title: GTP-dependent binding and nuclear transport of RNA polymerase II by Npa3 protein.
abstract: We identified XAB1 in a proteomic screen for factors that interact with human RNA polymerase II (RNAPII). Because XAB1 has a conserved Saccharomyces cerevisiae homologue called Npa3, yeast genetics and biochemical analysis were used to dissect the significance of the interaction. Degron-dependent Npa3 depletion resulted in genome-wide transcription decreases, correlating with a loss of RNAPII from genes as measured by chromatin immunoprecipitation. Surprisingly, however, transcription in vitro was unaffected by Npa3, suggesting that it affects a process that is not required for transcription in yeast extracts. Indeed, Npa3 depletion in vivo affects nuclear localization of RNAPII; the polymerase accumulates in the cytoplasm. Npa3 is a member of the GPN-LOOP family of GTPases. Npa3 mutants that either cannot bind GTP or that bind but cannot hydrolyze it are inviable and unable to support nuclear transport of RNAPII. Surprisingly, we were unable to detect interactions between Npa3 and proteins in the classical importin α/β pathway for nuclear import. Interestingly, Npa3-RNAPII binding is significantly increased by the addition of GTP or its slowly hydrolyzable analogue guanosine 5'-3-O-(thio)triphosphate (GTPγS). Moreover, the Npa3 mutant that binds GTP, but cannot hydrolyze it, binds RNAPII even in the absence of added GTP, whereas the mutant that cannot bind GTP is unable to bind the polymerase. Together, our data suggest that Npa3 defines an unconventional pathway for nuclear import of RNAPII, which involves GTP-dependent binding of Npa3 to the polymerase.
authors.affiliation: Mechanisms of Transcription Laboratory, Clare Hall Laboratories, Cancer Research UK London Research Institute, South Mimms EN6 3LD.
authors.forename: Lidija
authors.initials: L
authors.lastname: Staresincic
authors.affiliation: Mechanisms of Transcription Laboratory, Clare Hall Laboratories, Cancer Research UK London Research Institute, South Mimms EN6 3LD.
authors.forename: Jane
authors.initials: J
authors.lastname: Walker
authors.affiliation: Mechanisms of Transcription Laboratory, Clare Hall Laboratories, Cancer Research UK London Research Institute, South Mimms EN6 3LD.
authors.forename: A Barbara
authors.initials: AB
authors.lastname: Dirac-Svejstrup
authors.affiliation: Bioinformatics and Biostatistics Group, Cancer Research UK London Research Institute, 44 Lincoln's Inn Fields, London WC2A 3LY, United Kingdom.
authors.forename: Richard
authors.initials: R
authors.lastname: Mitter
authors.affiliation: Mechanisms of Transcription Laboratory, Clare Hall Laboratories, Cancer Research UK London Research Institute, South Mimms EN6 3LD. Electronic address: j.svejstrup@cancer.org.uk.
authors.forename: Jesper Q
authors.initials: JQ
authors.lastname: Svejstrup
mesh_terms: D021581:Active Transport, Cell Nucleus / Q000502:physiology; D002467:Cell Nucleus / Q000235:genetics / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism; D017353:Gene Deletion; D006801:Humans; D020559:Monomeric GTP-Binding Proteins; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding / Q000502:physiology; D012319:RNA Polymerase II / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D028901:alpha Karyopherins / Q000235:genetics / Q000378:metabolism; D028961:beta Karyopherins / Q000235:genetics / Q000378:metabolism
chemical_list: D009687:Nuclear Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D028901:alpha Karyopherins; D028961:beta Karyopherins; D012319:RNA Polymerase II; C472669:GPN1 protein, human; D019204:GTP-Binding Proteins; C568359:Npa3 protein, S cerevisiae; D020559:Monomeric GTP-Binding Proteins