Analysis of connexin32 expression in the nervous system: Discovery of an essential IRES element

Date of Award




Degree Name

Doctor of Philosophy (Ph.D.)

First Committee Member

Rudolf Werner - Committee Chair


Connexin32 is the primary gap junction protein expressed in Schwann cells. Mutations in the connexin32 gene have been associated with Charcot-Marie-Tooth disease, a neurodegenerative disorder. To better understand the regulation of expression of connexin32 in the nervous system, the effects of two naturally occurring mutations on gene expression were investigated.One mutation (mut-1) is in the neuro-specific promoter and the other (mut-2) is in the 5' untranslated region (UTR) of the connexin32 gene. The effects of these mutations on expression of a reporter construct were studied in transgenic mice. Transgenic mice were assayed for transcription (by RT-PCR) and translation (by luciferase assay) of the luciferase gene in the brain. No luciferase mRNA was detected in two independent lines of mut-1 mice, suggesting that the point mutation in the promoter is sufficient to abolish transcription. Evidence from gel mobility shift assays suggests that the mut-1 sequence may create a binding site for a factor that acts as a transcriptional inhibitor.The mut-2 mRNA was transcribed and spliced correctly, but no luciferase activity was detected in brain extracts. This result shows that the mutation in the 5' UTR connexin32 message was examined in an in vitro translation system. The 5 ' UTR, which contains upstream AUGs, inhibited translation of the connexin32 message. Since nerve cells apparently have no problem translating this mRNA in vivo they must have a mechanism that allows the ribosomes to bypass the upstream AUGs and initiate protein synthesis from the correct AUG codon, perhaps by the use of an internal ribosomal entry site (IRES). To investigate this possibility, the wildtype and mut-2 5'UTR sequences were tested in a bicistronic assay. The wildtype 5' UTR, but the mut-2 5 ' UTR, showed IRES activity. This supports the hypothesis that an IRES element in the 5' UTR mediates translation of connexin32 mRNA and that mut-2 abolishes the function of the IRES. This is the first evidence of an IRES mutation causing a human disease. In addition, the fact that the reporter gene is not translated in transgenic mice and that patients with this point mutation have a disease phenotype strongly suggests that the IRES element is essential for translation of the connexin32 mRNA in the nervous system.


Biology, Molecular; Biology, Neuroscience

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