Figure 1.
Nxf1CAST does not suppress mRNA deficit in AtrnmgL.
(A) Scale diagram of the mouse Atrn locus indicates the location and approximate size of the IAP insertion in mgL allele and the Northern blot probe. (B) Northern blot analysis of brain poly(A)+ RNA (5 µg per lane) from non-mutant and mutant littermates shows reduced level of full-length Atrn mRNA in mgL animals, independent of Nxf1 allele (B for C57BL/6, C for CAST/Ei). (C, D) Quantitative PCR (TaqMan) data shows nominal reduction in the abundance of spliced 5′ sequences, but ∼6-fold reduction of 3′ sequences, consistent with alternative splicing and 3′ end formation in the insertion, with no difference between Nxf1 genotypes. Error bars indicate standard deviation.
Figure 2.
Nxf1CAST suppresses pre-mRNA processing defects in Mgrnmd.
(A) Diagram of the Mgrn locus indicates locations and sizes of the md IAP insertion and probes. (B) Northern blot of brain poly(A)+ RNA (3 µg per lane) shows increased level of full-length Mgrn RNA and concomitantly reduced levels of mutant-specific transcripts in md mutant mice in the presence of Nxf1CAST. (C, D) Quantification of multiple independent Northern blot experiments is shown. Graphs show means of replicate experiments normalized to nonmutant control samples on each blot. Error bars indicate standard deviation. Presence of the lowest molecular weight band was inconsistent across experiments and not included in panel D. *p≤0.05, Wilcoxon signed-ranks test applied to paired (same-blot) samples. (E) Quantitative RT-PCR across the inserted intron indicates ∼2-fold increase in correctly spliced Mgrn1 RNA in the presence of Nxf1CAST. **p≤0.005, unpaired t-test with one tail.
Figure 3.
Nxf1CAST suppresses Ap3d1mh2J RNA, protein and visible phenotypes.
(A) Diagram of Ap3d1 shows location and size of the mh2J IAP insertion and Northern blot probes. (B) Northern blot of brain poly(A)+ RNA (5 µg per lane) shows that the level of full-length Ap3d1 RNA in mh2J mice is partially restored and levels of mutant-specific RNAs are reduced in the presence of Nxf1CAST. (C) Quantification of Northern blot experiments with paired samples, showing means and standard deviation of normalized values across all experiments. *p≤0.05, Wilcoxon signed-ranks test using 5 pairs (using one Nxf1 heterozygous sample to complete a pair) and p≤0.01, paired t-test with 4 pairs; each test has one tail. (D) Levels of mutant-specific RNAs appear decreased in the presence of Nxf1CAST, *p≤0.05, Wilcoxon signed-ranks test. (E) Quantitative RT-PCR analysis with primers spanning the inserted intron. **p = 0.0003, unpaired t-test with one tail. (F) Western blot of brain protein extracts shows increased level of Ap3d protein from mh2J in Nxf1CAST mice. (G) Quantification of replicate Western blots shows ∼2-fold increase in Ap3d with Nxf1CAST. *p = 0.03, paired t-test, one tail. (H) mh2J coat color dilution is attenuated in Nxf1CAST. (I) Average coat color scores, comparing mice to a printed grading matrix, and (J) tremor severity scores were assessed by observers blinded to genotype. Error bars indicate standard deviation. **p≤0.01, *p≤0.05, t-test with one tail.
Figure 4.
Nxf1CAST suppresses Usp14axJ expression and behavioral phenotypes.
(A) Diagram of Usp14 locus shows locations of the axJ IAP insertion and Northern blot probes. (B) Northern blot of brain poly(A)+ RNA (4 µg per lane) shows that the level of full-length Usp14 RNA in axJ mice is partially restored and levels of mutant-specific RNAs are reduced in the presence of Nxf1CAST. (C) Quantification of paired samples from multiple Northern blot experiments, showing means and standard deviations. *p≤0.05, Wilcoxon signed-ranks test. (D) Quantitative RT-PCR shows ∼2-fold difference in expression of correctly spliced Usp14 RNA in mutant brains homozygous for Nxf1CAST. *p≤0.05, unpaired t-test with one tail. (E) Western blot of brain protein extracts shows increased level of Usp14 protein from axJ in Nxf1CAST mice. *p≤0.05, Wilcoxon signed-ranks. (F) Quantification of replicate Western blots shows increased Usp14 expression in the presence of Nxf1CAST. *p≤0.05, Wilcoxon signed-ranks. (G) Neurological assessment scores for tremor assigned by observers blinded to genotype show highly significant improvement in animals homozygous for Nxf1CAST. Error bars indicate standard deviation. **p<0.01 unpaired t-test. See Videos S1 and S2.
Figure 5.
Nxf1CAST suppresses Atcayhes RNA, protein and behavioral phenotypes.
(A) Diagram of Atcay shows location of the hes IAP insertion, Northern blot probes and TaqMan assay. (B) Northern blot of brain poly(A)+ RNA (8 µg per lane) shows that level of mutant-specific Atcay transcripts in hes mice are reduced in the presence of Nxf1CAST. (C) Quantification of replicate Northern blot experiments shows reduced level of mutant-specific transcripts in Nxf1CAST brains. **p<0.01 paired t-test with one tail. (D) Quantitative PCR (TaqMan) analysis of Atcay RNA in hes homozygotes shows ∼2-fold increase in Nxf1CAST. *p<0.05 paired t-test with one tail. (E) Western blot of brain protein extracts shows increased level of Caytaxin protein from expression of Atcay in Nxf1CAST mice. (F) Quantification of replicate Western blots shows ∼2-fold increase in Caytaxin with Nxf1CAST. *p<0.05, t-test, one tail. (G) Average neurological assessment scores assigned by observers blinded to genotype show highly significant improvement in animals homozygous for Nxf1CAST. Error bars indicate standard deviation. **p<0.01, t-test, one tail. See Videos S3 and S4.
Figure 6.
Nxf1CAST does not suppress ETn-induced Zhx2Afr mutation.
(A) Diagram shows genomic organization of Zhx2, including location of the ETn-IIa insertion. Insertion is not to scale. (B) Quantitative RT-PCR shows reduced Zhx2 expression from mutant alleles (m), but no suppression by Nxf1CAST. Persistent Afp expression in adult liver in mutant animals is highly variable among F2 animals at P40, but not significantly different between Nxf1 genotypes. Error bars indicate standard deviation.
Figure 7.
Nxf1CAST does not suppress ETn-induced mutations in A/J.
(A) Genomic organization of five sense and three antisense-oriented ETn insertions monitored in F2 mice from A/J x B6–Nxf1CAST. Insertions are not to scale. (B) Quantitative PCR (SYBR green) on brain and/or muscle cDNA shows reduced expression of sense-oriented ETn alleles, but no significant differences between Nxf1 genotypes. m, insertion allele at each indicated locus. (C) Quantitative PCR shows modest (Atp9a) or no difference (Art3, Mark3) in expression of antisense-oriented ETn insertions, with no difference attributable to Nxf1. Error bars indicate standard deviation.
Figure 8.
The gag region and IΔ1 deletion differentiate AtrnmgL from other IAP insertions.
(A) Organization and pairwise percent identity of sequenced IAP elements from mutations in this study. RTE, RNA transport element; ppt, polypurine tract. Sequences have been deposited in GenBank (Accession numbers FJ854355–FJ854360). (B) Neighbor-joining trees for each of four aligned sequence blocks show similar topology for LTR and pol/RTE/ppt regions. For the undeleted portion of the gag gene, AtrnmgL clusters with Pitpnvb as a separate group. Recently described and sequenced IAP-IΔ1 elements inserted in Atp2b2jog and Gria4spkw1 mutations fall within the group of suppressed elements in all four sequence regions.
Table 1.
Summary of genetic crosses to test Nxf1CAST modifier effects.