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Abstract
Background Artemia eggs tend to develop ovoviviparously to yield nauplius larvae in good rearing conditions; while under adverse situations, they tend to develop oviparously and encysted diapause embryos are formed instead. However, the intrinsic mechanisms regulating this process are not well understood.Principal FindingThis study has characterized the function of cyclin K, a regulatory subunit of the positive transcription elongation factor b (P-TEFb) in the two different developmental pathways of Artemia. In the diapause-destined embryo, Western blots showed that the cyclin K protein was down-regulated as the embryo entered dormancy and reverted to relatively high levels of expression once development resumed, consistent with the fluctuations in phosphorylation of position 2 serines (Ser2) in the C-terminal domain (CTD) of the largest subunit (Rpb1) of RNA polymerase II (RNAP II). Interestingly, the cyclin K transcript levels remained constant during this process. In vitro translation data indicated that the template activity of cyclin K mRNA stored in the postdiapause cyst was repressed. In addition, in vivo knockdown of cyclin K in developing embryos by RNA interference eliminated phosphorylation of the CTD Ser2 of RNAP II and induced apoptosis by inhibiting the extracellular signal-regulated kinase (ERK) survival signaling pathway.Conclusions/SignificanceTaken together, these findings reveal a role for cyclin K in regulating RNAP II activity during diapause embryo development, which involves the post-transcriptional regulation of cyclin K. In addition, a further role was identified for cyclin K in regulating the control of cell survival during embryogenesis through ERK signaling pathways.
Highlights
RNA polymerase II (RNAP II) is a key enzyme involved in the synthesis of mRNA, and interruption of its function triggers apoptosis in human cells and induces abnormality in developing embryos [1,2]
Semiquantitative RT-PCR showed that the cyclin K transcript was detectable throughout the life-cycle of Artemia, and was most abundant during embryonic development (Fig. 2A)
The phospho-RNAP II could not be detected in the immunoprecipitates by cyclin K antibody (Fig. 3E). All of these results suggested that cyclin K is a possible key factor involved in regulating the activity of RNAP II during diapausedestined embryo development, even if cyclin K could not bound to C-terminal domain (CTD) Ser2 phosphorylated RNAP II
Summary
RNA polymerase II (RNAP II) is a key enzyme involved in the synthesis of mRNA, and interruption of its function triggers apoptosis in human cells and induces abnormality in developing embryos [1,2]. An in vitro kinase assay proved that the CDK9-cyclin K complex could functionally substitute the CDK9-cyclin T complex to phosphorylate CTD on Rpb of RNAP II without regard to the lower activity [15,16]; other research suggested that cyclin K is not involved in DNA transcription in vivo [17,18]. In the diapause-destined embryo, Western blots showed that the cyclin K protein was down-regulated as the embryo entered dormancy and reverted to relatively high levels of expression once development resumed, consistent with the fluctuations in phosphorylation of position 2 serines (Ser2) in the C-terminal domain (CTD) of the largest subunit (Rpb1) of RNA polymerase II (RNAP II). In vivo knockdown of cyclin K in developing embryos by RNA interference eliminated phosphorylation of the CTD Ser of RNAP II and induced apoptosis by inhibiting the extracellular signal-regulated kinase (ERK) survival signaling pathway
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