A bimodular nuclear localization signal assembled via an extended double-stranded RNA-binding domain acts as an RNA-sensing signal for transportin 1

Abstract

The human RNA-editing enzyme adenosine deaminase acting on RNA (ADAR1) carries a unique nuclear localization signal (NLS) that overlaps one of its double-stranded RNA-binding domains (dsRBDs). This dsRBD-NLS is recognized by the nuclear import receptor transportin 1 (Trn1; also called karyopherin-β2) in an RNA-sensitive manner. Most Trn1 cargos bear a well-characterized proline-tyrosine-NLS, which is missing from the dsRBD-NLS. Here, we report the structure of the dsRBD-NLS, which reveals an unusual dsRBD fold extended by an additional Nterminal α-helix that brings the N-and C-terminal flanking regions in close proximity. We demonstrate experimentally that the atypical ADAR1-NLS is bimodular and is formed by the combination of the two flexible fragments flanking the folded domain. The intervening dsRBD acts only as an RNA-sensing scaffold, allowing the two NLS modules to be properly positioned for interacting with Trn1. We also provide a structural model showing how Trn1 can recognize the dsRBD-NLS and how dsRNA binding can interfere with Trn1 binding.NMR | RNA-binding protein | nucleocytoplasmic shuttling |

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

“…(C) Cartoon representation of the dsRBD-NLS of human ADAR1 (PDB code 2mdr). 9 The atypical dsRBD-NLS of human ADAR1 is composed of the N-and C-terminal modules (in blue and in red, respectively) flanking the folded dsRBD. The additional N-terminal helix (helix a N , in purple) brings the 2 modules in close proximity.…”

“…29 We recently uncovered the molecular basis for the dsRBD-mediated nuclear import of the RNA-editing enzyme ADAR1. 9 Mammalian ADAR1 is a nucleocytoplasmic shuttling protein containing 3 dsRBDs. Nuclear import of ADAR1 is mediated by Transportin-1 (Trn1), a member of the karyopherin-b family, via an atypical NLS that overlaps the third dsRBD of the protein.…”

“…This clearly indicates that the dsRBD itself is not required for Trn1 interaction, but only helps juxtapose the 2 NLS-modules that are distantly spaced in the primary structure. 9 In addition, this impressive molecular construction was shown to act as an RNA-sensing NLS that can be switched on and off, depending on the presence of dsRNA associated with the dsRBD. 9 Indeed, the modulation of the import activity of ADAR1 directly depends on the RNA-binding status of the dsRBD-NLS.…”

“…9 Indeed, the modulation of the import activity of ADAR1 directly depends on the RNA-binding status of the dsRBD-NLS. 9,33 The dsRBD-NLS presents 2 functional and non-overlapping interaction surfaces: (i) a functional dsRNA-binding interface that involves the canonical elements of dsRBDs for dsRNA binding; and (ii) a functional Trn1-binding interface that consists of the N-and C-terminal modules flanking the folded dsRBD (Fig. 1E).…”

“…Even though these 2 interfaces are distinct, the dsRBD-NLS cannot bind to dsRNA and Trn1 simultaneously, most probably for steric reasons, and therefore constitutes an RNA-sensing NLS. 9 Conversely, RNA-binding enhances nuclear export of ADAR1, 33 which overall positions RNA as a central regulator of ADAR1 shuttling. The fact that dsRNA stimulates nuclear export suggests that ADAR1 might leave the nucleus bound to substrate RNAs, such as precursors of microRNAs.…”