The protein interaction network of the human transcription machinery reveals a role for the conserved GTPase RPAP4/GPN1 and microtubule assembly in nuclear import and biogenesis of RNA polymerase II.
RNA polymerase II (RNAPII), the 12-subunit enzyme that synthesizes all mRNAs and several non-coding RNAs in eukaryotes, plays a central role in cell function. Although multiple proteins are known to regulate the activity of RNAPII during transcription, little is known about the machinery that controls the fate of the enzyme before or after transcription. We used systematic protein affinity purification coupled to mass spectrometry (AP-MS) to characterize the high resolution network of protein interactions of RNAPII in the soluble fraction of human cell extracts. Our analysis revealed that many components of this network participate in RNAPII biogenesis. We show here that RNAPII-associated protein 4 (RPAP4/GPN1) shuttles between the nucleus and the cytoplasm and regulates nuclear import of POLR2A/RPB1 and POLR2B/RPB2, the two largest subunits of RNAPII. RPAP4/GPN1 is a member of a newly discovered GTPase family that contains a unique and highly conserved GPN loop motif that we show is essential, in conjunction with its GTP-binding motifs, for nuclear localization of POLR2A/RPB1 in a process that also requires microtubule assembly. A model for RNAPII biogenesis is presented.
Forget
Diane
D
Institut de recherches cliniques de Montréal, Montréal, Québec H2W1R7, Canada.
Lacombe
Andrée-Anne
AA
Cloutier
Philippe
P
Al-Khoury
Racha
R
Bouchard
Annie
A
Lavallée-Adam
Mathieu
M
Faubert
Denis
D
Jeronimo
Célia
C
Blanchette
Mathieu
M
Coulombe
Benoit
B
D002467:Cell Nucleus / Q000378:metabolism*; D002850:Chromatography, Gel; D002853:Chromatography, Liquid; D019204:GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D020868:Gene Silencing; D006367:HeLa Cells; D006801:Humans; D008870:Microtubules / Q000378:metabolism*; D021381:Protein Transport; D012319:RNA Polymerase II / Q000096:biosynthesis*; D034741:RNA, Small Interfering; D053719:Tandem Mass Spectrometry; D014158:Transcription, Genetic
D034741:RNA, Small Interfering; D012319:RNA Polymerase II; C472669:GPN1 protein, human; D019204:GTP-Binding Proteins