Beyond the Identification of Transcribed Sequences: Functional and Expression Analysis

9th Annual Workshop, October 28-31, 1999

Co-sponsored by the U.S. Department of Energy


MORSE

Substrate specificity of adenosine deaminases that act on RNA (ADARs) in C. elegans

Daniel P. Morse, Leath A. Tonkin, P. Joe Aruscavage, and Brenda L. Bass

Department of Biochemistry/HHMI, University of Utah, Salt Lake City, Utah, USA

Adenosine deaminases that act on RNA (ADARs) comprise a family of RNA editing enzymes that convert adenosines to inosines within double-stranded regions of RNA.  What are the biological functions for this type of RNA editing?  In mammals, one function is to alter codons allowing the production of more than one form of a protein from a single mRNA. 

To expand our knowledge of ADAR function, we have exploited the tools available in the nematode C. elegans.  We developed a method to identify new ADAR substrates (Morse and Bass, PNAS 96:6048-53) and, with the aid of the completed C. elegans genome sequence, have now found seven edited transcripts in the worm.  Unlike mammals, editing of these C. elegans RNAs does not alter codons.  Instead, five of the substrates are mRNAs edited in untranslated regions, and two are non-coding RNAs.  

Examination of the C. elegans genome sequence revealed two predicted open reading frames that potentially encode active ADARs, H15N14.1 and T20H4.4. We have obtained mutant worms containing deletions in one or the other of these genes.  Extracts from the H15N14.1 deletion strain exhibited reduced, but measurable, ADAR activity.  There was no detectable activity in extracts from the T20H4.4 deletion strain.   Consistent with these in vitro results, editing of the seven ADAR substrates was severely reduced or lacking in the T20H4.4 deletion strain, while there were more subtle effects on editing in the H15N14.1 deletion strain.  These results suggest that the two C. elegans ADARs cooperate to produce the wild-type editing pattern.  We hope that  correlation of mutant phenotypes with in vivo editing patterns will reveal specific functions for C. elegans ADARs. 


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