Analysis of the RNA binding specificity of the human tap protein, a constitutive transport element-specific nuclear RNA export factor.
The human Tap protein has been proposed to mediate Mason Pfizer monkey virus constitutive transport element (CTE)-dependent nuclear RNA export and may also play a role in global mRNA export. Here, we have used in vivo assays, in both yeast and human cells, together with in vitro assays, to further characterize the RNA binding properties of Tap, which has been proposed to contain a novel leucine-rich RNA binding motif. Using the yeast three hybrid assay, we selected RNA molecules that retain Tap binding activity from a pool of randomized CTE sequences. The recovered RNA sequences differed only minimally from the wild-type CTE yet all displayed lower affinity for Tap both in vivo and in vitro. Analysis of the RNA export activity of the recovered CTE variants revealed that Tap affinity was highly predictive of CTE biological activity. Together, these observations provide additional evidence supporting the identification of Tap as the direct cofactor for CTE function and demonstrate that RNA binding by Tap is highly sequence specific.
Kang
Y
Y
Department of Genetics, Duke University Medical Center, Durham, North Carolina 27710, USA.
Bogerd
H P
HP
Yang
J
J
Cullen
B R
BR
D000818:Animals; D001483:Base Sequence; D001692:Biological Transport / Q000235:genetics; D002467:Cell Nucleus / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D006801:Humans; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D009690:Nucleic Acid Conformation; D029543:Nucleocytoplasmic Transport Proteins; D011784:Quail; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D014407:Tumor Cells, Cultured
C106898:MEX67 protein, S cerevisiae; C122111:NXF1 protein, human; D009687:Nuclear Proteins; D029543:Nucleocytoplasmic Transport Proteins; D016601:RNA-Binding Proteins; D029701:Saccharomyces cerevisiae Proteins