Cloning of the first cDNA encoding a putative CCRFamide precursor: identification of the brain, eyestalk ganglia, and cardiac ganglion as sites of CCRFamide expression in the American lobster, Homarus americanus

Authors: Hull, Joe; Noble, Melissa; DICKINSON, PATSY - Bowdoin College; CHRISTIE, ANDREW - University Of Hawaii

Submitted to: Invertebrate Neuroscience
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/18/2020
Publication Date: 11/26/2020
Citation: Hull, J.J., Stefanek, M.A., Dickinson, P.S., Christie, A.E. 2020. Cloning of the first cDNA encoding a putative CCRFamide precursor: identification of the brain, eyestalk ganglia, and cardiac ganglion as sites of CCRFamide expression in the American lobster, Homarus americanus. Invertebrate Neuroscience. 20. https://doi.org/10.1007/s10158-020-00257-z.
DOI: https://doi.org/10.1007/s10158-020-00257-z

Interpretive Summary: Considerable progress has been made in the generation of genomic and transcriptomic resources for arthropod species. While many of these datasets were developed to help address specific research questions, they have proven to be powerful resources for unrelated gene discoveries including the identification of previously uncharacterized or unknown peptides. One example of this is the CCRFamide family of peptides, which are characterized by two disulfide bridges and an arginine-phenylalanine carboxyl terminus. Although a relatively well-conserved sequence has been found in diverse arthropod transcriptome/genome assemblies, no transcript has been directly cloned and sequenced. Using standard PCR methods, a CCRFamide transcript was amplified and validated from American lobster neural tissues. Additional transcript profiling established expression in the supraoesophageal ganglion (brain), eyestalk ganglia, and cardiac ganglion, suggesting the mature form of the peptide functions as a locally released neuropeptide. These findings provide the first non-in silico support for the existence of this peptide family in invertebrates and as such opens new avenues of research into the potential biological functions this peptide family may mediate in arthropods.

Technical Abstract: Over the past decade, many new peptide families have been identified via in silico analyses of genomic and transcriptomic datasets. While various molecular and biochemical methods have confirmed the existence of some of these new groups, others remain in silico discoveries of computationally assembled sequences only. An example of the latter are the CCRFamides, named for the predicted presence of two pairs of disulfide bonded cysteine residues and an amidated arginine-phenylalanine carboxyl-terminus in family members, which have been identified from annelid, molluscan, and arthropod genomes/transcriptomes, but for which no precursor protein-encoding cDNAs have been cloned. Using routine transcriptome mining methods, we identified four Homarus americanus (American lobster) CCRFamide transcripts that share high sequence identity across the predicted open reading frames but more limited conservation in their 5' terminal ends, suggesting the Homarus gene undergoes alternative splicing. RT-PCR profiling using primers designed to amplify an internal fragment common to all of the transcripts revealed expression in the supraoesophageal ganglion (brain), eyestalk ganglia, and cardiac ganglion. Variant specific profiling revealed a similar profile for variant 1, eyestalk ganglia specific expression of variant 2, and an absence of variant 3 expression in the cDNAs examined. The broad distribution of CCRFamide transcript expression in the H. americanus nervous system suggests a potential role as a locally released and/or circulating neuropeptide. This is the first report of the cloning of a CCRFamide-encoding cDNA from any species, and as such, provides the first non-in silico support for the existence of this invertebrate peptide family.