Mapping the functional domains of HAP95, a protein that binds RNA helicase A and activates the constitutive transport element of type D retroviruses.
The complex retroviruses such as human immunodeficiency virus, type 1, employ a virally encoded protein, Rev, to mediate the nuclear export of unspliced and partially spliced mRNA. In contrast, the simian type D retroviruses act through a cis-acting constitutive transport element (CTE) that presumably interacts directly with cellular export proteins. We first reported that RNA helicase A (RHA) is a shuttle protein that binds to functional CTE in vitro and in vivo. Recently, we isolated a novel protein, HAP95, that specifically binds to the nuclear transport domain of RHA and up-regulates CTE-mediated gene expression. Here, using truncation and deletion mutations, we mapped the domains of HAP95 that are important for RHA binding, transactivation of CTE, and nuclear cytoplasmic shuttling. We report evidence for a novel nuclear export signal in HAP95 and showed that the domains involved in RHA binding and nuclear localization are required for CTE activation. Finally, we showed that HAP95 synergizes significantly with RHA on CTE-mediated reporter gene expression and promotes nuclear export of unspliced mRNA in transfected cells. Taken together, these data support the proposal that HAP95 specifically facilitates CTE-mediated gene expression by directly binding to RHA.
Yang
J P
JP
Department of Biology, University of California, San Diego, La Jolla, California 92093-0665, USA.
Tang
H
H
Reddy
T R
TR
Wong-Staal
F
F
D021581:Active Transport, Cell Nucleus; D000595:Amino Acid Sequence; D001324:Autoantigens / Q000378:metabolism; D030121:Betaretrovirus / Q000378:metabolism*; D002352:Carrier Proteins / Q000737:chemistry* / Q000502:physiology; D053487:DEAD-box RNA Helicases; D004268:DNA-Binding Proteins; D006367:HeLa Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins; D009687:Nuclear Proteins / Q000737:chemistry* / Q000502:physiology; D012313:RNA / Q000378:metabolism; D020365:RNA Helicases / Q000378:metabolism
C490117:AKAP8L protein, human; D001324:Autoantigens; D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D047908:Intracellular Signaling Peptides and Proteins; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; D012313:RNA; C098487:DHX9 protein, human; D053487:DEAD-box RNA Helicases; D020365:RNA Helicases