Karyopherin alpha expression in porcine tissue
Abstract
The cell must modify expression of certain genes in order to respond appropriately to environmental and internal stimuli. In order for signaling molecules to exert their regulatory effects, they must first gain access to the nucleus, where transcription takes place. Transport occurs through the nuclear pore complexes embedded in the nuclear membrane. One of the best characterized nuclear transport systems is the classical nuclear transport complex, which consists primarily of two karyopherin proteins, alpha and beta, in addition to the cargo protein. While karyopherin beta manages nuclear entry via the nuclear pore, karyopherin alpha (KPNA) links the cargo and karyopherin beta. In mammals, there are seven known subtypes of KPNA. The subtypes are expressed at different levels in different tissues and vary in abundance throughout early development. While very similar in structure, evidence suggests that each subtype may bind a specific set of unique cargoes. KPNA plays essential roles in embryonic cleavage development, neural differentiation, and reproductive competence. To identify the unique functions of specific KPNA paralogs, I used two different methods of RNA interference to target specific paralogs within porcine fetal fibroblasts and germinal-vesicle stage oocytes. While neither microinjection of siRNA or induction of an miRNA-mimicking shRNA after lentiviral transfection affected the abundance of the targeted transcripts, further refinement of the lentiviral vector may provide a versatile platform for studying the effects of KPNA depletion. I also performed analysis of a data set of putative KPNA1- or KPNA7-interacting proteins identified within porcine ovarian tissue. Five out of twelve possible candidates identified on the basis of their size or the strength of their nuclear localization signal were repeatedly amplified from porcine cDNA; however two candidates were successfully cloned into the pENTR/SD/D-TOPO vector in an inverted orientation. In order to validate candidates, their ability to localize to the nucleus and interaction with KPNA1 or KPNA7 must be confirmed. Further characterization of the unique functions of each KPNA subtype and the transport patterns of their specific cargoes contributes to understanding the critical role of these transport proteins in precisely regulating gene expression during major differentiation events, including embryonic development and establishment of reproductive competence.
Degree
M.S.
Advisors
Cabot, Purdue University.
Subject Area
Molecular biology
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