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United States Department of Agriculture

Agricultural Research Service

Research Project: UTILIZATION OF GENOMICS FOR IMPROVING PRODUCTION TRAITS IN COOL AND COLD WATER AQUACULTURE

Location: Cool and Cold Water Aquaculture Research

Title: Importin alpha8 (KPNA7) interacts with STL3, an oocyte-specific protein in rainbow trout (Oncorhynchus mykiss)

Authors
item Wag, Lei -
item Ma, Hao -
item Yao, Jianbo -
item Rexroad, Caird

Submitted to: Society for the Study of Reproduction Annual Meeting
Publication Type: Abstract Only
Publication Acceptance Date: March 21, 2011
Publication Date: August 4, 2012
Citation: Wag, L., Ma, H., Yao, J., Rexroad III, C.E. 2012. Importin alpha8 (KPNA7) interacts with STL3, an oocyte-specific protein in rainbow trout (Oncorhynchus mykiss). Society for the Study of Reproduction Annual Meeting. 152.

Technical Abstract: Nuclear proteins are required for initiation of transcription in early embryos before embryonic genome activation. The regulation of transportation of nuclear proteins is mediated by transport factors. Importins (also known as karyopherins) are the major transport factors, which facilitate the transportation of proteins containing nuclear localization signals (NLSs) from the cytoplasm into the nucleus. To date, seven Importin alpha molecules (KPNA1 to 7) have been characterized in mammals, of which Importin alpha8 (KPNA7) is specifically expressed in oocytes and early embryos. Recent studies have shown that KPNA7 is an important maternal factor required for early embryonic development and KPNA7 knockout mice display abnormal gene expression and epigenetic modifications. We recently cloned and characterized rainbow trout KPNA7. The unique expression pattern of KPNA7 led us to hypothesize that the cargo proteins for KPNA7 are important at specific stages of oocyte maturation and embryonic development. The objective of this study was to further explore the functional network of KPNA7. Yeast two-hybrid system was used to search for KPNA7 interacting proteins. Using KPNA7 as bait, we screened a rainbow trout oocyte cDNA library and identified a strong interaction between KPNA7 and STL3 (rhamnose-binding lectin 3). This interaction was verified by successfully obtaining yeast clones with reintroduced STL3-AD and KPNA7-BD plasmids. Positive colonies were further tested by filter-lift assay and liquid culture assay for b-galactosidase activity. STL3 is an oocyte-specific protein, which has a variety of biological functions, such as prevention of polyspermy, regulation of carbohydrate metabolism and cross-linking of carbohydrate-rich proteins of the fertilization envelope. Real time PCR analysis revealed that expression of STL3 mRNA tends to increase gradually during early embryonic development. Using NLSmapper, a program for prediction of NLS specific to the importin alpha pathway, putative NLSs were detected within 60 amino acids at the C-terminus. Current work is focused on mapping the NLS on STL3 protein. Our data suggest that KPNA7 may function as a unique nuclear transport receptor for oocyte-specific proteins important for oocyte and embryonic development.

Last Modified: 10/24/2014
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