Ube3a mRNA and protein expression are not decreased in Mecp2R168X mutant mice

Abstract

Mutations in the transcriptional repressor methyl CpG binding protein 2 (MeCP2) are responsible for most cases of Rett Syndrome (RS), a severe neurodevelopmental disorder characterized by developmental regression, minimal speech, seizures, postnatal microcephaly and hand stereotypies. Absence of the maternal copy of ubiquitin protein ligase 3A (UBE3A) results in Angelman syndrome, also a severe developmental disorder that shares some clinical features with RS. As MeCP2 regulates gene expression, this has led to the hypothesis that MeCP2 may regulate UBE3A expression; however, there are conflicting reports regarding the expression of Ube3a in MeCP2 null mutant mice. We have generated a novel MeCP2 mutant knock-in mouse with the mutation R168X, one of the most common mutations in patients with RS. These mice show features similar to RS, including hypoactivity, forelimb stereotypies, breathing irregularities, weight changes, hind limb atrophy, and scoliosis. The male mice experience early death. Analysis of Ube3a mRNA and protein levels in the Mecp2(R168X) male mice showed no significant difference in expression compared to their wild type littermates.

Figure 1. Mecp2^R168X mutant mice

(A) An A to T point mutation was engineered to create a premature stop codon in place of an arginine at codon 168. (B) Confirmation of the mutant transcript by RTPCR. RTPCR was performed with primers flanking the mutation using cDNA reverse transcribed from WT mouse RNA (lane 1), cDNA reverse transcribed from mutant mouse RNA (lane 2), WT mouse RNA (lane 3), and mutant mouse RNA (lane 4). The expected 238bp product was detected in lanes 1 and 2 and confirmed as MeCP2 by sequencing. Larger bands are produced from genomic DNA in lanes 3 and 4 due to the presence of intron 3. (C) The Mecp2^R168X mutant RNA is relatively stabled compared to the WT transcript. Error bars show standard error of the mean. (D) Mecp2^R168X mutant mice (right lane) express a small protein which may be a prematurely truncated mutant MeCP2 protein, but do not express the full length WT protein. The WT mice (left lane) express the full length protein.

Figure 1. Mecp2^R168X mutant mice

(A) An A to T point mutation was engineered to create a premature stop codon in place of an arginine at codon 168. (B) Confirmation of the mutant transcript by RTPCR. RTPCR was performed with primers flanking the mutation using cDNA reverse transcribed from WT mouse RNA (lane 1), cDNA reverse transcribed from mutant mouse RNA (lane 2), WT mouse RNA (lane 3), and mutant mouse RNA (lane 4). The expected 238bp product was detected in lanes 1 and 2 and confirmed as MeCP2 by sequencing. Larger bands are produced from genomic DNA in lanes 3 and 4 due to the presence of intron 3. (C) The Mecp2^R168X mutant RNA is relatively stabled compared to the WT transcript. Error bars show standard error of the mean. (D) Mecp2^R168X mutant mice (right lane) express a small protein which may be a prematurely truncated mutant MeCP2 protein, but do not express the full length WT protein. The WT mice (left lane) express the full length protein.

Figure 1. Mecp2^R168X mutant mice

(A) An A to T point mutation was engineered to create a premature stop codon in place of an arginine at codon 168. (B) Confirmation of the mutant transcript by RTPCR. RTPCR was performed with primers flanking the mutation using cDNA reverse transcribed from WT mouse RNA (lane 1), cDNA reverse transcribed from mutant mouse RNA (lane 2), WT mouse RNA (lane 3), and mutant mouse RNA (lane 4). The expected 238bp product was detected in lanes 1 and 2 and confirmed as MeCP2 by sequencing. Larger bands are produced from genomic DNA in lanes 3 and 4 due to the presence of intron 3. (C) The Mecp2^R168X mutant RNA is relatively stabled compared to the WT transcript. Error bars show standard error of the mean. (D) Mecp2^R168X mutant mice (right lane) express a small protein which may be a prematurely truncated mutant MeCP2 protein, but do not express the full length WT protein. The WT mice (left lane) express the full length protein.

Figure 1. Mecp2^R168X mutant mice

(A) An A to T point mutation was engineered to create a premature stop codon in place of an arginine at codon 168. (B) Confirmation of the mutant transcript by RTPCR. RTPCR was performed with primers flanking the mutation using cDNA reverse transcribed from WT mouse RNA (lane 1), cDNA reverse transcribed from mutant mouse RNA (lane 2), WT mouse RNA (lane 3), and mutant mouse RNA (lane 4). The expected 238bp product was detected in lanes 1 and 2 and confirmed as MeCP2 by sequencing. Larger bands are produced from genomic DNA in lanes 3 and 4 due to the presence of intron 3. (C) The Mecp2^R168X mutant RNA is relatively stabled compared to the WT transcript. Error bars show standard error of the mean. (D) Mecp2^R168X mutant mice (right lane) express a small protein which may be a prematurely truncated mutant MeCP2 protein, but do not express the full length WT protein. The WT mice (left lane) express the full length protein.

Figure 2. Characterization of Mecp2^R168X mutant mice

(A) Lifespan of the Mecp2^R168X mutant mice is shortened, with a median lifespan of 83.5 days (n=45). (B) At 4 weeks of age, greater variance in the weight distribution of the affected males was apparent, with numerous low weight mice. (C) In the mice surviving to 11 weeks, weight variance was also apparent, but many more mice were obese. (D) At 3 weeks, an affected hemizygous Mecp2^R168X male (mouse on the right) had difficulty gaining weight while his WT littermate thrived. (E) At 7 weeks, an affected hemizygous Mecp2^R168X male (mouse on the right) showed greater weight gain his WT littermate. (F) 7 week affected male showing hindlimb clasping. (G) 7 week old WT male littermate showing normal function of hindlimbs.

Figure 2. Characterization of Mecp2^R168X mutant mice

(A) Lifespan of the Mecp2^R168X mutant mice is shortened, with a median lifespan of 83.5 days (n=45). (B) At 4 weeks of age, greater variance in the weight distribution of the affected males was apparent, with numerous low weight mice. (C) In the mice surviving to 11 weeks, weight variance was also apparent, but many more mice were obese. (D) At 3 weeks, an affected hemizygous Mecp2^R168X male (mouse on the right) had difficulty gaining weight while his WT littermate thrived. (E) At 7 weeks, an affected hemizygous Mecp2^R168X male (mouse on the right) showed greater weight gain his WT littermate. (F) 7 week affected male showing hindlimb clasping. (G) 7 week old WT male littermate showing normal function of hindlimbs.

Figure 2. Characterization of Mecp2^R168X mutant mice

(A) Lifespan of the Mecp2^R168X mutant mice is shortened, with a median lifespan of 83.5 days (n=45). (B) At 4 weeks of age, greater variance in the weight distribution of the affected males was apparent, with numerous low weight mice. (C) In the mice surviving to 11 weeks, weight variance was also apparent, but many more mice were obese. (D) At 3 weeks, an affected hemizygous Mecp2^R168X male (mouse on the right) had difficulty gaining weight while his WT littermate thrived. (E) At 7 weeks, an affected hemizygous Mecp2^R168X male (mouse on the right) showed greater weight gain his WT littermate. (F) 7 week affected male showing hindlimb clasping. (G) 7 week old WT male littermate showing normal function of hindlimbs.

Figure 2. Characterization of Mecp2^R168X mutant mice

(A) Lifespan of the Mecp2^R168X mutant mice is shortened, with a median lifespan of 83.5 days (n=45). (B) At 4 weeks of age, greater variance in the weight distribution of the affected males was apparent, with numerous low weight mice. (C) In the mice surviving to 11 weeks, weight variance was also apparent, but many more mice were obese. (D) At 3 weeks, an affected hemizygous Mecp2^R168X male (mouse on the right) had difficulty gaining weight while his WT littermate thrived. (E) At 7 weeks, an affected hemizygous Mecp2^R168X male (mouse on the right) showed greater weight gain his WT littermate. (F) 7 week affected male showing hindlimb clasping. (G) 7 week old WT male littermate showing normal function of hindlimbs.

Figure 2. Characterization of Mecp2^R168X mutant mice

(A) Lifespan of the Mecp2^R168X mutant mice is shortened, with a median lifespan of 83.5 days (n=45). (B) At 4 weeks of age, greater variance in the weight distribution of the affected males was apparent, with numerous low weight mice. (C) In the mice surviving to 11 weeks, weight variance was also apparent, but many more mice were obese. (D) At 3 weeks, an affected hemizygous Mecp2^R168X male (mouse on the right) had difficulty gaining weight while his WT littermate thrived. (E) At 7 weeks, an affected hemizygous Mecp2^R168X male (mouse on the right) showed greater weight gain his WT littermate. (F) 7 week affected male showing hindlimb clasping. (G) 7 week old WT male littermate showing normal function of hindlimbs.

Figure 2. Characterization of Mecp2^R168X mutant mice

(A) Lifespan of the Mecp2^R168X mutant mice is shortened, with a median lifespan of 83.5 days (n=45). (B) At 4 weeks of age, greater variance in the weight distribution of the affected males was apparent, with numerous low weight mice. (C) In the mice surviving to 11 weeks, weight variance was also apparent, but many more mice were obese. (D) At 3 weeks, an affected hemizygous Mecp2^R168X male (mouse on the right) had difficulty gaining weight while his WT littermate thrived. (E) At 7 weeks, an affected hemizygous Mecp2^R168X male (mouse on the right) showed greater weight gain his WT littermate. (F) 7 week affected male showing hindlimb clasping. (G) 7 week old WT male littermate showing normal function of hindlimbs.

Figure 2. Characterization of Mecp2^R168X mutant mice

(A) Lifespan of the Mecp2^R168X mutant mice is shortened, with a median lifespan of 83.5 days (n=45). (B) At 4 weeks of age, greater variance in the weight distribution of the affected males was apparent, with numerous low weight mice. (C) In the mice surviving to 11 weeks, weight variance was also apparent, but many more mice were obese. (D) At 3 weeks, an affected hemizygous Mecp2^R168X male (mouse on the right) had difficulty gaining weight while his WT littermate thrived. (E) At 7 weeks, an affected hemizygous Mecp2^R168X male (mouse on the right) showed greater weight gain his WT littermate. (F) 7 week affected male showing hindlimb clasping. (G) 7 week old WT male littermate showing normal function of hindlimbs.

Figure 3. Ube3a and Ube3a-ATS mRNA expression

Male hemizygous Mecp2^R168X mice no significant difference in Ube3a (A) or Ube3a-ATS (B) mRNA expression in cortical tissue as compared to their WT littermates when analyzed by real time qRT-PCR. Error bars show standard error of the mean.

Figure 3. Ube3a and Ube3a-ATS mRNA expression

Male hemizygous Mecp2^R168X mice no significant difference in Ube3a (A) or Ube3a-ATS (B) mRNA expression in cortical tissue as compared to their WT littermates when analyzed by real time qRT-PCR. Error bars show standard error of the mean.

Figure 4. Ube3a protein expression

(A) Male hemizygous Mecp2^R168X mice showed no change in Ube3a protein expression in cortical tissue as compared to their WT littermates when analyzed by Western blot. Error bars show standard error of the mean. (B) Western blot showing representative samples from 3 pairs of mice.

Figure 4. Ube3a protein expression

(A) Male hemizygous Mecp2^R168X mice showed no change in Ube3a protein expression in cortical tissue as compared to their WT littermates when analyzed by Western blot. Error bars show standard error of the mean. (B) Western blot showing representative samples from 3 pairs of mice.