The distribution of RNA polymerase II largest subunit (RPB1) in the Xenopus germinal vesicle

Abstract

We describe the localization of the largest subunit of RNA polymerase II (RPB1) in the oocyte nucleus of Xenopus laevis. A single oocyte nucleus contains 18 lampbrush chromosomes, ∼1500 extrachromosomal nucleoli, 50–100 Cajal bodies (CBs) and hundreds to thousands of B-snurposomes. CBs contain many factors involved in RNA transcription and processing, whereas B-snurposomes contain a subset of these factors involved in mRNA processing. Immunofluorescent staining demonstrates that most RPB1 is in the nucleoplasm and that the heptapeptide repeat comprising its carboxy-terminal domain (CTD) is not phosphorylated. A minor fraction of RPB1 is associated with the transcription units of the lampbrush chromosomes and is phosphorylated on serines 2 and 5 of the CTD. Another minor fraction occurs in CBs, which react with antibodies against unphosphorylated CTD repeats and repeats phosphorylated on serine 5. Although B-snurposomes are stained by an antibody against phosphorylated RPB1 (mAb H5), we present evidence that this stain is due to cross-reaction with one or more SR proteins. We show that constructs consisting of 15–17 CTD heptapeptide repeats fused to glutathione- S-transferase are targeted rapidly and specifically to CBs but not to B-snurposomes after injection into the nucleus. The staining and targeting data define at least three distinct populations of RPB1 in the GV with different states of phosphorylation. We suggest that CBs play a unique role in RPB1 metabolism, possibly as sites for assembly or modification of transcription complexes.