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Biophysical characterization of features of RNA helicase A that confer translational control of retroviral and selected cellular mRNAs
Author(s) -
Ranji Arnaz K,
Kvaratskhelia Mamuka,
BorisLawrie Kathleen A
Publication year - 2010
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.24.1_supplement.499.8
Subject(s) - rna , rna helicase a , helicase , biology , messenger rna , microbiology and biotechnology , untranslated region , rna binding protein , computational biology , genetics , gene
The 5′ untranslated region of some retroviruses and cellular mRNAs contain a cis‐acting Post‐transcriptional Control Element (PCE) that is necessary for efficient translation. Specific interaction of PCE with host RNA helicase A (RHA) is necessary for translation of the PCE‐containing viral and cellular mRNAs. This study sought to understand features of RHA necessary for selective interaction with the RNA switch. Site‐directed mutagenesis determined that the central DEIH helicase core is necessary for PCE activity, while the RGG domain is necessary for the nuclear localization. Here biophysical analyses tested the hypothesis that two redundant N‐terminal RNA binding domains are necessary to confer specific interaction with the Spleen Necrosis Virus (SNV) and junD PCE target RNA. Protein footprinting was employed to detect amino acids of RHA that interact with PCE RNA. Electrophoretic mobility shift assays and fluorescence polarization assays determined that the N‐terminal double‐stranded RNA binding domains specifically recognize structural features of PCE RNA. Loss‐of‐function point mutations in the double‐stranded RNA binding domains abolished binding to target mRNAs. The DEIH core does not exhibit affinity for the target mRNA, while the C‐terminal RGG domain binds non‐specifically to PCE RNA. Our results posit the model that PCE interaction with the N‐terminal domain of RHA exposes the DEIH domain, which catalyzes RNP rearrangement that facilitates polysome association. Molecular dissection of this new translational control axis may provide strategic drug targets for infectious disease and cancer. Funding: Glenn Barber fellowship of the OSU College of Veterinary Medicine and NIH R01 CA108882 and P01CA00730