GTP-dependent binding and nuclear transport of RNA polymerase II by Npa3 protein
Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › fagfællebedømt
Standard
GTP-dependent binding and nuclear transport of RNA polymerase II by Npa3 protein. / Staresincic, Lidija; Walker, Jane; Dirac-Svejstrup, A Barbara; Mitter, Richard; Svejstrup, Jesper Q.
I: Journal of Biological Chemistry, Bind 286, Nr. 41, 10.2011, s. 35553-61.Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › fagfællebedømt
Harvard
APA
Vancouver
Author
Bibtex
}
RIS
TY - JOUR
T1 - GTP-dependent binding and nuclear transport of RNA polymerase II by Npa3 protein
AU - Staresincic, Lidija
AU - Walker, Jane
AU - Dirac-Svejstrup, A Barbara
AU - Mitter, Richard
AU - Svejstrup, Jesper Q
PY - 2011/10
Y1 - 2011/10
N2 - 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 a/ß 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 (GTP¿S). 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.
AB - 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 a/ß 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 (GTP¿S). 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.
KW - Active Transport, Cell Nucleus
KW - Cell Nucleus
KW - GTP-Binding Proteins
KW - Gene Deletion
KW - Humans
KW - Nuclear Proteins
KW - Protein Binding
KW - RNA Polymerase II
KW - Saccharomyces cerevisiae
KW - Saccharomyces cerevisiae Proteins
KW - Transcription Factors
KW - alpha Karyopherins
KW - beta Karyopherins
U2 - 10.1074/jbc.M111.286161
DO - 10.1074/jbc.M111.286161
M3 - Journal article
C2 - 21844196
VL - 286
SP - 35553
EP - 35561
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 41
ER -
ID: 38419196