Research ArticleMolecular determinants of nucleolar translocation of RNA helicase A
Introduction
RNA helicase A (RHA), also named Nuclear DNA helicase II (NDH II), belongs to the DEAH-box family of nucleic acid helicases and is ubiquitously expressed in eukaryotic cells [1]. First isolated as an ATP-dependent RNA helicase [2], [3], RHA was later shown to possess DNA-unwinding activity as well [4]. RHA has been implicated in numerous cellular functions, based mainly on its interaction with a variety of proteins and RNA. The subcellular localization of RHA is dynamic, shuttling between the cytoplasm and the nucleus despite its steady-state nuclear localization [5], consistent with its potential functions in DNA transcription, RNA processing, translation and viral particle production [6], [7], [8], [9], [10], [11], [12], [13], [14], [15], [16], [17]. The structural domain responsible for this shuttling activity has been mapped to the C-terminus of the protein and designated the nuclear transport domain (NTD) for its ability to direct both nuclear import and export of RHA [5]. The nuclear import signal within the NTD interacts with importin-α3 and requires arginine methylation to direct the nuclear localization of RHA [18], [19]. The nuclear export mediated by NTD uses a yet to be identified pathway that is insensitive to leptomycin B, a drug that specifically blocks the CRM-1-dependent nuclear export pathway [5], [20], [21].
The localization of RHA is also dynamic within the nucleus. In human cells, RHA normally localizes in the nucleoplasm, excluded from the nucleolus in most cases [5], [22], [23]. However, a nucleolar accumulation of the protein was observed when these cells were treated with a transcription inhibitor or an agent that degrades F-actin [22], [23], suggesting an association between RHA and the nucleolus. Consistent with this result, RHA was found to be among the 30 or so RNA helicases that are part of the nucleolar proteome [24]. In addition, immunostaining of endogenous murine RHA in several mouse cell lines revealed an unexpected enrichment of RHA in the nucleolus even when cells were not treated with any transcription inhibitors [25]. Together, these results suggest that nucleolar localization and translocation could prove relevant for the cellular functions of RHA.
Like many other members of the helicase family, RHA has been implicated in the life cycles of viruses [12], [17], [26], [27], [28], [29]. The dynamic subcellular localization of RHA is likely to be relevant for its participation in the replication of these viruses, especially those that either replicate in the cytoplasm or involve the nucleolus during replication [12], [26], [28].
Here we present evidence that RHA, a protein that binds to the 3′ UTR of hepatitis C virus (HCV) [29], alters its subnuclear localization in response to HCV replication as well as transcription inhibition. Instead of its usual nucleoplasmic localization, RHA accumulates in the nucleolus or the perinucleolar region in HCV replicon cells. The same subnuclear redistribution of RHA was observed when the cells were treated with 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole (DRB) or cultured at 4 °C. Mapping experiments revealed that the NTD at the C-terminus is also important for nucleolar localization. Other important determinants include the ATPase motif and the second double-stranded RNA binding domain (dsRBD II), suggesting an involvement of RNA-binding in the nucleolar translocation of this protein.
Section snippets
Cells and treatments
Huh-7.5 (a human hepatocarcinoma cell line), 293-FT (derived from human kidney fibroblasts), 3T3 (mouse embryos fibroblast cell), BHK-21 (derived from Syrian hamster kidney), HeLa (a human cervical cancer cell line) and COS-1 (derived from African green monkey kidney) cells were maintained in standard Dulbecco's minimal Eagle's medium (Hyclone, Logan, UT) supplemented with 10% of Fetal Bovine Serum (Cell Generation), 10 U/ml of penicillin and 100 μg/ml of streptomycin (Sigma, St. Louis, MO).
Redistribution of RHA to the nucleolus and the perinucleolar region upon diverse cellular stress
RHA has been reported to interact with a number of viruses [12], [17], [26], [27], [28], [29]. In particular, it was identified as a member of a group of proteins that interacts with the genomes of two related RNA viruses: bovine viral diarrhea virus (BVDV) and hepatitis C virus [28], [29]. RNA interference demonstrated an essential role of RHA in the replication of BVDV [28]. Since both BVDV and HCV replicate in the cytoplasm and we previously found that RHA shuttles between the nucleus and
Discussion
Nucleolar localization and transport have been reported for other DEAD-box proteins. An3 protein, an RNA helicase expressed in Xenopus laevis oocytes, is a nucleo-cytoplasmic shuttling protein that associates with the nucleolus at certain stages of development [38], [39]. A similar development stage-dependent nucleolar association was also observed for RHA in bovine oocytes [40]. Another DEAD-box helicase, p68, has been shown to redistribute to the nucleolus in a cell cycle-dependent manner and
Acknowledgments
This work was supported by American Heart Association National Center. We thank Dr. Charlie Rice (Rockefeller University) for providing Clone B cells, Dr. Robert Tsai for B23-GFP plasmid and Dr. C-G Lee for the original RHA cDNA clone and anti-RHA sera. Dr. Kimberly Riddle helped with confocal imaging and the molecular cloning core facility of FSU biology department is acknowledged for assisting us with mutagenesis and plasmid construction. We would also like to thank Diana Lambert for
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