Kazuko Nishikura, Ph.D.
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 (7, 8). 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 (7, 8). 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 (10, 11). Studies by this laboratory revealed that ADAR1 null mutant mouse embryos died at midgestation due to massive and widespread apoptosis (10, 11). 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 (10, 11). Current efforts are focused on defining the molecular mechanism underlying the embryonic lethal phenotype of ADAR1 null mutant mice.
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 RISC (RNA-induced silencing complex), miRNAs block the translation of partially complementary targets located in the 3' UTR of specific mRNAs or guide the degradation of target mRNAs (1, 7, 8). 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.
Regulation of the host RNAi mechanism 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 RNA-induced silencing complex (RISC). A-to-I editing appears to be an adaptive mechanism that antagonizes miR-BART6 activities. Most importantly, miR-BART6 silences Dicer through multiple target sites located in the 3'UTR of Dicer mRNA. On-going studies are aimed to better understand functions of miR-BART6 RNAs and the role played by A-to-I RNA editing in the control of the host RNAi mecjanism.
ADAR1 stimulates Dicer functions in miRNA processing and RISC assembly
Although Dicer alone can cleave pre-miRNA to mature miRNA, its catalytic activity is modulated by TRBP and PACT (1). Studies of the laboratory revealed that ADAR1 forms a complex with Dicer and promotes Dicer activity. Michaelis-Menten plot analyses of steady-state enzyme kinetics data revealed that ADAR1 increases the Vmax of the Dicer cleavage of pre-miRNA by 4-fold in comparison to the reaction conducted with Dicer alone (9). ADAR1 appears to function in augmentation of Dicer cleavage of pre-miRNA, RISC assembly, and loading of miRNA (9). Next generation sequencing of small RNA confirmed global suppression of miRNA synthesis in ADAR1-null mouse embryos, which die around E12 (9). Together, these studies indicate that ADAR1 is required for promotion of rapid increase of miRNA production globally around E11–12, which is likely essential for embryo development (9). In ADAR1-null embryos, the rapid and significant upregulation of miRNA production cannot occur because of the lack of formation of the Dicer-ADAR1 complex. This seems to result in dysregulated expression of many genes, including those involved in cell death, cell proliferation, and organogenesis. Deficiency in the RNAi function rather than the RNA editing function of ADAR1 may underlie the apoptosis-prone and embryonic lethal phenotypes of ADAR1-null mice (9).
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 (3). 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 (3).
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.