Molecular recognition and RNA-regulation of a bimodular nuclear localization signal

Nucleo-cytoplasmic transport through nuclear pores is an essential and tightly regulated process in eukaryotic cells. Proteins and multi-component complexes are recognized by transport receptors and transported across the nuclear membrane. Cargo-receptor interactions are highly specific to ascertain that only fully assembled and matured complexes are being transported. Transportin-1 (Trn1) is a major nuclear import receptor, known to bind the so- called PY nuclear localization signal (NLS). However many Trn1 cargoes lack a PY-NLS, such as the human RNA-editing enzyme ADAR1. We recently uncovered how an RNA- binding domain in ADAR1 exposes a bimodular NLS, rendering its recognition by Trn1 RNA-sensitive. Here we aim at characterizing the molecular details and the RNA-regulation of this interaction using a combination of biophysical, structural, and cellular assays. Our studies will yield a detailed understanding of the rules governing the recognition of this novel, non-linear NLS by Trn1 and its regulation by RNA-binding. This study will also contribute to our understanding of the antiviral and anti-inflammatory role of the RNA- editing enzyme ADAR1 which modified cellular and viral RNAs in the nucleus and cytoplasm, respectively.

Adenosine deaminases acting on RNA convert adenosines to inosines in structured cellular RNAs. One of the enzymes performing this reaction is ADAR1. ADAR1 also has in important function in helping to distinguish cellular RNAs from foreign viral or bacterial RNAs. ADAR1 is produced in two isoforms. One isoform is active in the nucleus while the other is active in the cytoplasm. In this project we aimed at studying nuclear transport of the ADAR1 isoforms. We could show that the nuclear import factor Transportin 1 interacts strongly with ADAR1. Interestingly, we could show that the region in ADAR1 that interacts with Transportin 1 can also dimerize. The significance of this dimer formation is still unknown and is currently under investigation. As nuclear transport of ADAR1 is affected by the RNA-binding of the protein we have also isolated RNAs that interact specifically with the isoforms of ADAR1. Our studies show that the interaction of these RNAs is primarily regulated by the cellular localization of ADAR1 isoforms.