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Title: Identification of a functional splice variant of 14-3-3E1 in rainbow trout

item Rexroad, Caird

Submitted to: Marine Biotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/2/2009
Publication Date: 2/1/2010
Citation: Wanna, W., Rexroad III, C.E., Yao, J. 2010. Identification of a functional splice variant of 14-3-3E1 in rainbow trout. Marine Biotechnology. 12:70-80.

Interpretive Summary: The 14-3-3 family of proteins consists of small, acidic and highly conserved proteins that are present in all eukaryotic cells. To date, over 100 cellular proteins have been reported to interact with these proteins in a multitude of cellular pathways including metabolism, cell cycle, differentiation, cellular signaling, apoptosis, and neoplasic transformation. In fishes (Oncorhynchus mykiss, Danio rerio), the sequences of 10 and 11 14-3-3 proteins have been reported, respectively. We report the identification and characterization of a new protein of the rainbow trout 14-3-3E1 gene generated by genetic engineering. We have shown that this protein exhibits tissue and stage specific expression and appears to have a different cellular function compared to the wild type 14-3-3E1 protein (14-3-3E1 wt). Identifying and characterizing the full compliment of 14-3-3 proteins will enhance our ability to determine potential roles in biochemical processes that affect important aquaculture production traits.

Technical Abstract: The 14-3-3 proteins are a family of regulatory proteins involved in diverse cellular processes. The presence of 14-3-3 isoforms and the diversity of cellular processes regulated by 14-3-3 isoforms suggest functional specificity of the isoforms. In this study, we report the identification and characterization of a new isoform of the rainbow trout 14-3-3E1 gene generated by alternative splicing. The new isoform contains an insertion of 48 nucleotides (from intron 5) in the coding region of 14-3-3E1 which results in the introduction of a premature stop codon between exon 5 and exon 6. Thus the alternatively spliced form of 14-3-3E1 (14-3-3E1'C17) lacks 17 amino acid residues at the C terminus encoded by the last exon (exon 6). RT-PCR analysis revealed that the wild type 14-3-3E1 (14-3-3E1wt) is ubiquitously expressed, while 14-3-3E1'C17 shows tissue-specific as well as stage-specific expression during oogenesis and early embryogenesis. Analysis by yeast two hybrid system demonstrated that 14-3-3E1'17 interacts with a number of proteins including ATP synthase, ankyrin repeat domain 13b, cytochrome c subunit VIa, cytochrome c subunit VIb, 60s ribosomal protein L34, solute carrier family 17 member 6 (SLC17A6), troponin I and an unknown protein. Although all of these proteins except for SLC17A6 also interact with 14-3-3E1wt, 14-3-3E1'17 appears to have higher binding affinity with these proteins than 14-3-3E1wt. These findings suggest that alternative splicing affects the function and tissue-specific expression of 14-3-3E1.