Staresincic L, Walker J, Dirac-Svejstrup AB, Mitter R, Svejstrup JQ

We identified XAB1 in a proteomic screen for factors that interact with human RNA polymerase II (RNAPII). Since 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 ...

Read More

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-family of GTPases. Npa3 mutants that either cannot bind GTP, or which 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 hydrolysable analogue GTP-γS. Moreover, the Npa3 mutant that binds GTP, but cannot hydrolyze it, binds RNAPII even in the absence of added GTP, while 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.

Date: Aug. 15, 2011

View in: Pubmed Google Scholar

122282