Kazuko Nishikura, Ph.D.

Professor
Gene Expression and Regulation Program
215-898-3828, Office
kazuko@wistar.org

Introduction

RNA carries the genetic instructions of DNA to the cellular machinery responsible for synthesizing the proteins of the body. The Nishikura laboratory is exploring the phenomenon of RNA editing, a process by which a single gene can produce a number of closely related but distinct proteins. Recent studies suggest that disruptions in RNA editing may play a role in certain forms of depression and schizophrenia.

Research Summary

The editing of RNA prior to protein synthesis plays a critical role in the expression of certain gene products. One type of RNA editing involves the conversion of the nucleic acid bases adenosine into inosine. Members of the ADAR (adenosine deaminases acting on RNA) gene family are involved in this type of RNA editing. Three ADAR gene family members have been identified in humans and rodents by the Nishikura laboratory. The research focus of the laboratory is to better understand the functions of ADAR and the cellular processes regulated by double-stranded RNA and to identify possible human diseases caused by malfunction of these processes. Another research goal is ascertain the in vivo significance of RNA editing in the pathophysiology of psychiatric disorders such as depression and schizophrenia. Recent studies by the Nishikura laboratory and others indicate that G-protein coupling functions of serotonin 2C receptors (5-HT2CR) are dramatically altered by RNA editing. These studies raised the possibility that RNA editing of 5-HT2CR may play a role in certain human neuropsychiatric disorders. Despite their desired and profound effects on the immune system, interferons, particularly the alpha subtype, cause adverse effects, including depression. Using a human glioblastoma cell line expressing endogenous 5-HT2C receptors, the researchers have recently discovered significant alterations in the pattern of 5-HT2CR RNA editing in response to interferon treatment. Results support the hypothesis that cytokines may induce depression at least in part through their effects on the editing of 5-HT2CR RNA. In order to better understand biological functions of ADAR in vivo, the laboratory has also been analyzing mice with a mutation of the ADAR1 gene. The laboratory's current efforts focus on identifying ADAR1 target genes critical for development.

Recent Scientific Advances

RNA Editing Enzyme in Development and Disease: RNA editing plays a critical role in the expression of certain gene products by changing the sequence context of mRNAs, which results in synthesis of proteins not encoded in the gene sequence. One type of RNA editing involves the conversion of adenosine residues into inosine. Members of the ADAR (adenosine deaminases acting on RNA) gene family are involved in RNA editing that converts adenosine residues into inosine (A-to-I). This A-to-I RNA editing mediated by ADAR is essential for determining physiological properties such as calcium permeability or a receptor's sensitivity to a ligand of the targeted genes such as glutamate receptor channels and serotonin 2C receptors (5-HT2CR). ADAR may also be involved in other processes such as control of repetitive sequences and dsRNA (double-stranded RNA) mediated gene silencing (ref. 5, 6). Three separate ADAR gene family members (ADAR1-3) have been identified in humans and rodents(ref. 2, 6). The research focus of the Nishikura laboratory is to better understand the functions of ADAR and the cellular processes regulated by dsRNA and to identify possible human diseases caused by malfunction of these processes.

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. Recent studies by this laboratory revealed that ADAR1 null mutant mouse embryos died at midgestation with defects in the erythropoietic system, possibly due to deficiency in the metabolism of certain classes of dsRNA. Conditional inactivation of the ADAR1 gene will allow one to address specific questions with regard to the role of ADAR1. Accordingly, the laboratory has recently created a new floxed ADAR1-3LoxP allele in R1 ES cells using the LoxP/Cre system. These mice harboring the ADAR1^flox allele have been crossed with several Cre mouse lines. Despite previous difficulty in establishing the ADAR1^+/- heterozygous mouse line via chimeric mice, cross of the floxed ADAR1 mouse and the EIIa-Cre deletor mouse line generated ADAR1^wt/del (ADAR1^+/-) heterozygous mouse lines via in vivo deletion of the ADAR1 C-terminus by Cre recombinase. However, no ADAR1^del/del homozygote null mice were found among the progeny of ADAR1^wt/del intercrosses, and staged embryos were analyzed. The expected ratio of live wild-type, heterozygous, and homozygous embryos were recovered at E10.5 and E11.0. However, a large proportion of ADAR1^del/del (-/-) homozygote embryos die around E11.5-12.5. The severe anemia of the ADAR1^del/delhomozygote embryos was obvious by their very pale appearance of embryos collected live at E11.0. These findings together with previous studies on chimeric embryos indicate that abnormal proliferation and/or differentiation of erythroid cells may be caused by underediting of the RNA of currently unknown ADAR1 target gene(s). 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 (ref 3).

The research team has also investigated the interaction of ADAR proteins with other cellular proteins. Several specific ADAR interacting proteins such as spliceosome components Sm and SR proteins involved in splicing have been identified. It has been speculated that ADARs may form a large multicomponent protein complex. Possible candidates for such complexes are large nuclear ribonucleoprotein (lnRNP) particles. In collaboration with Dr. Joseph Sperling of the Weizmann Institute of Science and Dr. Ruth Sperling of the Hebrew University, the Nishikura team has investigated the association of ADAR proteins and lnRNP particles as physiological supramolecular complexes. The presence of ADARs in lnRNP particles was investigated by Western blot analysis using anti-ADAR antibodies and by indirect immunoprecipitation. Both ADAR1 and ADAR2 were found associated with the spliceosomal components Sm and SR proteins within the lnRNP particles. More recently the researchers have identified additional ADAR associated proteins, and are currently investigating their functional significance with regard to ADAR functions (ref 4). Current efforts are being made to understand the functional significance of various ADAR complexes.

Another research goal is to better understand the in vivo significance of 5-HT2CR RNA editing in the pathophysiology of psychiatric disorders such as depression and schizophrenia. Recent studies from the Nishikura laboratory and others indicate that G-protein coupling functions of 5-HT2CR are dramatically altered by RNA editing, which converts five adenosine residues to inosine, all located within the intracellular loop II of the receptor. Combinatorial and regionally uneven editing of the five sites resulted in the brain region-specific expression of 5-HT2CR isoforms carrying different amino acid residues at positions 156, 158, and 160. Pharmacological characterization revealed that the replacement of the gene-encoded Asn with Ser or Gly at position 158 results in a dramatic decrease of the basal level G-protein coupling activity, agonist affinity, and 5-HT potency (ref# 1). These previous studies raised the possibility that RNA editing of 5-HT2CR may have some causative relevance to certain human neuropsychiatric disorders. Interestingly, recombinant interferons have been used clinically for the treatment of chronic viral hepatitis, certain malignancies, and multiple sclerosis. Despite their desired and profound effects on the immune system, these cytokines, particularly the alpha subtype, cause adverse effects including depression. Using a human glioblastoma cell line expressing endogenous 5-HT2C receptors, the researchers have recently discovered significant alterations in the pattern of 5-HT2CR RNA editing in response to the interferon treatment. Results support the hypothesis that cytokines may induce depression at least in part through their effects on the editing of 5-HT2CR RNA.

Selected Publications

1) Wang, Q., Chen, C-X., Cho, D-S.C., O'Brien, P., Murray, J.M., and Nishikura, K. 2000. Altered G-protein coupling function of RNA editing and splicing variant serotonin 2C receptors. J. Neurochem. 74: 1290-1300.

2) Chen, C-X., Cho, D-S.C., Wang, Q., Lai, F., Carter, K.C., and Nishikura, K. 2000. A third member of the RNA-specific adenosine deaminase gene family, ADAR3, contains both single- and double-stranded RNA binding domains. RNA 6: 755-767.

3) 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.

4)Raitzkin, O., Cho, D-S., Sperling, J., Nishikura, K., and Sperling, R. 2001. RNA editing activity is associated with lnRNP particles : the nuclear pre-mRNA processing machinery. Proc. Natl. Acad. Sci. USA. 98: 6571-6576.

5) Nishikura, K. 2001. A short primer on RNAi: RNA-directed RNA polymerase acts as a key catalyst. Cell 107: 415-418.

6)Maas, M., Rich, A., and Nishikura, K. 2003. A-to-I RNA Editing: Recent news and residual mysteries. J. Biol. Chem. 278: 1391-1394.