Mediator subunits: gene expression pattern, a novel transcript identification and nuclear localization in human endothelial progenitor cells.
Mediator of RNA polymerase II transcription subunits (MEDs) serve to promote the assembly, activation, and regeneration of transcription complexes on core promoters during the initiation and re-initiation phases of transcription. There are no studies on the Mediator complex during development of endothelial progenitors (EPCs). Here, we have analysed all known MEDs during the differentiation of EPCs, by expression profile studies at RNA level and, for a limited subset of MED subunits, also at protein level. Since beneficial effects of L-arginine on EPCs have been described, we have also examined its effect on the expression of Mediator subunit coding genes. Through RT-PCR we have found increased expression for MED12 and decreased levels for MED30 after l-arginine treatment; Western blot analysis do not agree entirely with the RNA data in the identification of a putative protein product. Furthermore, we have analysed the three-dimensional nuclear positions of MED12 and MED30 genes in the presence of l-arginine treatment. Our major finding is the identification of a novel transcript of MED30, termed MED30 short (MED30s) generating by alternative splicing. Our results showed that the mRNA of this novel isoform is present only in circulating cells, but it is not expressed in cultured adherent cells. These findings are broadly relevant and will contribute to our understanding of the role of Mediator in eukaryotic gene expression. Despite the need to confirm the in vivo presence of the protein of this novel isoform, the presence of this novel RNA raises the possibility of regulating pathophysiological mechanism in progenitors.
Rienzo
Monica
M
Department of General Pathology, 1st School of Medicine, II University of Naples, 80138 Naples, Italy.
Nagel
Jens
J
Casamassimi
Amelia
A
Giovane
Alfonso
A
Dietzel
Steffen
S
Napoli
Claudio
C
D017398:Alternative Splicing; D001120:Arginine / Q000378:metabolism; D003001:Cloning, Molecular; D042783:Endothelial Cells / Q000166:cytology*; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D007963:Leukocytes, Mononuclear / Q000166:cytology; D056892:Mediator Complex / Q000378:metabolism; D008954:Models, Biological; D017434:Protein Structure, Tertiary; D012313:RNA / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013234:Stem Cells / Q000166:cytology*
C460107:MED12 protein, human; D056892:Mediator Complex; D012313:RNA; D001120:Arginine