DDX3는 RNA 운송과 번역에 관여하는 것으로 알려진 DEAD box RNA helicase 집단의 잘 보존된 도메인을 갖는 유전자 중 하나이다. 효모를 이용한 혼성화 분리법을 통해 DDX3의 카복실 말단 (501-662aa) 과 결합하는 56가지의 단백질을 분리해냈다. 그 단백질 중 DEAD box RNA helicase 유전자 p68 (DDX5)를 특성...
DDX3는 RNA 운송과 번역에 관여하는 것으로 알려진 DEAD box RNA helicase 집단의 잘 보존된 도메인을 갖는 유전자 중 하나이다. 효모를 이용한 혼성화 분리법을 통해 DDX3의 카복실 말단 (501-662aa) 과 결합하는 56가지의 단백질을 분리해냈다. 그 단백질 중 DEAD box RNA helicase 유전자 p68 (DDX5)를 특성화 시켰다. p68 역시 잘 보존된 도메인을 갖는 DEAD box RNA helicase 집단으로 RNA 성숙과 전사조절에 관여하는 것으로 알려져 있다. 단백질 수준에서의 DDX3와 p68의 유사성은 대략 60%정도이다. DDX3와 p68의 결합을 분석하기 위해 우리는 두 유전자의 다양한 구성을 이용하여 효모 혼성화 분리법과 면역조직화학 기법과 면역침강 기법을 이용하였다. 이 연구에서 우리는 DDX3의 카복실 말단이 p68의 카복실 말단과 결합하는 것을 보여줬고, 이 두 단백질의 결합을 더 확고히 하기위해 HeLa 세포에서 세포질 부분과 핵 부분을 따로 분리하여 DDX3 항체로 면역침강 실험을 하였고, 이에 DDX3가 세포질 부분에서만 p68과 면역침강됨을 확인하였다. 또한, 세포질에서는 DDX3가 비인산화된 형태로 존재하며, 이때 p68과 결합함을 확인하였다.
DDX3는 RNA 운송과 번역에 관여하는 것으로 알려진 DEAD box RNA helicase 집단의 잘 보존된 도메인을 갖는 유전자 중 하나이다. 효모를 이용한 혼성화 분리법을 통해 DDX3의 카복실 말단 (501-662aa) 과 결합하는 56가지의 단백질을 분리해냈다. 그 단백질 중 DEAD box RNA helicase 유전자 p68 (DDX5)를 특성화 시켰다. p68 역시 잘 보존된 도메인을 갖는 DEAD box RNA helicase 집단으로 RNA 성숙과 전사조절에 관여하는 것으로 알려져 있다. 단백질 수준에서의 DDX3와 p68의 유사성은 대략 60%정도이다. DDX3와 p68의 결합을 분석하기 위해 우리는 두 유전자의 다양한 구성을 이용하여 효모 혼성화 분리법과 면역조직화학 기법과 면역침강 기법을 이용하였다. 이 연구에서 우리는 DDX3의 카복실 말단이 p68의 카복실 말단과 결합하는 것을 보여줬고, 이 두 단백질의 결합을 더 확고히 하기위해 HeLa 세포에서 세포질 부분과 핵 부분을 따로 분리하여 DDX3 항체로 면역침강 실험을 하였고, 이에 DDX3가 세포질 부분에서만 p68과 면역침강됨을 확인하였다. 또한, 세포질에서는 DDX3가 비인산화된 형태로 존재하며, 이때 p68과 결합함을 확인하였다.
DDX3 is a highly conserved member of the DEAD-box RNA helicase family and known to be involved in translation and RNA transport. From yeast two-hybrid screening with DDX3 C-terminal domain (501-656 aa) as a bait, we found 56 candidate proteins. Among them, DEAD-box RNA helicase gene DDX5 was charact...
DDX3 is a highly conserved member of the DEAD-box RNA helicase family and known to be involved in translation and RNA transport. From yeast two-hybrid screening with DDX3 C-terminal domain (501-656 aa) as a bait, we found 56 candidate proteins. Among them, DEAD-box RNA helicase gene DDX5 was characterized. DDX5 is also a highly conserved member of DEAD-box RNA helicase family and involved in the RNA processing and transcriptional regulation. The homology of both genes, DDX3 and DDX5, was approximately 60 % at the protein level. To analyze the interaction between DDX3 and DDX5, I have performed yeast two-hybrid assay with deleted constructs of both genes and immunohistochemistry (IH) and immunoprecipitation (IP) analysis with polyclonal antibodies. From the study, DDX3 interacts with DDX5: the C-terminal domain of DDX3 is necessary to interact with DDX5 and the C-terminal region of DDX5 is also involved in the interaction with DDX3. To analyze the localization of DDX3 and DDX5, IH analysis was performed with polyclonal DDX3 and DDX5 antibodies. DDX3 mainly localized in the cytoplasm; DDX5 mainly localized in the nucleus and minorly in the cytoplsam. In the cytoplasm, DDX3 and DDX5 were colocalized. To confirm the interaction between DDX3 and DDX5 in the cytoplasm, IP was performed with nuclear fraction and cytoplasmic fraction of HeLa cells. DDX3 was immunoprecipitated with DDX5 in the cytoplsamic fraction, not nuclear fraction eventhough DDX3 and DDX5 were detected in both lysates. To chraracterize this phenomenon, phosphorylation of DDX3 was analyzed with pTyr, pSer, and pThr-specific antibodies. DDX3 in the nucleus was highly phosphorylated than cytoplasmic DDX3. Dephosphorylation of DDX3 with PP2A and PTP1B reduced the interaction with DDX5 in the cytoplasm. From the study, DEAD-box RNA helicase gene DDX3 interacted with DDX5 in the cytoplasm and phosphorylation of DDX3 had an effect on the interaction with DDX5.
DDX3 is a highly conserved member of the DEAD-box RNA helicase family and known to be involved in translation and RNA transport. From yeast two-hybrid screening with DDX3 C-terminal domain (501-656 aa) as a bait, we found 56 candidate proteins. Among them, DEAD-box RNA helicase gene DDX5 was characterized. DDX5 is also a highly conserved member of DEAD-box RNA helicase family and involved in the RNA processing and transcriptional regulation. The homology of both genes, DDX3 and DDX5, was approximately 60 % at the protein level. To analyze the interaction between DDX3 and DDX5, I have performed yeast two-hybrid assay with deleted constructs of both genes and immunohistochemistry (IH) and immunoprecipitation (IP) analysis with polyclonal antibodies. From the study, DDX3 interacts with DDX5: the C-terminal domain of DDX3 is necessary to interact with DDX5 and the C-terminal region of DDX5 is also involved in the interaction with DDX3. To analyze the localization of DDX3 and DDX5, IH analysis was performed with polyclonal DDX3 and DDX5 antibodies. DDX3 mainly localized in the cytoplasm; DDX5 mainly localized in the nucleus and minorly in the cytoplsam. In the cytoplasm, DDX3 and DDX5 were colocalized. To confirm the interaction between DDX3 and DDX5 in the cytoplasm, IP was performed with nuclear fraction and cytoplasmic fraction of HeLa cells. DDX3 was immunoprecipitated with DDX5 in the cytoplsamic fraction, not nuclear fraction eventhough DDX3 and DDX5 were detected in both lysates. To chraracterize this phenomenon, phosphorylation of DDX3 was analyzed with pTyr, pSer, and pThr-specific antibodies. DDX3 in the nucleus was highly phosphorylated than cytoplasmic DDX3. Dephosphorylation of DDX3 with PP2A and PTP1B reduced the interaction with DDX5 in the cytoplasm. From the study, DEAD-box RNA helicase gene DDX3 interacted with DDX5 in the cytoplasm and phosphorylation of DDX3 had an effect on the interaction with DDX5.
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