Intracellular Trafficking of ADAR1

Adenosine deaminases that act on RNA (ADARs) are RNA editing enzymes that convert adenosines to inosines in double-stranded RNA substrates. In mRNAs this editing event can lead to a codon exchange thus allowing the formation of diverse proteins from a single gene. ADAR-mediated editing has been detected in all metazoa tested and seems to be essential for normal life and development in most organisms. Editing of endogenous substrates seemingly occurs in the nucleus where the majority of constitutively expressed ADARs are found. However, several lines of evidence also suggest a cytoplasmic function of at least one ADAR family member in defeating viral infection. Editing of endogenous substrates is very specific and in most cases only affects a few adenosines in any given substrate. In vitro, however, editing is more promiscuous and can affect the majority of adenosines in artificial double-stranded substrates. This fact and other biological evidence suggests that editing is highly regulated in vivo. On the one hand regulation of editing warrants that only a fraction of substrate molecules become edited, on the other hand most structured cellular RNAs are not edited and thus are protected from inadvertent editing. Recently, several mechanisms capable to regulate ADAR activity have been uncovered that differ amongst organisms, cell types and ADAR isoforms. In Drosophila for instance, ADAR edits its own pre-mRNA giving rise to an enzymatically less active enzyme. Flies unable to downregulate their editing activity are inviable. In vertebrates ADAR activity is regulated at the transcriptional level, via alternative splicing and by controlling the level of freely available ADAR in the nucleoplasm. We could show recently that human ADAR1 is a nucleo-cytoplasmic shuttling protein with an atypical nuclear localization signal in one of its double stranded RNA-binding domains. Additionally, nuclear accumulation is regulated via several signals found in this protein in an RNA-binding dependent manner. Here we propose to study the regulatory mechanisms that control the levels of freely available ADAR1 in the nucleus. Cis- and trans- acting elements will be characterized and their influence on editing activity will be determined.

Adenosine deaminases that act on RNA (ADARs) are RNA editing enzymes that convert adenosines to inosines in double-stranded RNA substrates. In mRNAs this editing event can lead to a codon exchange thus allowing the formation of diverse proteins from a single gene. ADAR-mediated editing has been detected in all metazoa tested and seems to be essential for normal life and development in most organisms. Editing of endogenous substrates seemingly occurs in the nucleus where the majority of constitutively expressed ADARs are found. However, several lines of evidence also suggest a cytoplasmic function of at least one ADAR family member in defeating viral infection. Editing of endogenous substrates is very specific and in most cases only affects a few adenosines in any given substrate. In vitro, however, editing is more promiscuous and can affect the majority of adenosines in artificial double-stranded substrates. This fact and other biological evidence suggests that editing is highly regulated in vivo. On the one hand regulation of editing warrants that only a fraction of substrate molecules become edited, on the other hand most structured cellular RNAs are not edited and thus are protected from inadvertent editing. Recently, several mechanisms capable to regulate ADAR activity have been uncovered that differ amongst organisms, cell types and ADAR isoforms. In Drosophila for instance, ADAR edits its own pre-mRNA giving rise to an enzymatically less active enzyme. Flies unable to downregulate their editing activity are inviable. In vertebrates ADAR activity is regulated at the transcriptional level, via alternative splicing and by controlling the level of freely available ADAR in the nucleoplasm. We could show recently that human ADAR1 is a nucleo-cytoplasmic shuttling protein with an atypical nuclear localization signal in one of its double stranded RNA-binding domains. Additionally, nuclear accumulation is regulated via several signals found in this protein in an RNA-binding dependent manner. Here we propose to study the regulatory mechanisms that control the levels of freely available ADAR1 in the nucleus. Cis- and trans- acting elements will be characterized and their influence on editing activity will be determined.