The bombesin-like peptides were originally characterized in frog skin, then later found to have a wide distribution and range of actions in mammals. The bombesin-like peptides have classically been divided into three subfamilies, the bombesin subfamily, of which gastrin-releasing peptide (GRP) is the mammalian form; the ranatensin subfamily, of which neuromedin-B (NMB) is the mammalian form; and the phyllolitorin subfamily, which to date has only been characterized in amphibians. As a first step in characterizing mammalian phyllolitorin-like peptides, we have cloned complementary DNAs (cDNAs) encoding Leu8 and Phe8 phyllolitorin from Phyllomedusa sauvagei. Sequence analysis revealed that the amphibian phyllolitorin messenger RNA (mRNA) encodes a precursor of 90 amino acids containing a signal peptide sequence, an amino-terminal extension peptide, the phyllolitorin peptide of nine amino acids, and a carboxy-terminal extension peptide. Northern blot, reverse transcriptase-polymerase chain reaction (PCR), and in situ hybridization analysis showed that the mRNA was present at highest levels in skin, at lower levels in brain, and at lowest levels in gut. Phylogenetic analysis of bombesin-like peptide prohormone sequences showed that the phyllolitorin prohormones are much more closely related to the bombesin and ranatensin prohormones than to the GRP and NMB prohormones. This analysis suggests that the bombesin-like peptides should be reclassified into the GRP subfamily, the NMB subfamily, and the skin peptide subfamily. Surprisingly, the cDNAs encoding Phe8 and Leu8 phyllolitorins were identical except for a single T to C difference in the codon coding for the Phe or Leu residue of phyllolitorin. PCR analysis of the skin, stomach, and brain phyllolitorin cDNAs showed that skin contained equal proportions of Phe8 and Leu8 phyllolitorin, that Phe8 phyllolitorin predominated in stomach, and that only Phe8 phyllolitorin could be identified in brain. PCR analysis of genomic subclones showed that all 52 subclones sequenced used the codon TTC encoding only Phea phyllolitorin. Genomic Southern blot analysis was consistent with a single phyllolitorin gene. This suggests that Leu8 and Phe8 phyllolitorins are encoded by the same gene and that a regulated posttran-scriptional RNA-editing mechanism converts a U to a C to generate the two phyllolitorin mRNAs. This is the first report of potential U to C RNA editing in eucaryotes and suggests a new mechanism for generating neuropeptide diversity.
ASJC Scopus subject areas
- Molecular Biology