1. P-TEFb Kinase Activity Is Essential for Global Transcription, Resumptionof Meiosis and Embryonic Genome Activation in Pig
- Author
-
Dong Il Jin, Hyun Young Shin, Ki Myung Choi, Reza K. Oqani, Tao Lin, and Jae Eun Lee
- Subjects
0301 basic medicine ,Embryology ,Positive Transcriptional Elongation Factor B ,Transcription, Genetic ,Swine ,Molecular biology ,lcsh:Medicine ,RNA polymerase II ,Immunostaining ,Biochemistry ,0302 clinical medicine ,Animal Cells ,Cell Cycle and Cell Division ,Post-Translational Modification ,Phosphorylation ,lcsh:Science ,P-TEFb ,RNA transcription labeling ,Mammals ,Staining ,Multidisciplinary ,Genome ,biology ,Cyclin T ,Agriculture ,Cell biology ,Nucleic acids ,Meiosis ,Ribosomal RNA ,Cell Processes ,030220 oncology & carcinogenesis ,OVA ,Vertebrates ,Female ,Cellular Types ,Research Article ,Cellular structures and organelles ,Livestock ,Embryonic Development ,Fertilization in Vitro ,03 medical and health sciences ,Cyclins ,Animals ,Kinase activity ,Non-coding RNA ,Germinal vesicle ,lcsh:R ,Cyclin-dependent kinase 2 ,Embryos ,Organisms ,Biology and Life Sciences ,Proteins ,Cell Biology ,Embryo, Mammalian ,Cyclin-Dependent Kinase 9 ,In Vitro Oocyte Maturation Techniques ,Research and analysis methods ,030104 developmental biology ,Germ Cells ,Molecular biology techniques ,Specimen Preparation and Treatment ,Amniotes ,Cyclin-dependent kinase complex ,biology.protein ,Oocytes ,RNA ,lcsh:Q ,Cyclin-dependent kinase 9 ,Nucleic acid labeling ,Ribosomes ,Developmental Biology ,Cell labeling - Abstract
Positive transcription elongation factor b (P-TEFb) is a RNA polymerase II carboxyl-terminal domain (Pol II CTD) kinase that phosphorylates Ser2 of the CTD and promotes the elongation phase of transcription. Despite the fact that P-TEFb has role in many cellular processes, the role of this kinase complex remains to be understood in mammalian early developmental events. In this study, using immunocytochemical analyses, we found that the P-TEFb components, CDK9, Cyclin T1 and Cyclin T2 were localized to nuclear speckles, as well as in nucleolar-like bodies in pig germinal vesicle oocytes. Using nascent RNA labeling and small molecule inhibitors, we showed that inhibition of CDK9 activity abolished the transcription of GV oocytes globally. Moreover, using fluorescence in situ hybridization, in absence of CDK9 kinase activity the production of ribosomal RNAs was impaired. We also presented the evidences indicating that P-TEFb kinase activity is essential for resumption of oocyte meiosis and embryo development. Treatment with CDK9 inhibitors resulted in germinal vesicle arrest in maturing oocytes in vitro. Inhibition of CDK9 kinase activity did not interfere with in vitro fertilization and pronuclear formation. However, when in vitro produced zygotes were treated with CDK9 inhibitors, their development beyond the 4-cell stage was impaired. In these embryos, inhibition of CDK9 abrogated global transcriptional activity and rRNA production. Collectively, our data suggested that P-TEFb kinase activity is crucial for oocyte maturation, embryo development and regulation of RNA transcription in pig.
- Published
- 2016