Phosphorylation of the RNA Polymerase II Largest Subunit during Xenopus laevis Oocyte Maturation

Abstract

Xenopus laevis oogenesis is characterized by an active transcription which ceases abruptly upon maturation. To survey changes in the characteristics of the transcriptional machinery which might contribute to this transcriptional arrest, the phosphorylation status of the RNA polymerase II largest subunit (RPB1 subunit) was analyzed during oocyte maturation. We found that the RPB1 subunit accumulates in large quantities from previtellogenic early diplotene oocytes up to fully grown oocytes. The C-terminal domain (CTD) of the RPB1 subunit was essentially hypophosphorylated in growing oocytes from Dumont stage IV to stage VI. Upon maturation, the proportion of hyperphosphorylated RPB1 subunits increased dramatically and abruptly. The hyperphosphorylated RPB1 subunits were dephosphorylated within 1 h after fertilization or heat shock of the matured oocytes. Extracts from metaphase II-arrested oocytes showed a much stronger CTD kinase activity than extracts from prophase stage VI oocytes. Most of this kinase activity was attributed to the activated Xp42 mitogen-activated protein (MAP) kinase, a MAP kinase of the ERK type. Making use of artificial maturation of the stage VI oocyte through microinjection of a recombinant stable cyclin B1, we observed a parallel activation of Xp42 MAP kinase and phosphorylation of RPB1. Both events required protein synthesis, which demonstrated that activation of p34 cdc2 kinase was insufficient to phosphorylate RPB1 ex vivo and was consistent with a contribution of the Xp42 MAP kinase to RPB1 subunit phosphorylation. These results further support the possibility that the largest RNA polymerase II subunit is a substrate of the ERK-type MAP kinases during oocyte maturation, as previously proposed during stress or growth factor stimulation of mammalian cells.In Xenopus laevis, as in many species, transcription is arrested in the matured gametes. During oogenesis, proteins and RNAs accumulate to support early embryogenesis. Although the fully grown oocyte contains as much RNA polymerase II (RNAPII) activity as 10 5 individual somatic cells (43), little is known about its properties at maturation, after the germinal vesicle breakdown. From this perspective, we decided to investigate the phosphorylation of the RNAPII largest subunit (RPB1) in the developing and maturing oocytes. The RNAPII core enzyme is an assembly of 12 subunits (29, 45). Extensive studies have assigned an important role to the phosphorylation of the C-terminal domain (CTD) of RPB1 in regulating the initiation of transcription (11, 18). The hypophosphorylated RPB1 (IIa subunit) binds to the TATA box binding protein (TBP) within a preinitiation complex of transcription, this interaction being abolished by phosphorylation of the CTD (28, 52). The steady-state distribution between the hypophosphorylated IIa and the hyperphosphorylated IIo forms of RPB1 results from the antagonistic activity of CTD kinases and CTD phosphatases. A CTD phosphatase has recently been purified and characterized, but few data are available concerning...

Cyclin C/CDK8 and cyclin H/CDK7/p36 are biochemically distinct CTD kinases

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N6-Methyladenosine Sequencing Highlights the Involvement of mRNA Methylation in Oocyte Meiotic Maturation and Embryo Development by Regulating Translation in Xenopus laevis

“…During oogenesis in animals like Xenopus laevis, mouse, and human, germinal vesicle (GV)-stage 3 oocytes gradually achieve maximum size, and genomic transcription activity is silenced (1,2). After that, the fully grown GV oocytes resume meiosis and develop to metaphase of the second meiosis (MII) stage.…”

Phospho-Signaling at Oocyte Maturation and Fertilization: Set Up for Embryogenesis and Beyond Part II. Kinase Regulators and Substrates

“…In C. elegans, embryonically transcribed gene products are required for gastrulation initiation where a large subunit of RNAPII is involved (Powell-Coffman et al 1996). In Xenopus, the largest subunit of RNA polymerase II (RPB1) accumulates in large quantities from previtellogenic early diplotene oocytes up to fully grown oocytes where the C-terminal domain (CTD) was essentially hypophosphorylated in growing oocytes from stage IV to VI (Bellier et al 1997). Upon maturation, RPB1 is hyperphosphorylated dramatically and abruptly but dephosphorylated within 1 h after fertilization (Bellier et al 1997).…”

“…In Xenopus, the largest subunit of RNA polymerase II (RPB1) accumulates in large quantities from previtellogenic early diplotene oocytes up to fully grown oocytes where the C-terminal domain (CTD) was essentially hypophosphorylated in growing oocytes from stage IV to VI (Bellier et al 1997). Upon maturation, RPB1 is hyperphosphorylated dramatically and abruptly but dephosphorylated within 1 h after fertilization (Bellier et al 1997). Metaphase II-arrested oocytes showed a much stronger CTD kinase activity than that of prophase stage VI and this kinase activity were attributed to the activated MAPK i.e.…”

“…RPB1 could be a substrate of MAPKs (e.g. p42) during Xenopus oocyte maturation (Bellier et al 1997). …”