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Novel Alternative Splice Variants of Mouse Cdk5rap2.

Kraemer N, Issa-Jahns L, Neubert G, Ravindran E, Mani S, Ninnemann O, Kaindl AM - PLoS ONE (2015)

Bottom Line: Autosomal recessive primary microcephaly (MCPH) is a rare neurodevelopmental disorder characterized by a pronounced reduction of brain volume and intellectual disability.A current model for the microcephaly phenotype invokes a stem cell proliferation and differentiation defect, which has moved the disease into the spotlight of stem cell biology and neurodevelopmental science.Homozygous mutations of the Cyclin-dependent kinase-5 regulatory subunit-associated protein 2 gene CDK5RAP2 are one genetic cause of MCPH.

View Article: PubMed Central - PubMed

Affiliation: Institute of Cell Biology and Neurobiology, Charité -Universitätsmedizin Berlin, Berlin, Germany; Department of Pediatric Neurology, Charité -Universitätsmedizin Berlin, Berlin, Germany.

ABSTRACT
Autosomal recessive primary microcephaly (MCPH) is a rare neurodevelopmental disorder characterized by a pronounced reduction of brain volume and intellectual disability. A current model for the microcephaly phenotype invokes a stem cell proliferation and differentiation defect, which has moved the disease into the spotlight of stem cell biology and neurodevelopmental science. Homozygous mutations of the Cyclin-dependent kinase-5 regulatory subunit-associated protein 2 gene CDK5RAP2 are one genetic cause of MCPH. To further characterize the pathomechanism underlying MCPH, we generated a conditional Cdk5rap2 LoxP/hCMV Cre mutant mouse. Further analysis, initiated on account of a lack of a microcephaly phenotype in these mutant mice, revealed the presence of previously unknown splice variants of the Cdk5rap2 gene that are at least in part accountable for the lack of microcephaly in the mice.

No MeSH data available.


Related in: MedlinePlus

mCdk5rap2 splice variants.Schematic representation of mCdk5rap2, mCdk5rap2-V1, and mCdk5rap2-V2 mRNA (top) and protein (bottom).
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pone.0136684.g002: mCdk5rap2 splice variants.Schematic representation of mCdk5rap2, mCdk5rap2-V1, and mCdk5rap2-V2 mRNA (top) and protein (bottom).

Mentions: The lack of a phenotype of conditional Cdk5rap2 knockout mice lead us to analyze Cdk5rap2 mRNA in mutant (hom KO) and wild-type (WT) mice in detail. We thereby identified one mRNA variant (mCdk5rap2-V1) with an additional exon 3a, which is expressed both in the WT as well as in the hom KO mice. Given that mCdk5rap2-V1 is expressed only at very low levels and is not upregulated in the mutant mice, it is unlikely that this variant is solely responsible for the rescue of the phenotype in the Cdk5rap2 cKO mice. Moreover, we did not detect immunopositivity in hom KO cortical sections when applying the N-terminal antibody N1, which should recognize mCdk5rap2-V1. In addition to this, the detection of Cdk5rap2 in hom KO mice cortex with the more C-terminal antibody A1 indicates the existence of an additional variant. In this regard, our data further suggest a second Cdk5rap2 mRNA variant (mCdk5rap2-V2) which lacks the N-terminal part of the full length Cdk5rap2 mRNA sequence from bp 1 to 638 (exon 1 to 6) and uses an alternative start codon in exon 7. Since in WT mice translation of mCdk5rap2-V1 results in a truncated 85 aa protein it is most likely that this variant is physiologically redundant (Fig 2). The suggested variant mCdk5rap2-V2 lacks the γTuRC binding domain, which has been reported to be important for the γTuRC attachment to the centrosome and therefore for the microtubule organizing function of the centrosome [12]. However, mCdk5rap2-V2 localizes to the centrosome in mouse neocortex, as shown in immunohistological stainings. This allows speculation that potentially targeting Cdk5rap2 to the centrosome is not exclusively dependent of the yTuRC site but rather on interaction with other proteins such as pericentrin. The functional significance of these variants as well as those predicted in genome datasets (S3 Fig) and the human variants (S4 Fig) will need to be addressed in further functional studies.


Novel Alternative Splice Variants of Mouse Cdk5rap2.

Kraemer N, Issa-Jahns L, Neubert G, Ravindran E, Mani S, Ninnemann O, Kaindl AM - PLoS ONE (2015)

mCdk5rap2 splice variants.Schematic representation of mCdk5rap2, mCdk5rap2-V1, and mCdk5rap2-V2 mRNA (top) and protein (bottom).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4556188&req=5

pone.0136684.g002: mCdk5rap2 splice variants.Schematic representation of mCdk5rap2, mCdk5rap2-V1, and mCdk5rap2-V2 mRNA (top) and protein (bottom).
Mentions: The lack of a phenotype of conditional Cdk5rap2 knockout mice lead us to analyze Cdk5rap2 mRNA in mutant (hom KO) and wild-type (WT) mice in detail. We thereby identified one mRNA variant (mCdk5rap2-V1) with an additional exon 3a, which is expressed both in the WT as well as in the hom KO mice. Given that mCdk5rap2-V1 is expressed only at very low levels and is not upregulated in the mutant mice, it is unlikely that this variant is solely responsible for the rescue of the phenotype in the Cdk5rap2 cKO mice. Moreover, we did not detect immunopositivity in hom KO cortical sections when applying the N-terminal antibody N1, which should recognize mCdk5rap2-V1. In addition to this, the detection of Cdk5rap2 in hom KO mice cortex with the more C-terminal antibody A1 indicates the existence of an additional variant. In this regard, our data further suggest a second Cdk5rap2 mRNA variant (mCdk5rap2-V2) which lacks the N-terminal part of the full length Cdk5rap2 mRNA sequence from bp 1 to 638 (exon 1 to 6) and uses an alternative start codon in exon 7. Since in WT mice translation of mCdk5rap2-V1 results in a truncated 85 aa protein it is most likely that this variant is physiologically redundant (Fig 2). The suggested variant mCdk5rap2-V2 lacks the γTuRC binding domain, which has been reported to be important for the γTuRC attachment to the centrosome and therefore for the microtubule organizing function of the centrosome [12]. However, mCdk5rap2-V2 localizes to the centrosome in mouse neocortex, as shown in immunohistological stainings. This allows speculation that potentially targeting Cdk5rap2 to the centrosome is not exclusively dependent of the yTuRC site but rather on interaction with other proteins such as pericentrin. The functional significance of these variants as well as those predicted in genome datasets (S3 Fig) and the human variants (S4 Fig) will need to be addressed in further functional studies.

Bottom Line: Autosomal recessive primary microcephaly (MCPH) is a rare neurodevelopmental disorder characterized by a pronounced reduction of brain volume and intellectual disability.A current model for the microcephaly phenotype invokes a stem cell proliferation and differentiation defect, which has moved the disease into the spotlight of stem cell biology and neurodevelopmental science.Homozygous mutations of the Cyclin-dependent kinase-5 regulatory subunit-associated protein 2 gene CDK5RAP2 are one genetic cause of MCPH.

View Article: PubMed Central - PubMed

Affiliation: Institute of Cell Biology and Neurobiology, Charité -Universitätsmedizin Berlin, Berlin, Germany; Department of Pediatric Neurology, Charité -Universitätsmedizin Berlin, Berlin, Germany.

ABSTRACT
Autosomal recessive primary microcephaly (MCPH) is a rare neurodevelopmental disorder characterized by a pronounced reduction of brain volume and intellectual disability. A current model for the microcephaly phenotype invokes a stem cell proliferation and differentiation defect, which has moved the disease into the spotlight of stem cell biology and neurodevelopmental science. Homozygous mutations of the Cyclin-dependent kinase-5 regulatory subunit-associated protein 2 gene CDK5RAP2 are one genetic cause of MCPH. To further characterize the pathomechanism underlying MCPH, we generated a conditional Cdk5rap2 LoxP/hCMV Cre mutant mouse. Further analysis, initiated on account of a lack of a microcephaly phenotype in these mutant mice, revealed the presence of previously unknown splice variants of the Cdk5rap2 gene that are at least in part accountable for the lack of microcephaly in the mice.

No MeSH data available.


Related in: MedlinePlus