Analysis of mono-ADP-ribosyltransferase 4 gene expression in human monocytes: splicing pattern and potential regulatory elements.
Mono-ADP-ribosyltransferase (ART) 4 belongs to a family of ectoenzymes that catalyze the transfer of ADP-ribose from NAD+ to a target protein. ART4 could be detected on HEL cells and erythrocytes by FACS analysis while it was absent from activated monocytes, despite the presence of ART4 mRNA in these cells. The predicted glycosylphosphatidylinositol (GPI) linkage of ART4 could be verified by showing that treatment of erythrocytes, HEL cells and ART4-transfected HEK-293-T cells with phosphatidylinositol-specific phospholipase C results in a decrease in ART4 expression. Furthermore, an ART4 construct carrying an Ala285Val mutation that is critical for the formation of a GPI anchor failed to be expressed in transfected C-33A cells. Analysis of the gene structure revealed that the first of the three exons was at least 236 bp longer than previously published and that splicing occurred in the coding region of the mRNA from HEL cells and monocytes. When carrying out 5' inverse RACE-PCR we confirmed the existence of 5 ATGs in the 5' untranslated region (5'UTR). By deletion and site-directed mutagenesis of the ATGs, we showed that the first two ATGs impair translation and that both the 3rd and 5th ATG can be used for translation initiation after expression in C-33A cells. On analysis of the 3'UTR, which contains 2 adenylate/uridylate-rich elements (AREs), we detected one variant in monocytes that would be devoid of a GPI-anchor signal and thus could represent a secreted form of ART4. Thus, alternative splicing and the use of regulatory elements in the 5'UTR and 3'UTR represent means to control ART4 expression.
Grahnert
Andreas
A
Institute of Biology II, Dept. of Immunobiology, University of Leipzig, Talstrasse 33, D-04103 Leipzig, Germany.
Friedrich
Maik
M
Engeland
Kurt
K
Hauschildt
Sunna
S
D020413:3' Untranslated Regions / Q000737:chemistry / Q000235:genetics; D020121:5' Untranslated Regions / Q000737:chemistry / Q000235:genetics; D036002:ADP Ribose Transferases / Q000032:analysis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017398:Alternative Splicing; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001483:Base Sequence; D002460:Cell Line; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D003001:Cloning, Molecular; D018387:Codon, Initiator; D018388:Codon, Terminator; D005091:Exons; D016650:Fluorescein-5-isothiocyanate; D019084:Fluorescent Antibody Technique, Indirect; D005456:Fluorescent Dyes; D015870:Gene Expression; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000201:enzymology* / Q000378:metabolism*; D008565:Membrane Proteins / Q000032:analysis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D014633:Valine / Q000378:metabolism
D020413:3' Untranslated Regions; D020121:5' Untranslated Regions; D018387:Codon, Initiator; D018388:Codon, Terminator; D005456:Fluorescent Dyes; D008565:Membrane Proteins; D012333:RNA, Messenger; D036002:ADP Ribose Transferases; C105234:ART4 protein, human; D014633:Valine; D016650:Fluorescein-5-isothiocyanate