In vitro and in vivo analysis of the processing and fate of the peptide products of the short proopiomelanocortin mRNA.
Many peripheral tissues express the proopiomelanocortin (POMC) gene as an 800-base mRNA that lacks the 5' end of the 1200-base pituitary transcript. The missing region encodes the peptide signal sequence, and thus, it is unlikely that any translation product would be secreted. We have found that a RNA transcript equivalent to this short message, generated by transcription in vitro from a T7 polymerase promoter, is translatable in a rabbit reticulocyte lysate, generating peptides of 27.5, 22.5, and 15.5 kD. None of these peptides appears to be processed or protected from proteinase-K digestion by a microsomal membrane fraction. In vivo studies were undertaken by transfecting into GH3 cells one of two expression vectors containing sequences that would produce either a full-length mRNA or a short (800-base) mRNA. The neomycin resistance gene was cotransfected with these plasmids, and 30 permanent cell lines were produced after selection in G418. Cell lines containing the full-length RNA secreted large quantities of ACTH and beta-endorphin immunoreactivity, whereas those expressing the short transcript secreted neither of these peptides. However extractable peptide was present in this latter type of cell line, thereby suggesting that the 800-base mRNA was translated, and that no peptide reached the secretory vesicle. These findings raise important questions about the role of peripheral POMC gene expression.
Clark
A J
AJ
Department of Endocrinology, St. Bartholomew's Hospital Medical College, London, Great Britain.
Lavender
P M
PM
Coates
P
P
Johnson
M R
MR
Rees
L H
LH
D000324:Adrenocorticotropic Hormone / Q000494:pharmacology; D000818:Animals; D002460:Cell Line; D004247:DNA / Q000737:chemistry; D004351:Drug Resistance / Q000235:genetics; D005786:Gene Expression Regulation / Q000187:drug effects; D008861:Microsomes / Q000187:drug effects / Q000378:metabolism; D009355:Neomycin / Q000494:pharmacology; D011333:Pro-Opiomelanocortin / Q000235:genetics*; D011401:Promoter Regions, Genetic; D012323:RNA Processing, Post-Transcriptional; D012333:RNA, Messenger / Q000378:metabolism*; D011817:Rabbits; D051381:Rats; D012156:Reticulocytes / Q000378:metabolism; D001615:beta-Endorphin / Q000494:pharmacology
D012333:RNA, Messenger; D001615:beta-Endorphin; D011333:Pro-Opiomelanocortin; D000324:Adrenocorticotropic Hormone; D004247:DNA; D009355:Neomycin