Abstract
BackgroundNuclear transport factor 2 and small GTPase Ran participate in the nucleo-cytoplasm transport of macromolecules, but their function in the 20-hydroxyecdysone (20E) signal transduction pathway are not well known.ResultsA 703 bp encoding Ntf2 and a 1233 bp encoding Ran full-length cDNAs were cloned from Helicoverpa armigera, and named Ha-Ntf2 and Ha-Ran, respectively. Northern blot and immunoblotting revealed that Ha-Ntf2 had an obviously higher expression levels in the head-thorax and integument of the metamorphically committed larvae. In contrast, the expression of Ha-Ran did not show obvious variation at various developmental stages in four tissues by immunoblotting analysis, except in the midgut, which showed increased expression from 5th-36 h (molting) to 6th-48 h. Both expressions of Ha-Ntf2 and Ha-Ran could be upregulated by 20E in vitro. Immunohistochemistry revealed that Ha-Ntf2 and Ha-Ran were primarily localized in the nucleus of various tissues. Protein binding assay and co-immunoprecipitation indicated that Ha-Ntf2 and Ha-Ran can combine with each other in vitro and in vivo. Knock down of Ha-Ntf2 or Ha-Ran by RNAi resulted in the suppression of other 20E regulated genes including EcR-B1, USP1, E75B, BR-CZ2, HHR3 and Ha-eIF5c. In addition, the knockdown of Ha-Ntf2 resulted in Ha-Ran being prevented in the cytoplasm. The nuclear location of the ecdysone receptor b1 (EcR-B1) was also blocked after the knockdown of Ha-Ntf2 and Ha-Ran.ConclusionThese evidences suggested that Ha-Ntf2 and Ha-Ran participated in the 20E signal transduction pathway by regulating the location of EcR-B1.
Highlights
Nuclear transport factor 2 and small GTPase Ran participate in the nucleo-cytoplasm transport of macromolecules, but their function in the 20-hydroxyecdysone (20E) signal transduction pathway are not well known
Using RNAi technique in the H. armigera epidermis (HaEpi) cell line, we found that after interfering Ha-Nuclear transport factor 2 (Ntf2) and Ha-Ran, the expression of other genes, such as ecdysone receptor b1 (EcR-B1), ultraspiracle protein 1 (USP1), ecdysone induced protein E75b (E75b), broad-complex Z2 (BRCZ2), and hormone receptor 3 (HHR3) decreased
Results cDNA cloning and sequence analysis of Ha-Ntf2 and Ha-Ran The full-length of Ha-Ntf2 and Ha-Ran cDNA were cloned using the strategies described in Materials and Methods section
Summary
Nuclear transport factor 2 and small GTPase Ran participate in the nucleo-cytoplasm transport of macromolecules, but their function in the 20-hydroxyecdysone (20E) signal transduction pathway are not well known. Macromolecules, such as cargo proteins, require some soluble nucleic or cytosolic factors for active transport [1,2] This nucleocytoplasmic transport locate proteins in the cytoplasm or nucleus through export ribosomes, mRNAs and tRNAs to the cytoplasm, or import nuclear proteins from the cytoplasm[3], and functions as a key step in signal transduction pathways and in the regulation of cell cycle progression [4]. RanGTP is transported from the nucleus into the cytoplasm by importinb and is converted to RanGDP by RanGAP, a cytoplasmic GTPase-activating protein [13] This cause Ran to act as a major regulator of nucleocytoplasmic transport and to regulate the interaction between proteins
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