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, 23 (2), 293-302

Evaluating the Effects of CELF1 Deficiency in a Mouse Model of RNA Toxicity

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Evaluating the Effects of CELF1 Deficiency in a Mouse Model of RNA Toxicity

Yun Kyoung Kim et al. Hum Mol Genet.

Abstract

Myotonic dystrophy type 1 (DM1), the most common form of adult-onset muscular dystrophy, is caused by an expanded (CTG)n repeat in the 3' untranslated region of the DM protein kinase (DMPK) gene. The toxic RNA transcripts produced from the mutant allele alter the function of RNA-binding proteins leading to the functional depletion of muscleblind-like (MBNL) proteins and an increase in steady state levels of CUG-BP1 (CUGBP-ETR-3 like factor 1, CELF1). The role of increased CELF1 in DM1 pathogenesis is well studied using genetically engineered mouse models. Also, as a potential therapeutic strategy, the benefits of increasing MBNL1 expression have recently been reported. However, the effect of reduction of CELF1 is not yet clear. In this study, we generated CELF1 knockout mice, which also carry an inducible toxic RNA transgene to test the effects of CELF1 reduction in RNA toxicity. We found that the absence of CELF1 did not correct splicing defects. It did however mitigate the increase in translational targets of CELF1 (MEF2A and C/EBPβ). Notably, we found that loss of CELF1 prevented deterioration of muscle function by the toxic RNA, and resulted in better muscle histopathology. These data suggest that while reduction of CELF1 may be of limited benefit with respect to DM1-associated spliceopathy, it may be beneficial to the muscular dystrophy associated with RNA toxicity.

Figures

Figure 1.
Figure 1.
CELF1 levels correlate with muscle histopathology in skeletal muscles of patients with DM1 and in the 5-313 mice. Graded muscle tissues from (A) human samples (normal = 3 unaffected adults, DM1 = 3 female and 3 male, ages between 47 and 55 years) and (B) the 5-313 mouse model (2 months old, 2 weeks induction, paraspinal muscles) was subjected to western blot for CELF1. CELF1 levels increased with severity of muscle histopathology in both DM1 individuals and the 5-313 mice. GAPDH was used as loading control. (C and D) Quantification for CELF1 protein levels using western blots for human (C) and mouse (D) tissues (n ≥ 3/group). A t-test was used to compare the results from normal tissues with each of the other groups (*P < 0.05, **P < 0.01, ***P < 0.005). Representative images provided in Supplementary Material, Figure S1A.
Figure 2.
Figure 2.
Levels of relevant RNA-binding proteins. Protein extracts were prepared from paraspinal muscle tissues of mice and examined by western blots. Each lane represents a different mouse with the indicated genotype and induction condition. CELF1 was not detected in Celf1−/− mice. Western blot for CELF2, MBNL1 showed no change due to absence of CELF1 or presence of RNA toxicity. Western blot for eGFP confirmed transgene induction. GAPDH was used as loading control (see Supplementary Material, Fig. S4 for graphical representation of quantifications).
Figure 3.
Figure 3.
Preservation of muscle function in Celf1−/−/5-313+/− mice. Celf1+/+/5-313+/− mice (n = 7) and Celf1−/−/5-313+/− mice (n = 5) were induced with 0.2% of doxycycline for 5 months. Uninduced mice were used as control (Celf1+/+/5-313+/− (n = 4), Celf1−/−/5-313+/− (n = 3)). (A) Mice were subjected to treadmill running and distance run was measured. The measurements were converted to % retained, as compared with a baseline measurement for each mouse. (B) Grip strength was assessed using a grip-strength meter. Data were expressed as force in grams and converted to % of grip-strength retained as compared with baseline. t-Tests for statistical significance were performed between Celf1+/+/5-313+/− (−dox) mice and either Celf1+/+/5-313+/− (+dox) mice or Celf1−/−/5-313+/− (−dox) mice. Comparison between Celf1+/+/5-313+/− (+dox) mice and Celf1−/−/5-313+/− (+dox) mice is as marked by brackets. *P < 0.05, **P < 0.01, ***P < 0.005.
Figure 4.
Figure 4.
Absence of CELF1 has no beneficial effects on the RNA splicing defects caused by RNA toxicity. (A) ClCn-1 exon 7a, Tnnt3-fetal exon and Smyd1 exon 39 were analyzed as mRNA splicing targets of DM1. (B) Nrap exon 12 and Fxr1h exons15-16 were assessed as CELF1-specific targets. Both Celf1+/+/5-313 and Celf1−/−/5-313 mice were tested in uninduced (−dox) and induced (+dox) conditions (n = 5 per group). The percentage of exon inclusion is graphically represented. (C) Expression of embryonic myosin heavy chain (Myh3) was examined by qRT–PCR. Significant differences were calculated and indicated as described in Figure 3. *P < 0.05, **P < 0.01 and ***P < 0.005 (t-test).
Figure 5.
Figure 5.
Improved muscle histopathology in CELF1−/−/5-313 and correlation with levels of Nkx2-5 mRNA. (A) Quadriceps femoris muscles were stained with H&E. Both uninduced Celf1+/+/5-313 and Celf1−/−/5-313 mice have normal muscle histology. Celf1−/−/5-313 (+dox) mice have milder histopathology compared with Celf1+/+/5-313(+dox) mice. (B) Histopathology grading shows Celf1−/− has a beneficial effect. Mann–Whitney U-test was used for statistical significance. Celf1+/+/5-313 (−dox) mice (n = 4) were compared with Celf1+/+/5-313 (+dox) mice (n = 5) or Celf1−/−/5-313 (−dox) mice (n = 3). Comparisons between induced (+dox) Celf1+/+/5-313 and Celf1−/−/5-313 mice (n = 5), and Celf1−/−/5-313 (−dox) mice and Celf1−/−/5-313 (+dox) mice are marked by brackets. *P < 0.05, **P < 0.01 and ***P < 0.005 (Mann–Whitney U-test). (C) Nkx2-5 mRNA level was assessed by qRT–PCR and relative expression is presented. Celf1−/−/5-313 mice (+dox) have five times less Nkx2-5 mRNA than Celf1+/+/5-313 mice (+dox) (n = 5); **P < 0.01 (t-test).
Figure 6.
Figure 6.
MEF2A and C/EBPβ protein levels and expression of MuRF1 mRNA are affected by the absence of CELF1 in RNA toxicity. (A) Western blot for MEF2A. Numbers indicate relative intensity of bands (n = 4). MEF2A levels are not significantly affected by CELF1 deficiency in uninduced mice (P > 0.2, t-test). MEF2A increases significantly in Celf1+/+/5-313 with RNA toxicity (*P < 0.05, t-test). This is not seen in CELF1 deficient mice with RNA toxicity. (B) C/EBPβ LAP and (C) LIP isoforms were measured from western blots and relative protein expression are graphically represented. Both LAP and LIP levels were increased by RNA toxicity in induced 5-313 mice but not in Celf1−/−/5-313 mice (n = 5 per group). (D) Relative MuRF1 mRNA expressions were measured by qRT–PCR. Celf1+/+/5-313(+dox) mice (n = 6) have 5-fold more MuRF1 mRNA than uninduced mice (n = 7) and these levels are reduced to 1.6-fold in Celf1−/−/5-313(+dox) (n = 5). Significant differences were calculated and indicated as described in Figure 3. *P < 0.05, **P < 0.01 and ***P < 0.005 (t-test).

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