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General Comment |
NCBI Summary:
This gene encodes the largest subunit of RNA polymerase II, the polymerase responsible for synthesizing messenger RNA in eukaryotes. The product of this gene contains a carboxy terminal domain composed of heptapeptide repeats that are essential for polymerase activity. These repeats contain serine and threonine residues that are phosphorylated in actively transcribing RNA polymerase. In addition, this subunit, in combination with several other polymerase subunits, forms the DNA binding domain of the polymerase, a groove in which the DNA template is transcribed into RNA. [provided by RefSeq, Jul 2008]
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Comment |
Unique subcellular distribution of RPB1 with a phosphorylated C-terminal domain (CTD) in mouse oocytes during meiotic division and its relationship with chromosome separation. Wei H et al. (2015) Polymerase (RNA) II (DNA directed) polypeptide A (RPB1) is the largest subunit of RNA polymerase II (RNAPII), and phosphorylation of its C-terminal domain (CTD) is required for transcription initiation, elongation and RNA processing. Little is known about the CTD phosphorylation pattern and potential function during cell division when transcription is silenced. In this study, we assessed the protein expression and subcellular distribution of RPB1 during mouse oocyte meiotic division. Western blot analysis revealed that the RPB1 CTD was highly phosphorylated on Ser2 (pRPB1(Ser2)), Ser5 (pRPB1(Ser5)) and Ser7 (pRPB1(Ser7)). High and stable expression of pRPB1(Ser2) and pRPB1(Ser5) was detected from germinal vesicle (GV) to Metaphase II (MII) stage. In contrast, pRPB1(Ser7) only emerged after germinal vesicle breakdown (GVBD) and gradually increased to its peak level at metaphase I (MI) and MII. Immunofluorescence demonstrated that pRPB1(Ser2), pRPB1(Ser5) and pRPB1(Ser7) were pronouncedly aggregated within the nucleus of GV oocytes with a non-surrounded nucleolus (NSN) but very faintly labeled in oocytes with a surrounded nucleolus (SN). After meiotic resumption, pRPB1(Ser2) was again detected at spindle poles and co-localized with key microtubule organizing center (MTOC) components, pericentrin and γ-tubulin. pRPB1(Ser5) and pRPB1(Ser7) were assembled as filamentous aggregates and co-localized with microtubules throughout the spindle structure, responding to spindle-disturbing drugs, nocodazole or taxol, in pattern strongly similar to microtubules. pRPB1(Ser2) and pRPB1(Ser5) were constantly localized on chromosomes, with a relatively high concentration in centromere areas. Taken together, our data suggest that the CTD is highly phosphorylated and may be required for accurate chromosome segregation in mouse oocytes during meiosis.//////////////////
Global Gene Silencing is Caused by the Dissociation of RNA Polymerase II from DNA in Mouse Oocytes. Abe KI et al. As mouse oocytes approach maturity, a global repression of gene transcription occurs. Here, we investigated the involvement of RPB1, the largest subunit of RNA polymerase II (RNAP II), in the regulation of this transcriptional silencing mechanism. Using BrUTP to follow transcription in an in vitro run-on assay, we observed an abrupt decrease in transcriptional activity when oocytes reached their full size (approximately 80 mum). Immunoblotting using antibodies specific for the phosphorylated and unphosphorylated forms of RPB1 revealed that RPB1 is phosphorylated at Ser-2 and Ser-5 in the small growing oocytes in which active transcription occurs. By contrast, in transcriptionally inactive, full-grown oocytes, RPB1 is predominantly unphosphorylated. When we permeabilized the nuclear membrane using Triton X-100 during fixation for immunocytochemistry, the unphosphorylated form of RPB1 diffused out of the nucleus in the full-grown oocytes but still remained there in the small growing oocytes, indicating that RPB1 is not bound to DNA in full-grown oocytes. These results suggest that the immediate cause of global transcriptional silencing is the dissociation of RNAP II from the DNA. We also observed dissociation of RPB1 from the DNA in full-grown oocytes treated with trichostatin A to decondense their chromatin, suggesting that chromatin condensation is not an essential process in gene silencing during oocyte growth.
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