen injections and experimental timeline.

(C) Lower body weights of Polr3a-tamKI (red) versus WT (black) mice (males n=18/group, nested t test p<0.0001****, females n= 20/group, nested t test p<0.0001****). Data show mean ± SD.

(D) Body length differences at P56. Images are representative.

(E) LFB-staining of WT and KI brain sections at P42 and P90. The cerebral cortex, hippocampus and thalamus are marked by red, blue and black lines, respectively. Scale bar, 1000 µm.

(F) H&E-stained WT and KI pancreata at P42 and P90 shows a dramatic loss of acinar cells. Pancreatic islets are marked with a black arrowhead. Scale bar,100 µm.

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 (Orient sum) is the sum of three orientation behaviors (see Supplemental Fig. S3).

(E) Exploratory behavior (Rear sum) is the sum of three rearing behaviors (see Supplemental Fig. S3).

(F) Leg grooming. Data were collected at weekly intervals beginning at P42 (WT n= 9 and KI n= 5). Mice were tested for nine minutes with recording in three intervals of three minutes each. Values show the mean ± SEM at six- and nine-minute timepoints. Multiple t tests and one-way ANOVA, * p<0.05, ** p < 0.01, *** p < 0.001.

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 hybridization using transcript-specific oligonucleotide probes. All blots represent sequential probing of a single gel. The cropped images frame the relevant regions.

(B) Quantitation of Pol III transcripts in panel A (mean ± SEM, n= 5 biological replicates).

(C) RT-qPCR of Pol III transcript abundance (mean ± SEM, n= 3-6 biological replicates).

(D) RT-qPCR of selected ATF4-regulated ISR genes (mean ± SEM, n= 3-5 biological replicates).

(E) Global gene expression quantified by RNA-seq. The volcano plot shows gene expression changes (KI/WT, n= 4 biological replicates). Differentially expressed (DE) ATF4-regulated genes (green) (p-adj <0.05, log2FC >|0.58|) and mitochondrially-encoded transcripts (blue). ATF4-regulated genes assayed in panel D and Ddit3/Chop are labeled.

(F) Top ten GO bioprocesses among up-regulated DE genes are shown with a heatmap of Z-scores for DE genes annotated to the GO:0002376 immune system process. For all graphs: WT, gray bars and circles; KI, red bars and circles. ns, not significant; * p ≤0.05; ** p ≤0.01; *** p ≤0.005.

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 et al. 2018.

(D-E) Immunostaining of oligodendrocytes at the midline of the corpus callosum. Scale bar, 40 μm. Mean fluorescence intensities of MBP and CC1, MBP staining density and CC1 cell counts represent mean ± SD, n= 3.

(F) NeuN immunostaining of neurons in the motor cortex. Scale bar, 40 μm. Mean fluorescence intensities and cell counts represent mean ± SD of bilaterally symmetric regions (n= 3).

(G-H) Iba1 staining of microglia in the striatum (G) and cerebral cortex (H). Insets show larger magnifications of individual microglia. Note the striking shortening of processes and enlargement of the cell body in the striatum. Scale bar, 200 μm. Cell counts represent mean ± SD of bilaterally symmetric regions, (n= 2). For all graphs: WT, black; KI, red; ns, not significant; * p ≤0.05; ** p ≤0.01; *** p ≤0.005; **** p < 0.0001.

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 Fig. 3A.

(B) Quantitation of Pol III transcripts in panel A. Mean ± SEM, n= 5 biological replicates.

(C) RT-qPCR of Pol III transcript abundance. Mean ± SEM, n= 3-5 biological replicates.

(D) Venn diagram of the overlap between P75 and P42 DE genes (p-adj <0.05, log2FC >|0.58|).

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 biological replicates, p = 0.0003.

(B) tRNA fold change (KI/WT) is plotted against Log mean WT reads. Symbols show tRNAs encoded by unique loci (gray), identical tRNAs encoded by multiple loci (hollow black), iMet tRNAs (red) and tRNAArg-TCT-4-1 (green).

(C) tRNA fold change (KI/WT) for all decoder families. Individual tRNAs are ordered from most to least fold change and grouped by codon recognition (tRNA decoder family). The amino acid for each tRNA decoder family is indicated.

(D) Violin plots of tRNA decoder fold changes (KI/WT) for cerebra (Ca) and cerebella (Cb) at P42. tRNA reads were summed for each tRNA decoder family and normalized to spike-in and mitochondrial tRNA reads, p = 0.0250. For Ca, n= 4 (KI) and n= 6 (WT) biological replicates and for Cb, n= 5 (KI) and n= 6 (WT) biological replicates.

(E) The cytoplasmic tRNA profile for KI cerebra is plotted against the WT profile. tRNA decoder reads are expressed as a percentage of their respective total cytoplasmic decoder pool, mean ± SEM. tRNA decoders that are significantly lower in the KI compared to WT (p ≤ 0.05) fall below the regression line and are labeled. The inset shows tRNA decoders representing <0.2% of the total tRNA pool.

(F) DE tRNA decoders in cerebra (Ca).

(G) DE tRNA decoders in cerebella (Cb). Heatmaps represent Z scores of normalized read counts for significant DE genes (p-adj <0.05).

(H) Venn diagram of the overlap between DE tRNA decoders in Ca and Cb (p-adj <0.05).