Abstract
The constitutive transport element (CTE) of type D retroviruses mediates the nuclear export of unspliced viral transcripts. We previously showed that RNA helicase A functionally interacts with CTE and contains a bidirectional nuclear transport domain at the carboxyl terminus. Here we report the identification of a novel human protein, helicase A-binding protein 95 (HAP95), which specifically binds to the carboxyl terminus of RNA helicase A. HAP95 is partially homologous to AKAP95, a member of the A kinase-anchoring protein family, but lacks the protein kinase A binding domain characteristic of this family. HAP95 is a nuclear protein at steady state but shuttles between the nucleus and cytoplasm. Overexpression of HAP95 significantly increases CTE-dependent gene expression but has no effect on general gene expression or that mediated by the Rev/Rev response element of human immunodeficiency virus type 1.
Highlights
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AF199414
We previously showed that RNA helicase A functionally interacts with constitutive transport element (CTE) and contains a bidirectional nuclear transport domain at the carboxyl terminus
We report the identification of a novel human protein, helicase A-binding protein 95 (HAP95), which binds to the carboxyl terminus of RNA helicase A
Summary
Yeast Two-hybrid Screenings—The sequence corresponding to the CTD of RHA (nt 1150 –1259) was amplified with PCR and cloned into the vector pGBT9, in phase with the DNA binding domain of the yeast GAL4 gene This vector was transformed into the yeast strain HF7C along with the human leukocyte cDNA library contained in the GAL4 activation domain-expressing plasmid, pGAD10, as described previously (CLONTECH, Palo Alto, CA). Verification, and identification of plasmid clones that code for RHA-binding protein peptides was done as described previously [11]. Cells were washed with PBS and incubated on ice with lysis buffer (150 mM NaCl, 10 mM Tris, pH 7.8, 1.5 mM MgCl2, 20 units/ml RNasin (Promega)). Cell extracts were prepared and tested for CAT activity through standard assay procedures as described previously [12]
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