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Endocrinology Vol. 138, No. 1 169-181
Copyright © 1997 by The Endocrine Society

ARTICLES

The Human Gene for the Regulatory Subunit RI{alpha} of Cyclic Adenosine 3',5'-Monophosphate-Dependent Protein Kinase: Two Distinct Promoters Provide Differential Regulation of Alternately Spliced Messenger Ribonucleic Acids1

Rigmor Solberg, Mårten Sandberg, Vasanti Natarajan, Peter A. Torjesen, Vidar Hansson, Tore Jahnsen and Kjetil Taskén

Institute of Medical Biochemistry, University of Oslo (R.S., M.S., V.N., V.H., T.J., K.T.); the Department of Anesthesiology, Ullevål Hospital (M.S.); and the Hormone Laboratory, Aker Hospital (P.A.T.), Oslo, Norway

Address all correspondence and requests for reprints to: Rigmor Solberg, Ph.D., Institute for Surgical Research, Rikshospitalet-The National Hospital, N-0027 Oslo, Norway. E-mail: rigmor.solberg{at}basalmed.uio.no

The present study reports the exon-intron organization of the human RI{alpha} gene of cAMP-dependent protein kinase and approximately 2 kilobases (kb) of the 5'-flanking region obtained by isolation and sequencing of several phage clones from human genomic libraries. The RI{alpha} gene is composed of nine coding exons of varying lengths, separated by introns, giving the gene a total length of at least 21 kb. Our recent cloning of a processed RI{alpha} pseudogene with a 5'-noncoding region different from the previously reported RI{alpha} complementary DNA indicated that the RI{alpha} gene may have multiple leader exons giving rise to alternately spliced messenger RNAs (mRNAs). Reverse transcription of human testis RNA followed by PCR identified two different RI{alpha} mRNA species (RI{alpha}1a and RI{alpha}1b) containing distinct 5'-sequences due to alternately splicing the gene. The previously known RI{alpha}1b mRNA revealed low constitutive expression in a human B lymphoid cell line (Reh) and was stimulated only 4- to 6-fold by treatment with cAMP. In contrast, very low levels of the novel RI{alpha}1a mRNA were present in untreated Reh cells, but were stimulated 40- to 50-fold by cAMP. The 5'-flanking sequence of the RI{alpha} gene was G/C rich and did not contain any TATA box. Several putative transcription initiation sites were identified in front of each leader exon (exons 1a and 1b) by the 5'-rapid amplification of complementary DNA ends technique. To determine whether the sequences 5' of both leader exons had promoter activities, the 5'-flanking sequences of exons 1a and 1b were inserted in front of a chloramphenicol acetyltransferase reporter gene, and their ability to direct transcription were examined. Transfection of these constructs into rat GH4C1 cells demonstrated that both constructs had promoter activities, as evidenced by high levels of chloramphenicol acetyltransferase activity.




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