Dr Mary O'Connell: Chromosomes and Gene Expression
Nucleic Acid Editing:
From Germline to the CNS
Summary
Rob Benne first coined the term RNA editing in 1986 when he found four nucleotides in the coxII transcript from trypanosome mitochondria that were not encoded in the DNA. Since then other types of RNA editing have been found. The conversion of adenosine to inosine is the major type of RNA editing found in mammals; it is very widespread and is present in all metazoans. More recently DNA editing has been discovered and the enzymes involved are important for generating the mature immune system and for the innate immune response.
Approach, Progress and Future Work
At a biochemical level we are analysing ADAR proteins from both vertebrates and Drosophila and the pre-mRNAs that they edit to elucidate what controls the specificity of this editing reaction. We make extensive use of the yeast Pichia pastoris for ADAR protein production. Mammalian cell culture systems are being used to study sub-cellular localization of ADARs, interactions with substrates and interactions between RNA editing and RNA interference.
RNA editing in Drosophila
To take advantage of the power of genetics as well as biochemistry we are studying RNA editing in Drosophila melanogaster One major advantage is that Drosophila melanogaster contains only a single Adar gene. Null alleles of the gene were generated by our collaborator Robert Reenan. This was the first published example of the generation of an organism completely lacking ADAR activity. Mutant flies underwent normal development, were morphologically wild-type and had near normal life-spans. Mutant flies exhibit extreme behavioural deficits that include locomotor uncoordination and mating defects. A video shows the Adar 1F4 mutant flies on the left. Notice the severe leg tremors of the mutant flies. Flies rescued by driving ADAR expression using the GAL4-UAS system in the middle panel and the wild-type flies are on the right. These phenotypes are also accompanied by age-dependent neurodegeneration. Frontal sections through aged adult heads reveal large lesions in the brain and the retina is disorganised. These results suggest that the main targets of A to I editing in Drosophila are expressed in the CNS. One of the long-term goals of this project is to rescue both the locomotion and neurodegenerative phenotypes of the null flies by injecting into flies the edited version of the pre-mRNA encoding the receptor that underlies this defect. We also rescue Adar mutant flies by expressing human ADARs and use this system to study disease-relevant aspects of human ADAR function.
3-D Imaging of Drosophila
Whole adult Drosophila can be bleached and mounted in Murray’s Clear to produce a transparent object suitable for imaging by Optical Projection Tomography (OPT), or by confocal microscopy. This was published in PLoS ONE. 2007 Sep 5;2(9):e834. The lab member that was involved in this work is Leeanne McGurk.
Virtual Section of cha>GFP Flies
Collaborations and Funding
- Robert Reenan Brown University, USA.
- Angela Gallo Ospedale Pediatrico 'Bambino Gesù', Rome, Italy.
- Joshua Rosenthal Institute of Neurobiology, University of Puerto Rico.
Lab Members
Current lab members involved in this work are:
- Dr Mary O'Connell (Principal Investigator)
- James Brindle (Research Assistant)
- Dr Liam Keegan (Scientist Investigator)
- Dr Hui Sun (Post Doctoral Fellow)
- Simona Paro (Graduate Student)
- Nucleic Acid Editing: From Germline to the CNS (this page)
- Detailed Publication Listings
- OPT Videos
- Download Quicktime
- Download Flash Player
RNA editing is defined as an alteration in the coding capacity of mRNA other than splicing or 3'-processing. The conversion of adenosine to inosine is by hydrolytic deamination that occurs in pre-mRNA. Inosine is read as if it were guanosine by the translational machinery, therefore editing often results in the incorporation of a different amino acid at the edited position. By changing the information content of mRNA, RNA editing generates increased diversity in proteins. Most of the transcripts that are recoded by editing are expressed in the CNS. This preference for recoding CNS transcripts is widespread, and is observed in both vertebrates and invertebrates.
Recoding can have major physiological affects on the function of the protein as for example editing of the pre-mRNA encoding the glutamate-gated ion channel receptor B subunit (GluR-B) at the Q/R sites renders the glutamate receptor impermeable to Ca2+ ions, which is necessary for the function of the receptor. It also controls the trafficking of the subunit through the EM. RNA editing has also been implicated in neurological disorders in particular motorneuron diseases such as ALS and this one of our research topics.
Various group found that Alu transcripts are highly edited and that the high content of inosine found in the mammal transcripts is due to editing repetitive elements. However even though this is very common, the biological relevance of this has yet to be established.
ADAR1 and ADAR2 are members of a family of adenosine deaminases that catalyse this type of RNA editing in double-stranded (ds) RNA. Peter Seeburg’s group generated mice that are null for ADAR2 activity. A transgenic mouse lacking ADAR2 is heterozygous viable and normal. Homozygous mice have apparently normal embryonic development but die during or soon after weaning and are prone to seizures. ADAR2 is the enzyme responsible for editing the Q/R site of GluR-B pre-mRNA. In the ADAR2 knockout, the edited form of GluR-B(R) was generated whereby an arginine residue was genomically encoded rather than introduced by editing. This bypass of the editing requirement at this position, rescued the phenotype of the ADAR2 knockout and showed that the most important transcript that is edited by ADAR2 is that encoding GluR-B. Different groups have also generated ADAR1 deficient mice. The knockout of ADAR1 is homozygous lethal at day E12.5 but the pre-mRNA that is edited by ADAR1 that causes this lethality is unknown.
There are two other members of the ADAR family in mammals, ADAR3 and Tenr. ADAR3 is expressed in the brain, is evolutionary well conserved whereas Tenr is expressed in the testis and mice lacking it are infertile with problems in spermatogenesis. No substrate for either of these two enzymes has been found so far. In addition there is now growing evidence that ADARs play a role in the RNA interference (RNAi) pathway though the exact mechanism has yet to be elucidated. This is also one of the research topics in the group.
H.E. Staining of wt Drosophila Sections
Purpose
The aim of our work is to determine what is the biological role of RNA editing. We want to elucidate how the ADAR enzymes identify the specific adenosine that is edited. We want to determine what is the role of ADAR1, is it an RNA editing enzyme or is its major function in the RNAi pathway? We also want to determine if a decrease in editing by ADAR2 at the Q/R site in GluR-B contributes to ALS. Drosophila that are null for Adar display an ataxic phenotype with problems in locomotion (see movie clip) and have age-related neurodegeneration. We are investigating what type of neurodegeneration this is a potential model for human neurodegenerative diseases. Finally we want to determine what is the role of ADAR3 in the brain and Tenr in the testis.