Figure 4. GCN2 activation in the B6J-Gtpbp2^nmf205-/- brain is independent of uncharged tRNA^Arg_UCU and unprocessed n-Tr20 tRNA.

(A) Northern blot analysis of cerebellar RNA from 3-week-old B6J and B6J-Gtpbp2^nmf205-/- (B6J-nmf205^-/-) mice using pooled probes to n-Tr21/22/23/25 tRNAs to assess the expression levels of the tRNA^Arg_UCU isodecoder family. 5S was used as internal control. (B) Charged (pH 5) and uncharged (pH 9) tRNA^Arg_UCU levels in the 3-week-old B6J and B6J-Gtpbp2^nmf205-/- cerebellum. Note that the levels of uncharged tRNA^Arg_UCU are negligible in both the B6J-Gtpbp2^nmf205-/- and B6J cerebellum. A short (right) and a longer (left) exposure are shown. (C–E) RT-qPCR analysis of expression of ATF4 target genes. (C) Overexpression of the mutant n-Tr20 tRNA does not change expression of ATF4 targets in the 3-week-old cerebellum although increased levels of unprocessed n-Tr20 are present. (n=3 mice per genotype) (D) Overexpression of the mutant n-Tr20 tRNA does not change expression of ATF4 targets even in the Gtpbp2^nmf205-/- mutant cerebellum at 3 weeks of age. (n=3) (E) ATF4 targets are significantly upregulated in the P0 brain of B6J-n-Tr20^-/-; Gtpbp2^nmf205-/- mice, although no unprocessed or uncharged forms of n-Tr20 are present. (n=4 mice per genotype) Error bars = SEM. *p<0.05, **p<0.01, and ***p<0.001 (Student's unpaired two-tailed t tests, C, E; one-way ANOVA, D).

DOI: http://dx.doi.org/10.7554/eLife.14295.022

Figure 4—figure supplement 1. Generation of mice overexpressing mutant n-Tr20.

(A) Schematic showing 1.2-kb long genomic region around n-Tr20 containing the B6J SNP (indicated with an asterisk) that was used to produce transgenic mice. (B) Northern blot analysis using whole brain RNA comparing the expression pattern of n-Tr20 in B6N and B6J and the transgenic founder lines. Based on the amount of the 115-nt unprocessed form, line Tg681 has the highest expression of the mutant transgene and hence was chosen for the further experiments described in this paper.

DOI: http://dx.doi.org/10.7554/eLife.14295.023

Figure 4—figure supplement 2. Generation of mice with the deleted n-Tr20 allele.

(A) LoxP sites were introduced flanking the n-Tr20 locus were introduced by homologous recombination as shown in the schematic. n-Tr20 was deleted in the germline by crossing B6J mice carrying the floxed allele to B6J-EIIa-Cre mice. (B) PCR analysis of genomic DNA using the indicated primers confirmed Cre-excision of n-Tr20. (C) Northern blot analysis showing complete loss of n-Tr20 expression in B6J-n-Tr20^-/- brain. For comparison, levels of n-Tr20 are shown in the B6J and B6N brain.

DOI: http://dx.doi.org/10.7554/eLife.14295.024