Research
The editing of RNA plays a critical role in the expression of certain gene products by changing the sequence context of mRNAs. One type of RNA editing involves the conversion of adenosine residues into inosine specifically in double-stranded RNA (dsRNA) (7, 8). This A-to-I RNA editing is catalyzed by members of the ADAR (adenosine deaminases acting on RNA) gene family. When A-to-I RNA editing occurs within a coding sequence, synthesis of proteins not encoded by the genome and consequent alteration of their functions can result, as demonstrated with transcripts of glutamate receptor (GluR) ion channels and 5-HT2C serotonin receptors, both of which play important roles in brain functions.
In addition, A-to-I RNA editing is involved in control of the expression and function of microRNA (miRNA), which is a small RNA essential for the RNAi mediated gene silencing mechanism. The research focus of the laboratory is to better understand the functions of ADAR and the cellular processes regulated by A-to-I RNA editing and to identify possible human diseases caused by malfunction of these processes.
Recent Scientific Advances
Embryonic lethality of ADAR1 null mice caused by massive apoptosis: Three separate ADAR gene family members (ADAR1-3) have been identified in humans and rodents. In order to better understand biological functions of ADAR in vivo, the laboratory has been analyzing phenotypes of mice with a mutation of the ADAR1 gene (9, 10). Studies by this laboratory revealed that ADAR1 null mutant mouse embryos died at midgestation due to massive and widespread apoptosis (10). Examination of liver-specific ADAR1 null mouse livers revealed that many hepatocytes undergo apoptosis. In addition, fibroblast cells derived from ADAR1 null embryos were found to be prone to apoptosis when subjected to serum deprivation.
Taken together, the results imply that ADAR1 functions to promote survival of numerous tissues by editing a currently unknown dsRNA molecule(s) required for protection against stress-induced apoptosis. Close examination of dying embryos also revealed defects in the erythropoietic system as well as malformation of vital organs such as liver and heart, indicating a possibility of the ADAR1 involvement in human congenital diseases affecting these organs. Current efforts are focused on defining the molecular mechanism underlying the embryonic lethal phenotype of ADAR1 null mutant mice and to identify the ADAR1 target dsRNA critical for development.
Modulation of miRNA expression and function by A-to-I RNA editing: Primary transcripts of miRNA genes (pri-miRNAs) are processed sequentially by Drosha and Dicer. Nuclear Drosha cleaves pri-miRNAs, releasing 60- to 70-nt pre-miRNAs. Recognition of correctly processed pre-miRNAs and their nuclear export is carried out by Exportin-5 and RanGTP. Cytoplasmic Dicer then processes pre-miRNAs into 20- to 22-nt mature miRNAs. Following integration into miRISC, miRNAs block the translation of partially complementary targets located in the 3' UTR of specific mRNAs or guide the degradation of target mRNAs (1). Recent studies of the laboratory revealed that primary transcripts of miRNA are subject to A-to-I RNA editing and demonstrated that miRNA editing results in inhibition of miRNA processing pathway at the Drosha or Dicer cleavage steps (5, 12) or leads to expression of edited mature miRNAs that silence genes different from those targeted by unedited miRNAs (6). The findings revealed a previously unknown role for A-to-I RNA editing in the control of miRNA biogenesis as well as the miRNA-mediated gene silencing mechanism. Global survey of human and mouse pri-miRNA editing sites revealed that approximately 20% of mammalian pri-miRNAs are subject to A-to-I RNA editing (4).
It is tempting to speculate that the apoptosis-prone phenotype of ADAR1 null mouse embryos may be a manifestation of deficiencies in this particular ADAR1 function of controlling the generation of miRNAs. Comparative analysis of high throughput RNA sequencing data obtained with a set of wildtype and ADAR1 null mouse embryos is currently under way in attempt of identifying the critical dsRNA.
Regulation of viral replication and latency by editing of EBV miR-BART6 RNAs: Epstein-Barr Virus (EBV) is associated with a variety of human cancers such as Burkitt's lymphoma, Hodgkin's disease, and nasopharyngeal carcinoma. The EBV genome encodes multiple miRNA genes of its own. Recent studies of the laboratory revealed that primary transcripts of ebv-miR-BART6 (pri-miR-BART6) are edited by ADAR1 in latently EBV-infected cells (2). Editing dramatically reduced expression and loading of miR-BART6 onto the miRNA-induced silencing complex (miRISC). A-to-I editing appear to be an adaptive mechanism that antagonize miR-BART6 activities. Most importantly, miR-BART6 silences Dicer through multiple target sites located in the 3'UTR of Dicer mRNA. Finally, it was found that miR-BART6 RNAs suppress the expression of several EBV genes essential for control of lytic replication and latency (2).
On-going stuies are aimed to better understad functions of miR-BART6 RNAs and the role played by A-to-I RNA editing in EBV infection and latency.
Role of 5-HT2c receptor mRNA editing in fat and glucose metabolism: 5-HT2CR is a G-protein coupling receptor detected exclusively in the central nervous system and plays roles in various physiological and behavioral processes. Five A-to-I RNA editing sites were identified in 5-HT2CR pre-mRNAs (11). Combinatorial editing at the five sites would change three amino acids Ile156 (I), Asn158 (N), and Ile160 (I) to Val (V), Gly (G), and Val (V), respectively. The regionally uneven editing efficiency of five sites results in the region-specific expression of 24 different 5-HT2CR isoforms carrying different amino acid residues at positions 156, 158, and 160. RNA editing of 5-HT2CR mRNAs significantly alters the G-protein coupling functions of the receptor (11).
To evaluate the significance of 5-HT2CR mRNA editing in vivo, a mutant mouse line, VGV, which resulted in sole expression of the fully edited VGV receptor, has been created in the Nishikura laboratory (3). VGV mice had a severely reduced fat mass, in spite of compensatory hyperphagia, due to dramatic increase in energy expenditure. In addition, VGV mice have reduced insulin levels and increased greater glucose tolerance. These findings provided the first direct evidence that editing of 5-HT2CR miRNAs plays a critical role in the regulation of energy expenditure and lipid and glucose metabolism (3). VGV mice are likely to provide a useful animal model for studies on metabolic rate, diabetes, and obesity regulated via A-to-I editing of 5-HT2CR mRNA. Furthermore, editing of HT2CR mRNA can be a future target of great promise for development of drugs to control obesity.
1. Chendrimada TP, Gregory RI, Kumaraswamy E, Norman J, Cooch N, Nishikura K and Shiekhattar R. 2005. TRBP recruits the Dicer complex to Ago2 for microRNA processing and gene silencing. Nature 436: 740-744.
2. Iizasa H, Wulff B-E, Alla NR, Maragkakis M, Megraw M, Hatzigeorgiou A, Iwakiri D, Takada K, Wiedmer A, Showe L, Lieberman P and Nishikura K. 2010. Editing of Epstein-Barr virus-encoded BART6 microRNAs controls their dicer targeting and consequently affects viral latency. J Biol Chem 285: 33358-33370.
3. Kawahara Y, Grimberg A, Teegarden S, Mombereau C, Liu S, Bale TL, Blendy JA and Nishikura K. 2008. Dysregulated editing of serotonin 2C receptor mRNAs results in energy dissipation and loss of fat mass. J Neurosci 28: 12834-12844.
4. Kawahara Y, Megraw M, Kreider E, Iizasa H, Valente L, Hatzigeorgiou AG and Nishikura K. 2008. Frequency and fate of microRNA editing in human brain. Nucleic Acids Res 36: 5270-5280.
5. Kawahara Y, Zinshteyn B, Chendrimada TP, Shiekhattar R and Nishikura K. 2007. RNA editing of microRNA-151 blocks cleavage by the Dicer-TRBP complex. EMBO Reports 8: 763-769.
6. Kawahara Y, Zinshteyn B, Sethupathy P, Iizasa H, Hatzigeorgiou AG and Nishikura K. 2007. Redirection of silencing targets by adenosine-to-inosine editing of miRNAs. Science 315: 1137-1140.
7. Nishikura K. 2006. Editor meets silencer: crosstalk between RNA editing and RNA interference. Nat Rev Mol Cell Biol 7: 919-931.
8. Nishikura K. 2010. Functions and regulation of RNA editing by ADAR deaminases. Annu Rev Biochem 79: 321-349.
9. Wang Q, Khillan J, Gadue P and Nishikura K. 2000. Requirement of the RNA editing deaminase ADAR1 gene for embryonic erythropoiesis. Science 290: 1765-1768.
10. Wang Q, Miyakoda M, Yang W, Khillan J, Stachura DL, Weiss MJ and Nishikura K. 2004. Stress-induced apoptosis associated with null mutation of ADAR1 RNA editing deaminase gene. J Biol Chem 279: 4952-4961.
11. Wang Q, O'Brien PJ, Chen CX, Cho DS, Murray JM and Nishikura K. 2000. Altered G protein-coupling functions of RNA editing isoform and splicing variant serotonin2C receptors. J Neurochem 74: 1290-1300.
12. Yang W, Chendrimada TP, Wang Q, Higuchi M, Seeburg PH, Shiekhattar R and Nishikura K. 2006. Modulation of microRNA processing and expression through RNA editing by ADAR deaminases. Nat Struct Mol Biol 13: 13-21.
