Rudolf Werner

Professor of Biochemistry and Molecular Biology

Dr. rer.nat. (1963) University of Freiburg, Germany.

Regulation of connexin gene expression by IRES elements
and miRNA

Tel: (305) 243-6998, Fax: (305) 243-3065

r.werner@miami.edu

Research in my laboratory was concerned with the regulation of gene expression, both at the transcriptional and translational level. We were  studying connexin genes. Connexins are proteins that form cell-cell channels. They are found in most animal cells in the form of intermembrane clusters, called  gap junctions, that contain many individual channels . There is a family of different connexins expressed in different cell types. For example, connexin43 is found in heart muscle and in uterus. It functions in the propagation of electrical signals that result in the contraction of the muscle. The gene for connexin43 is expressed constitutively in heart, whereas it is induced by estrogen in uterus. We are studying the connexin43 promoter to understand the mechanism of this differential regulation. In the course of these studies, one of the graduate students in my lab, Elisa Oltra, has identified a novel transcription factor "Ini". The gene for Ini is highly conserved across different species and produces a lethal phenotype when mutated in yeast.

More recently we discovered that connexin43 is regulated at the translational level by a microRNA. miR206 specifically blocks translation of connexin43 mRNA. This inhibition is essential for the differentiation of myoblasts into myotubes and muscle fibers.

Another connexin, connexin32, is found in liver, pancreas and in nervous tissue. To study its promoter we have linked various portions of it to the reporter gene for luciferase and introduced these constructs into transgenic mice. Luciferase expression studies in these mice revealed that connexin32 is transcribed from two different promoters in a tissue-specific manner. This results in two mRNA species that have the same coding sequence but different 5’-untranslated regions. When the two mRNAs are added to an in vitro reticulocyte translation system, only the liver mRNA is translated efficiently. This observation led to the discovery of an internal ribosome entry site (IRES) in the 5'-UTR of the nerve-specific mRNA.  Mutations in the human connexin32 gene have been associated with Charcot-Marie-Tooth (CMTX1) disease, a neuropathy that affects peripheral nerves leading to paralysis. We have identified mutations in the nerve-specific promoter region of the connexin32 gene in two families with CMTX1. One of these mutations is located in the 5’-UTR of the mRNA, the other in the promoter itself. The 5'-UTR mutation knocks out the IRES when tested in a bicistronic reporter system. This is one of the first demonstrations that the IRES of a cellular mRNA is essential for function. It is also one of the first IRES mutations that causes a human disease. 

In addition to the work on connexin32 gene expression, we are also studying the differential expression of another connexin, connexin43, found in heart muscle and in uterine smooth muscle. In heart muscle, connexin43 is expressed constitutively, whereas in uterus the same gene is under the control of the hormones estrogen and progesterone. We are trying to understand the mechanism of this differential regulation using a variety of experimental approaches, including promoter analysis with reporter genes in both transfected cells and in transgenic mice. In the process of this analysis, Elisa Oltra, a postdoc in my lab, has discovered a novel transcription factor called Ini. This factor has not been described previously in Genebank, but a search in the EST database revealed that this protein is highly conserved and found in other species including plants and even yeast. Elisa has recently isolated the yeast gene for ini and mutated it by knockout. The ini mutation is lethal in yeast, indicating that it has a vital function in the cell. Adam Schiavi, a former graduate student in my laboratory, has created a large number of deletion mutations in the connexin43 promoter and is analyzing their effects on the induction of the gene by estrogen. He found a non-canonical estrogen responsive element in the proximal promoter, which when mutated reduces estrogen inducibility by 50%. The other 50% estrogen inducibility is located in the 5'-UTR of the connexin43 mRNA. Actually, the 5'-UTR of connexin43 contains a very active IRES element that appears to be regulated by a translational repressor that is removed by estrogen.

Essentially all connexin genes contain their 5'-UTR in a separate exon. It seems to suggest that during evolution, a specific kind of 5'-UTR was selected for connexin genes. With the recent discovery of IRES elements in two connexin genes I propose the following hypothesis for this selection. Stem cells do not express gap junctions while most differentiated cells do. This suggests that gap junctions serve a vital function in the maintenance of the differentiated state. This would require that gap junctions be expressed at all times, even in conditions where cap-mediated translation is impaired, such as during mitosis, apoptosis or stress (e.g., heat shock). It is proposed that  IRES elements are present in the 5'-UTRs of most connexin mRNAs ensuring their translation under all conditions. This hypothesis is testable, and we are in the process of looking for IRES elements in other connexin genes. We are also interested in the structure of the IRES elements and their mode of regulation.

I do not do research anymore. I am only involved in teaching medical students.

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