The Influence of RNA Editing on miRNA Processing and Gene Regulation

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Brent Shepherd and Dr. Evan Johnson, Statistics

The emergence of high-throughput DNA sequencing within the past decade has redefined the world of molecular research. Mapping a human genome, a project that cost the United States government almost $3 billion and took 13 years to complete, is now speculated to soon be less than a week-long procedure costing $1000 or less1,2. With high-throughput DNA sequencing machines, we can rapidly process genomic data for relatively cheap amounts of money. As a result, genomic information is becoming more widely available for many different species. In addition to DNA, high-throughput machines can also be used to sequence other nucleotide-based molecules, including small RNA.

The University of Utah Brenda Bass Lab in the department of biochemistry used high-throughput sequencing methods to obtain the sequences of millions of small RNAs ranging from 15 to 30 nucleotides in length. These small RNAs all came from the nematode worm Caenorhabditis Elegans (C. Elegans), and are separated into two different categories. The first group of RNAs came from a wild-type worm. The second group of RNAs came from worms having a mutant allele that inhibits RNA editing by the ADAR enzyme. Using these two groups of small RNAs, our goal was to determine computationally the impact of RNA editing on the production of microRNA (miRNA) molecules. We also wanted to know which miRNAs that RNA editing affects most. We aimed to determine this using an asymptotic multinomial approximation combined with a likelihood ratio test.

In RNA editing, certain RNA sequences are altered by special enzymes after being transcribed from the original DNA template. These alterations include deletions, insertions, and base modifications. RNA editing has been found exclusively in eukaryotes, and its deregulation has been linked in humans to many diseases of the central nervous system, including depression, epilepsy, schizophrenia and others3. Adenosine deaminase enzymes acting on RNA (ADAR) account for a large proportion of RNA editing. ADAR enzymes target specific adenine bases and chemically alter them into inosine, which acts functionally identical to a guanine base during translation. Because of this alteration often an entirely new protein results with a completely different function. A worm that is lacking these particular enzymes we shall call ADAR worms. In the mutant ADAR C. Elegans, the genes that code for ADAR enzyme production within the cell do not get expressed, thus suppressing RNA editing.

RNA editing has recently been shown to cause alterations in the processing of miRNAs4. MiRNAs are short RNA sequences about 21 to 23 nucleotides in length. Since the recent discovery of miRNA, they have already been shown to be vital to the cell in helping regulate gene expression. A miRNA, when formed, will attach to a complementary mRNA sequence and inhibit translation.

Though RNA editing has been shown to affect miRNA, the experiments have been carried out on only a handful of the many miRNAs known. We proceeded to answer the large-scale effects of RNA editing on miRNA processing computationally. We hypothesized that by comparing the wild-type and mutant worms and the proportions of each type’s small RNAs that map to known miRNAs, we could determine computationally the RNA editing site locations on miRNAs. We also planned to target the specific adenine bases to which these edits occurred.

After writing, altering, correcting, and re-correcting numerous coding scripts, the results we obtained were quite surprising. We found that in the nematode worm C. Elegans, little, if any, RNA editing takes place within the miRNA locations. Only one adenine base showed a significant editing percentage, much lower than our original estimate. We repeated this procedure on different C. Elegans wild-type and ADAR mutant datasets, and the results were the same. Only one location showed significant editing.

Because no official editing results at these locations had ever been analyzed, we tested the functionality of our code at another location within the C. Elegans genome that had been shown to have significant amounts of RNA editing. Our results caught various editing sites, and indicated that our code was, in fact, working properly. This information further validated our conclusions made previously regarding the miRNAs.

Though this information was interesting, it has proved to be only the beginning. Since drawing these conclusions, we have been working closely with the University of Utah to further evaluate RNA editing effects on different types of RNA molecules within the C. Elegans nematode at different genomic locations. We are also using larger datasets of small RNAs they have collected to strengthen the validity of our results. Also, now that we are quite confident with our methodology and our coding pipeline, we plan to do these same analyses across the entire human genome and look for editing sites there.

References

  • Kahvejian A., Quackenbush J., Thompson J.F. What would you do if you could sequence everything? Nat. Biotechnol. 26 1125-1133 (2008).
  • Schloss J.A. How to get genomes at one ten-thousandth the cost. Nat. Biotechnol. 26 1113-1115 (2008).
  • Kwak S., Kawahara Y. Deficient RNA editing of GluR2 and neuronal death in amyotropic lateral sclerosis. J Mol Med 83 110-120 (2005).
  • Yang W., et al. Modulation of microRNA processing and expression through RNA editing by ADAR deaminases. Nat Struct Biol 13 13-21 (2006).