Analysis of the 3' untranslated regions of alpha-tubulin and S-crystallin mRNA and the identification of CPEB in dark- and light-adapted octopus retinas.
OBJECTIVE
We previously reported the differential expression and translation of mRNA and protein in dark- and light-adapted octopus retinas, which may result from cytoplasmic polyadenylation element (CPE)-dependent mRNA masking and unmasking. Here we investigate the presence of CPEs in alpha-tubulin and S-crystallin mRNA and report the identification of cytoplasmic polyadenylation element binding protein (CPEB) in light- and dark-adapted octopus retinas.


METHODS
3'-RACE and sequencing were used to isolate and analyze the 3'-UTRs of alpha-tubulin and S-crystallin mRNA. Total retinal protein isolated from light- and dark-adapted octopus retinas was subjected to western blot analysis followed by CPEB antibody detection, PEP-171 inhibition of CPEB, and dephosphorylation of CPEB.


RESULTS
The following CPE-like sequence was detected in the 3'-UTR of isolated long S-crystallin mRNA variants: UUUAACA. No CPE or CPE-like sequences were detected in the 3'-UTRs of alpha-tubulin mRNA or of the short S-crystallin mRNA variants. Western blot analysis detected CPEB as two putative bands migrating between 60-80 kDa, while a third band migrated below 30 kDa in dark- and light-adapted retinas.


CONCLUSIONS
The detection of CPEB and the identification of the putative CPE-like sequences in the S-crystallin 3'-UTR suggest that CPEB may be involved in the activation of masked S-crystallin mRNA, but not in the regulation of alpha-tubulin mRNA, resulting in increased S-crystallin protein synthesis in dark-adapted octopus retinas.
Kelly
Shannan
S
Department of Biology, California State University, Dominguez Hills, Carson, CA 90747, USA.
Yamamoto
Hideki
H
Robles
Laura J
LJ
D020413:3' Untranslated Regions / Q000235:genetics*; D000222:Adaptation, Physiological / Q000528:radiation effects; D000818:Animals; D015153:Blotting, Western; D003459:Crystallins / Q000235:genetics*; D003624:Darkness; D004587:Electrophoresis, Agar Gel; D008027:Light; D008957:Models, Genetic; D008970:Molecular Weight; D049831:Octopodiformes / Q000235:genetics / Q000378:metabolism / Q000528:radiation effects*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012160:Retina / Q000378:metabolism / Q000528:radiation effects*; D014404:Tubulin / Q000235:genetics*; D039221:mRNA Cleavage and Polyadenylation Factors / Q000378:metabolism*
D020413:3' Untranslated Regions; D003459:Crystallins; D012333:RNA, Messenger; D014404:Tubulin; D039221:mRNA Cleavage and Polyadenylation Factors