Unique subcellular distribution of RPB1 with a phosphorylated C-terminal domain (CTD) in mouse oocytes during meiotic division and its relationship with chromosome separation.
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.
Department of Histology and Embryology, School of Basic Medical Sciences, Capital Medical University, Beijing 100069, China.
HaoJie
H
Wei
Qian
Q
Wang
Juan
J
DU
Xin
X
Li
Nana
N
Zhang
Yan
Y
Cao
Wei
W
Ma
D000818:Animals; D015153:Blotting, Western; D002503:Centromere / Q000737:chemistry; D003593:Cytoplasm / Q000737:chemistry; D005260:Female; D005455:Fluorescent Antibody Technique; D008297:Male; D008540:Meiosis / Q000502:physiology*; D051379:Mice; D008815:Mice, Inbred Strains; D022101:Microtubule-Organizing Center / Q000737:chemistry; D009865:Oocytes / Q000737:chemistry*; D010766:Phosphorylation; D012319:RNA Polymerase II / Q000032:analysis*
D012319:RNA Polymerase II; C522173:Rpb1 protein, mouse