The double-stranded RNA binding domain of human Dicer functions as a nuclear localization signal

Abstract

Dicer is a key player in microRNA (miRNA) and RNA interference (RNAi) pathways, processing miRNA precursors and doublestranded RNA into ∼21-nt-long products ultimately triggering sequence-dependent gene silencing. Although processing of substrates in vertebrate cells occurs in the cytoplasm, there is growing evidence suggesting Dicer is also present and functional in the nucleus. To address this possibility, we searched for a nuclear localization signal (NLS) in human Dicer and identified its C-terminal double-stranded RNA binding domain (dsRBD) as harboring NLS activity. We show that the dsRBD-NLS can mediate nuclear import of a reporter protein via interaction with importins β, 7, and 8. In the context of full-length Dicer, the dsRBD-NLS is masked. However, duplication of the dsRBD localizes the full-length protein to the nucleus. Furthermore, deletion of the N-terminal helicase domain results in partial accumulation of Dicer in the nucleus upon leptomycin B treatment, indicating that CRM1 contributes to nuclear export of Dicer. Finally, we demonstrate that human Dicer has the ability to shuttle between the nucleus and the cytoplasm. We conclude that Dicer is a shuttling protein whose steady-state localization is cytoplasmic.

Functions of double-stranded RNA-binding domains in nucleocytoplasmic transport

“…Indeed, similarly to human Dicer, for which the C-terminal dsRBD was shown to be involved in the regulation of the sub-cellular localization of the protein, and especially its transient nuclear import (see ref 28 and text above), the C-terminal dsRBD of the yeast Schizosaccharomyces pombe Dicer (Dcr1) is also critical for the nuclear localization of the protein. 41 In contrast to human Dicer, S. pombe Dcr1 is predominantly nuclear and localizes at the nuclear periphery, near the nuclear pore complexes (NPCs).…”

“…The atypical dsRBD-NLS of human Dicer is composed of a cluster of positively charged residues represented as sticks in red (namely K1891, R1910 and R1907). 28 Noteworthy, these residues do not belong to the canonical region of interaction with dsRNA. (C) Cartoon representation of the dsRBD-NLS of human ADAR1 (PDB code 2mdr).…”

“…28 However, in the fulllength protein, the dsRBD-NLS is masked and Dicer localizes primarily to the cytoplasm. Therefore, even though the dsRBD-NLS mediates nuclear import, human Dicer constantly shuttles between the cytoplasm and the nucleus, and is primarily localized to the cytoplasm under steady-state conditions.…”

“…Therefore, even though the dsRBD-NLS mediates nuclear import, human Dicer constantly shuttles between the cytoplasm and the nucleus, and is primarily localized to the cytoplasm under steady-state conditions. 28 The shuttling properties of the dsRBD-NLS might be regulated by another domain of the protein situated in its N-terminal part, namely the helicase domain, and also by the presence of dsRNA. 28 It will therefore be interesting to investigate whether the localization of human Dicer is sensitive to variation of external conditions, a layer of regulation that was shown to enable a rapid response to stress conditions in the fission yeast.…”

“…28 The shuttling properties of the dsRBD-NLS might be regulated by another domain of the protein situated in its N-terminal part, namely the helicase domain, and also by the presence of dsRNA. 28 It will therefore be interesting to investigate whether the localization of human Dicer is sensitive to variation of external conditions, a layer of regulation that was shown to enable a rapid response to stress conditions in the fission yeast. 29 We recently uncovered the molecular basis for the dsRBD-mediated nuclear import of the RNA-editing enzyme ADAR1.…”