Abundance of the largest subunit of RNA polymerase II in the nucleus is regulated by nucleo-cytoplasmic shuttling.
Eukaryotic RNA polymerase II is a complex enzyme composed of 12 distinct subunits that is present in cells in low abundance. Transcription of mRNA by RNA polymerase II involves a phosphorylation/dephosphorylation cycle of the carboxyl-terminal domain (CTD) of the enzyme's largest subunit. We have generated stable murine cell lines expressing an alpha-amanitin-resistant form of the largest subunit of RNA polymerase II (RNA Pol II LS). These cells maintained transcriptional activity in the presence of alpha-amanitin, indicating that the exogenous protein was functional. We observed that over-expressed RNA Pol II LS was predominantly hypophosphorylated, soluble and accumulated in the cytoplasm in a CRM1-dependent manner. Our results further showed that the transcriptionally active form of RNA Pol II LS containing phosphoserine in position 2 of the CTD repeats was restricted to the nucleus and its levels remained remarkably constant. We propose that nucleo-cytoplasmic shuttling of RNA Pol II LS may provide a mechanism to control the pool of RNA polymerase subunits that is accessible for assembly of a functional enzyme in the nucleus.
Custódio
Noélia
N
Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Av. Prof. Egas Moniz, 1649-028 Lisboa, Portugal.
Antoniou
Michael
M
Carmo-Fonseca
Maria
M
D021581:Active Transport, Cell Nucleus; D000546:Amanitins / Q000494:pharmacology; D000818:Animals; D002467:Cell Nucleus / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism; D006801:Humans; D028884:Karyopherins / Q000378:metabolism; D051379:Mice; D010766:Phosphorylation / Q000187:drug effects; D021381:Protein Transport; D012319:RNA Polymerase II / Q000378:metabolism*; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism; D012995:Solubility; D014407:Tumor Cells, Cultured; D000097606:Exportin 1 Protein
D000546:Amanitins; D028884:Karyopherins; D012319:RNA Polymerase II; D018160:Receptors, Cytoplasmic and Nuclear; D000097606:Exportin 1 Protein; C410949:RNA polymerase II largest subunit