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Title	Expression analysis of Prx1 and Prx2 homeodomain proteins leads to the identification of an internal ribosomal entry site in Prx1 transcripts
Author	Chesterman, Elizabeth S.
Date	2002
Affiliation/College	College of Graduate Studies
Department	Molecular and Cellular Biology and Pathobiology
Extent	xi, 200 leaves : ill.; 29 cm
Source	QH447.8 H65 C525e 2002
Subject.MeSH	Academic Dissertations Academic Dissertations--South Carolina Heart--embryology Homeodomain Proteins Genes
Collection	MUSC Theses and Dissertations
Contributing Institution	Waring Historical Library (MUSC)
Rights	Copyright is held by the author.
Language	English
Type	Text
Format	application/pdf
Media Type	Manuscripts
Resource Identifier	mtd2002_chesterman_es
Abstract	Gene targeting experiments have previously defined that the Prxl and Prx2 homeobox genes are essential for normal bone development in the craniofacial and limb regions, as well as development of the aortic arch region. Prxl and Prx2 transcripts have been previously detected in the embryonic and adult heart by Northern analysis and in situ hybridization. Therefore, the most surprising result of these gene-targeting experiments was that cardiac morphology was normal. In order to define if Prxl or Prx2 protein is expressed in the heart, novel Prx 1 and Prx2 antibodies were developed. During murine development, Prxl and Prx2 immunoreactivity was generally consistent with previously published data on l)NA localization within craniofacial and limb mesenchyme. However, despite the expression of Prxl and Prx2 transcripts in the endocardial cushions and valves, no Prxl or Prx2 protein was detectable in the murine embryonic and adult heart. This suggests that Prx land Prx2 expression in the heart is posttranscriptionally regulated. Parallel studies with embryonic chicken tissue demonstrate that no Prxl protein was detectable, suggesting conservation of a posttranscriptional regulatory mechanism for Prx 1 expression in the hearts of vertebrates. While the vast majority of eukaryotic ribosome docking for scanning occurs at the 5' cap, there is a second mode that is not cap-dependent. This alternative mechanism is called the Internal Ribosome Entry Site (IRES). The IRES offers a unique mode of entry and, therefore, the potential for a more highly regulated mechanism of initiation of scanning than that of the 5' cap. Moreover, the presence of an IRES allows the transcript to escape normal regulation at the 5' cap. Other cellular transcripts that contain a functional IRES are regulated in development, in differentiation, cell cycle-specifically, and tissue-specifically via this mode of ribosome docking. Using a bicistronic reporter assay in transient transfections, we have shown that the 5' untranslated region (UTR) of the murine Prxl transcript exhibits IRES activity in several cell lines. Further analysis has demonstrated conserVation of this activity in the human Prxl 5' UTR, and has defined the T_ -40< sequences essential for IRES function and presented a testable model of its mode of action. We hypothesize that utilization of the IRES element in the Prxl transcript is tissue specifically regulated and that its presence in the Prx 1 transcript offers a unique point of regulation that is likely critical for cardiac development.

Expression analysis of Prx1 and Prx2 homeodomain proteins leads to the identification of an internal ribosomal entry site in Prx1 transcripts

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