Helicase associated 2 domain is essential for helicase activity of RNA helicase A |
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Authors: | Li Xing Xia Zhao Meijuan NiuLawrence Kleiman |
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Institution: | Lady Davis Institute for Medical Research, McGill AIDS Centre, Jewish General Hospital, Montreal, Quebec H3T 1E2, Canada; Department of Medicine, McGill University, Montreal, Quebec H3T 1E2, Canada |
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Abstract: | RNA helicase A (RHA), a DExD/H box protein, plays critical roles in a wide variety of cellular or viral functions. RHA contains a conserved core helicase domain that is flanked by five other domains. Two double-stranded RNA binding domains (dsRBD1 and dsRBD2) are at the N-terminus, whereas HA2 (helicase associated 2), OB-fold (oligonucleotide- or oligosaccharide-binding fold), and RGG (repeats of arginine and glycine–glycine residues) domains are at the C-terminus. The role of these domains in the helicase activity of RHA is still elusive due to the difficulty of obtaining enzymatically active mutant RHA. Here, we purified a series of mutant RHAs containing deletions in either N-terminus or C-terminus. Analysis of these mutant RHAs reveals that the dsRBDs are not required for RNA unwinding, but can enhance the helicase activity by promoting the binding of RHA to substrate RNA. In contrast, deletion of C-terminal domains including RGG, OB-fold, and HA2 does not significantly affect the binding of RHA to substrate RNA. However, HA2 is essential for the RNA unwinding by RHA whereas the RGG and OB-fold are dispensable. The results indicate that the core helicase domain alone is not enough for RHA to execute the unwinding activity. |
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Keywords: | ADAR adenosine deaminase acting on RNA dsRNA double-stranded RNA dsDNA double-stranded DNA dsRBD double-stranded RNA binding domain FTSC fluorescein-5-thiosemicarbazide FP fluorescence polarization GST Glutathione S-transferase HA2 helicase associated 2 NTP nucleotide triphosphate nt nucleotide OB-fold oligonucleotide/Oligosaccharide binding fold PKR dsRNA-dependent protein kinase RHA RNA helicase A RISC RNA-induced silencing complex RGG Arg&ndash Gly&ndash Gly repeats ssRNA single-stranded RNA 32pCp [5&prime -32P]Cytidine 3&prime 5&prime -bis(phosphate) |
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