Induction of multiple pituitary adenylate cyclase activating polypeptide (PACAP) transcripts through alternative cleavage and polyadenylation of proPACAP precursor mRNA.

Abstract

Many regulated events guide neuropeptide biosynthesis, processing, and secretion. For PACAP peptides, these events have not been well examined. In our studies of PACAP expression in sympathetic neurons, we discovered that neuronal depolarization not only increased the levels of the 2.2 kb form of proPACAP mRNA identified in neuronal tissues, but also induced a novel 0.9 kb PACAP transcript, which appeared similar in size to a form present in testes. Using reverse-transcription PCR and 3' RACE studies, we demonstrated that the 0.9 kb PACAP mRNA in depolarized SCG neurons was not identical to the testicular PACAP mRNA, but represented shortened, more stable, forms of the 2.2 kb transcript resulting from alternative upstream polyadenylation site usage. These results demonstrate that post-transcriptional mechanisms play important roles in determining cellular PACAP levels and provide several important insights. For example, alternative upstream polyadenylation can elicit a major influence on the amount of bioactive peptide that can by synthesized, since short 3' UTR transcripts are usually more stable due to elimination of destabilizing elements present in the longer messages. In cells such as testicular germ cells, which have restricted transcriptional periods, stable mRNAs allow longer translational events and extended periods of peptide production. The neuronal PACAP system adopts a similar post-transcriptional strategy following neuronal depolarization, and although the roles of PACAP remain unclear, this suggests important roles for PACAP peptides during increased neuronal activity. Additionally, unlike alternative polyadenylation described for many genes, alternative site usage in the proPACAP transcript does not result from alternative splicing. The mechanism of alternative site usage may be related to changes in the expression and binding of polyadenylation factors to the short and long 3' UTR proPACAP sites leading to production of more stable transcripts and increased PACAP precursor biosynthesis. The implications of increased PACAP production following altered neurophysiological states and the mechanisms underlying alternative polyadenylation site choice are important considerations for future inquiries.