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, 7 (3), e33218

Muscleblind-like 1 Knockout Mice Reveal Novel Splicing Defects in the Myotonic Dystrophy Brain


Muscleblind-like 1 Knockout Mice Reveal Novel Splicing Defects in the Myotonic Dystrophy Brain

Koichi Suenaga et al. PLoS One.


Myotonic dystrophy type 1 (DM1) is a multi-systemic disorder caused by a CTG trinucleotide repeat expansion (CTG(exp)) in the DMPK gene. In skeletal muscle, nuclear sequestration of the alternative splicing factor muscleblind-like 1 (MBNL1) explains the majority of the alternative splicing defects observed in the HSA(LR) transgenic mouse model which expresses a pathogenic range CTG(exp). In the present study, we addressed the possibility that MBNL1 sequestration by CUG(exp) RNA also contributes to splicing defects in the mammalian brain. We examined RNA from the brains of homozygous Mbnl1(ΔE3/ΔE3) knockout mice using splicing-sensitive microarrays. We used RT-PCR to validate a subset of alternative cassette exons identified by microarray analysis with brain tissues from Mbnl1(ΔE3/ΔE3) knockout mice and post-mortem DM1 patients. Surprisingly, splicing-sensitive microarray analysis of Mbnl1(ΔE3/ΔE3) brains yielded only 14 candidates for mis-spliced exons. While we confirmed that several of these splicing events are perturbed in both Mbnl1 knockout and DM1 brains, the extent of splicing mis-regulation in the mouse model was significantly less than observed in DM1. Additionally, several alternative exons, including Grin1 exon 4, App exon 7 and Mapt exons 3 and 9, which have previously been reported to be aberrantly spliced in human DM1 brain, were spliced normally in the Mbnl1 knockout brain. The sequestration of MBNL1 by CUG(exp) RNA results in some of the aberrant splicing events in the DM1 brain. However, we conclude that other factors, possibly other MBNL proteins, likely contribute to splicing mis-regulation in the DM1 brain.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.


Figure 1
Figure 1. MBNL1 splicing targets in the mouse brain.
RT-PCR analysis of wild-type (wt, n = 3, 3–6 months of age) versus Mbnl1 ΔE3/ΔE3 (ko, n = 3, 3–6months of age) brain splicing. (A) Representative PCR products. (B) Percent exon inclusion measured by phosphoimager analysis. Loss of MBNL1 expression results in reduced exon inclusion for Sorbs1 exon 25, Spag9 exon 31, Sorbs1 exon 6 and Dclk1 exon 19 in both the hippocampus (hp) and cerebellum (cb). The inclusion of these exons is enhanced in the adult (P42) forebrain (fb) compared to the neonatal (P6) forebrain. Sorbs1 exon 25 splicing in the heart (ht) and Mprip splicing in the brain are included as positive and negative splicing controls. Both unpaired t-test and permutation test were used for calculating p value (*p value<0.05, **p value<0.01). Averages and standard deviations were generated by unpaired t-test.
Figure 2
Figure 2. Spatial and temporal differences in the expression of Camk2d.
(A) Transcripts of Camk2d. (B) Representative PCR products. Six spliced isoforms were detected by using primer pairs located in exons 14a and 17. Right panel shows isoform switching from δ9 to δ4 during the fetal (P1, P6) to adult (P42) transition in whole brain (wb), forebrain (fb) and hindbrain (hb) whereas the δ9 isoform was undetectable in adult hindbrain. Left panel shows a detectable increase in the δ9 isoform in Mbnl1 ΔE3/ΔE3 hippocampus (hp) compared with wild-type sibs. However, no δ9 isoform could be detected in the cerebellum of either wild-type or Mbnl1 knockout mice. (C) The percentage of Camk2d isoform δ9 to δ1+4+9+3+2. Both unpaired t-test and permutation test were used for calculating p value (n = 3, *p value<0.05). Averages and standard deviations were generated by unpaired t-test.
Figure 3
Figure 3. Aberrant splicing events in Mbnl1 ΔE3/ΔE3 brain are also observed in the human DM1 brain.
We compared brain RNA from normal control temporal cortex (control, tp, n = 4), disease control temporal cortex (disease, tp, n = 9), myotonic dystrophy type 1 temporal cortex (DM1, tp, n = 12), fetal control whole brain (fetal, wb, n = 1), disease control cerebellum (disease, cb, n = 4), and DM1 cerebellum (DM1, cb, n = 5). (A) Representative RT-PCR products detected with microchip electrophoretic separation using SV1210 software on the Hitachi SV1210 from exclusion and inclusion of exon 26 of SORBS1 (upper), exon 19 of DCLK1 (middle), and exon 9 of MPRIP (bottom). (B) Graphical representation of RT-PCR analysis depicting percentages of exon 26 inclusion of SORBS1 (upper), exon 19 inclusion of DCLK1 (middle), and exon 9 inclusion of MPRIP (bottom). (C) Transcripts of CAMK2D. (D) Representative RT-PCR products from CAMK2D isoforms: δ2(−exon14b−exon15), δ9(−exon14b+exon15), and δ1(+exon14b+exon15) indicated by each arrow. (E) Graphical representation of RT-PCR analysis depicting percentages of δ2 (upper), δ9 (middle) and δ1 (bottom). Mann-Whitney U test was used for calculating the p value. Statistically significant differences (p<0.05) are indicated by an asterisk.

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