Corticotropin-releasing factor upregulates expression of two truncated pro-opiomelanocortin transcripts in murine lymphocytes.
Lymphocytes harbor a pro-opiomelanocortin (POMC) mRNA. In this report, a novel procedure was used to study the exonic arrangement of this transcript in lymphocytes. Poly(A)+ mRNA, purified from both corticotropin-releasing factor (CRF)-treated and nontreated lymphocytes, was selectively reverse-transcribed using an antisense oligonucleotide primer complementary to the 3' junction of the translated/nontranslated region of exon 3 of POMC. Alkaline agarose gel analysis of first-strand cDNA synthesis showed an upregulation of POMC transcripts in CRF-treated cells. This first-strand cDNA was amplified in a polymerase chain reaction (PCR) using the complementary antisense primer and selective sense primers homologous to the 5' ends of exons 1, 2, and 3, as well as a region immediately 5' to the ACTH/beta-lipotropin coding region of exon 3 of pituitary POMC. Primers directed at exons 1 and 2 did not amplify a POMC product in nontreated control or CRF-treated cells. However, with both treated and nontreated cells, the internal exon 3 primer amplified the expected size exon 3 DNA fragment (approximately 549 bp). Interestingly, a primer directed at the 5' end of exon 3 apparently did not amplify a POMC product in nontreated cells but did amplify a full-size POMC exon 3 from CRF-treated cells (approximately 615 bp). However, upon reamplification of the original PCR products from nontreated cells, full-length exon 3 product was also observed. Southern gel analysis using a pituitary POMC cDNA probe showed that all of the above PCR products were POMC-related. The results of this study show that lymphocytes basally transcribe at least two POMC transcripts that are upregulated by CRF. These two transcripts lack exons 1 and 2 but contain either part or all of exon 3. The smaller exon 3 transcript was the most abundant transcript under all conditions examined.
Galin
F S
FS
Department of Physiology and Biophysics, University of Alabama, Birmingham 35294.
LeBoeuf
R D
RD
Blalock
J E
JE
D000324:Adrenocorticotropic Hormone / Q000096:biosynthesis; D000818:Animals; D003346:Corticotropin-Releasing Hormone / Q000494:pharmacology*; D004247:DNA / Q000096:biosynthesis; D005091:Exons; D005260:Female; D008214:Lymphocytes / Q000378:metabolism*; D051379:Mice; D008809:Mice, Inbred C3H; D011333:Pro-Opiomelanocortin / Q000235:genetics*; D012333:RNA, Messenger / Q000032:analysis*; D014158:Transcription, Genetic; D015854:Up-Regulation
D012333:RNA, Messenger; D011333:Pro-Opiomelanocortin; D000324:Adrenocorticotropic Hormone; D004247:DNA; D003346:Corticotropin-Releasing Hormone