Nuclear import and substrate recognition by dsRBDs

Adenosine deaminases that act on RNA (ADARs) convert adenosines to inosines in double-stranded and structured RNAs. The consequences of RNA editing are numerous going from changes of codons, splice patterns, altered stability or localization of RNA molecules. Substrate recognition and editing can be specific in some substrates and rather non-specific in others. RNA-binding and recognition is mediated via multiple double-stranded RNA-binding domains (dsRBDs) that recognize the structure, rather than the sequence of the substrate RNA. Mammalian ADAR1 is a nucleo-cytoplasmic shuttling protein that harbors three dsRBDs. Specific editing of coding RNAs occurs in the nucleus. For other substrates such as pri- and pre-miRNAs it is less clear whether editing occurs exclusively in the nucleus or whether the enzyme might also act in the cytoplasm, possibly accompanying its substrates from their site of transcription to the cytoplasm. Nucleo-cytoplasmic transport of ADAR1 is mediated via its double-stranded RNA-binding domains. The third dsRBD of ADAR1 acts as a nuclear localization signal. RNA-binding interferes with nuclear import and might even stimulate nuclear export. dsRBD#3 is specifically recognized by the nuclear import receptor transportin-1. Here we propose to determine the atomic structure of dsRBD#3 in ADAR1 and to identify regions and features that are required for NLS activity using NMR spectroscopy. We also aim at determining the molecular basis of RNA recognition by dsRBD#3 and by the other dsRBDs of ADAR1 in order to understand how ADAR1 discriminates its substrates and also how RNA binding by dsRBD#3 prevents protein import. Finally, our study will help identifying nuclear localization signals in other dsRBDs, a motif recurrently found in proteins triggering the many facettes of double-stranded RNA response.

The RNA-modifying enzyme ADAR1 is an important factor in the defense against viral RNAs and mobile genetic elements. ADAR1 acts both in the nucleus and cytoplasm of animal cells. When no RNA is bound to ADAR1 the protein shuttles continuously through the nuclear pores- little channels in the nuclear membrane- between the nucleus and cytoplasm. During viral infection or when RNA is bound to ADAR1, however, the protein accumulates in the cytoplasm and nuclear import is inhibited. Using cell biological and structural analysis in a collaboration with the ETH Zürich we could show how nuclear import is being regulated by RNA: for nuclear transport to occur, transport proteins bind to a regions that flanks one of the three RNA binding domains found in ADAR1. The RNA binding domain is important for the proper positioning of the nuclear import elements. However, when RNA is bound to the RNA binding domain the interaction with the import proteins is inhibited therefore leading to a cytoplasmic accumulation of ADAR1. This way, nuclear import of viral RNAs bound to ADAR1 can be prevented.When ADAR1 has bound to RNA, the protein modifies individual nucleotides thereby converting adenosines to inosines. As inosines are interpreted as guanosines by the cell the modifications introduced by ADAR1 can change or even destroy the genetic information stored in the RNA. This way ADAR1 can inhibit viral replication.