The seeming vagaries of the transcriptome may become more explicable, thanks to validation efforts that are distinguishing true RNA editing events from artifacts. One such effort, made by researchers centered at Brown University, involved the painstaking, genome-wide validation of 1,799 RNA editing sites in Drosophila melanogaster, plus the prediction of another 1,782 sites. The result: a master list of 3,581 sites in which adenosine-to-inosine (A-to-I) RNA editing is accomplished by adenosine deaminases that act on RNA (ADAR).
The researchers used the tried-and-true, decades-old Sanger method of sequencing to double-check all the candidate editing sites that they had found using the high-throughput technology called single-molecule sequencing. They compared the sequenced RNA of a population of fruit flies to their sequenced DNA and to the RNA of another population of flies engineered to lack the ADAR editing enzyme. By comparing these three sequences they were able to see the RNA editing changes that could not be attributed to anomalies in DNA (that is, mutations, or single-nucleotide polymorphisms) and that never occurred in flies incapable of editing.
The researcher team published its results in Nature Structural & Molecular Biology on September 29. The team’s paper, “Genome-wide analysis of A-to-I RNA editing by single-molecule sequencing in Drosophila,” presents a discovery pipeline that has demonstrated an accuracy of about 87%. According to the team, this accuracy is sufficient to reveal the global patterns underlying biological functions of RNA editing in their Drosophila model.
“Drosophila serves as a model for all the organisms where people are studying transcriptomes,” said the paper’s corresponding author Robert Reenan, Ph.D., professor of biology in the department of molecular biology, cell biology, and biochemistry at Brown. “But in the early days of RNA editing research, the catalog of these sites was determined completely by chance—people working on genes of interest would discover a site. The number of sites grew slowly.”
Several more recent attempts to catalog RNA editing sites have yielded larger catalogs, but those contained many errors (the paper provides a comparison between the new list and previous efforts such as ModENCODE).
“For anyone who wants to do the same experiment under the same conditions, the sites [that we validated] should be there,” said co-author and postdoctoral researcher Yiannis Savva, Ph.D. “In other papers, they just did sequencing to say there is an editing site there, but when you check, it's not there.”
When they examined the implications of their results, the research team arrived at several insights. One was that a considerable amount of editing occurs in sections of RNA that do not code for making proteins. Editing is concentrated in a small number of RNAs, raising the question of what accounts for that selectivity.
Where editing is found, the researchers discovered, there is usually more alternative splicing, which means the body is more often assembling a different recipe from its genetic instructions to make certain proteins.
The researchers also found that the RNAs that are most heavily edited tend to be expressed to a lesser extent, decreasing how often they are put into action in the body.
RNA editing helps explain why organisms are even more different from each other—and from themselves at different times—than DNA differences alone would suggest. “RNA editing has emerged as a way to diversify not just the proteome but the transcriptome overall,” Dr. Reenan said.
Although the editing changes considered in the study may seem subtle, they are significant because they change how genetic instructions in DNA are put into action in the fly body, affecting many fundamental functions including proper neural and gender development. In humans, perturbed RNA editing has been strongly implicated in the diseases ALS and Acardi-Gutieres disease.