Lab In The News
Dr. Kazuko Nishikura: the RNA Explorer
Kazuko Nishikura has been a pillar of Wistar science for the past 37 years with a career overlapping with the rise and expansion of the RNA biology field, which explores the alternative functions of RNA in the cell, besides carrying the genetic information from DNA to proteins.
The Nishikura Laboratory
The Nishikura laboratory explores the phenomenon of RNA editing, which regulates 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). This A-to-I RNA editing is catalyzed by members of the ADAR (adenosine deaminases acting on RNA) gene family, discovered in the lab. 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 team has also investigated the interaction of ADAR proteins with other cellular proteins. In particular, they are interested in the tendency of ADAR to form large multipart protein complexes, and in the exact role these complexes play in the biology of the cell. For example, they discovered that ADAR1 forms a complex with Dicer to play a major role in the control of RNA interference mechanism. More recently, they also found that ADAR1 promotes survival of stressed cells by protecting a class of anti-apoptotic gene transcripts from Staufen1 mediated mRNA decay mechanism. Interestingly, this anti-apoptotic function of ADAR1 is regulated through its phosphorylation by MAP kinases.
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.
Moeko Minakuchi, Ph.D.
Yusuke Shiromoto, Ph.D.
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. There are two ADAR1 isoforms, an interferon inducible full length p150 and a shorter N-terminus truncated p110. In order to better understand biological functions of ADAR in vivo, the laboratory has analyzed the effects of ADAR1 gene mutations in mouse models. These studies revealed that ADAR1 null mutant mouse embryos dye at mid-gestation due to widespread apoptosis and overwhelming interferon responses caused by activation of the dsRNA sensing mechanism mediated by MDA5-MAVS signaling. ADAR1, specifically p150 isoform, suppresses this MDA5-MAVS-IFN signaling pathway by editing and masking a currently unknown dsRNA molecule(s), which, if remained unedited, triggers the dsRNA sensing mechanism. Recent studies indicate that deficiency in this particular function of ADAR1p150 results in Aicardi–Goutières syndrome (AGS), a severe human inflammatory disorder most typically affecting the brain and skin.
Current efforts are focused on defining the molecular mechanism underlying the embryonic lethal phenotype of ADAR1 null mutant mice and identification of the dsRNA(s), which triggers the MDA5-MAVS-IFN pathway.
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 from the laboratory revealed that primary transcripts of ebv-miR-BART6 (pri-miR-BART6) are edited by ADAR1 in latently EBV-infected cells. Editing dramatically reduced expression and loading of miR-BART6 into the 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. Interestingly, suppression of Dicer activities is reported to result in promotion of epithelial-mesenchymal transistion and metastatic transformation of cancer cells.
On-going studies are aimed at examining a hypothesis that A-to-I editing of miR-BART6 RNAs antagonizes the EBV viral strategy of manipulating the host cell RNAi mechanism and consequently promoting metastasis of certain cancers such as breast cancer.
ADAR1 stimulates Dicer functions in miRNA processing and RISC assembly
The laboratory has shown that ADAR1 forms a complex with Dicer and promotes its catalytic activity, increasing Dicer cleavage of pre-miRNA, RISC assembly, and loading of miRNA. Next generation sequencing of small RNA confirmed global suppression of miRNA and endogenous siRNA synthesis in ADAR1-null mouse embryos. These studies indicate that ADAR1 is required for promotion of rapid increase of miRNA synthesis during embryogenesis, when the expression of TRBP, a canonical partner of Dicer, remains low. In ADAR1-null embryos, the lack of formation of the Dicer-ADAR1 complex results in dysregulated expression of many genes involved in cell death, cell proliferation, and organogenesis, which is likely to contribute at least in part to their embryonic lethality.
Age-Related Macular Degeneration (AMD) is a leading cause of vision loss in elderly people. Accumulation of cytotoxic non-coding Alu repetitive RNAs in macular cells seems to underlie AMD pathology. Recent studies revealed that loss of Dicer function to process Alu RNAs to small fragmented RNAs (Alu-siRNAs) underlie the AMD pathology. On-going research of the Nishikura laboratory is to examine whether the Dicer promoting function of ADAR1 has any relevance to AMD.
Stress response functions or ADAR1 and their relevance to cancer
Although the ADAR1p110 isoform usually localizes in the nucleus, recent Nishikura laboratory studies revealed that ADAR1p110 moves to the cytoplasm in response to stress such as UV irradiation and heat shock. They discovered that stress-activated phosphorylation of ADAR1p110 by MKK6-p38-MSK MAP kinases promotes its binding to Exportin-5 and export from the nucleus. Once translocated to the cytoplasm, ADAR1p110 suppresses apoptosis of stressed cells by protecting many anti-apoptotic gene transcripts that contain 3’UTR dsRNA structures primarily made from inverted Alu repeats. ADAR1p110 competitively inhibits binding of Staufen1 to the 3’UTR dsRNAs and antagonizes the Staufen1-mediated mRNA decay. Their studies revealed a new stress response mechanism, in which human ADAR1p110 and Staufen1 regulate surveillance of a set of mRNAs required for survival of stressed cells.
Apoptosis is one way to inhibit transformation of stressed and DNA damaged cells to cancer
ADAR1p110 promotes survival of stressed cells. Thus, ADAR1 may be considered to be pro-oncogenic. Ongoing studies aim at defining the relevance of stress response functions of ADAR1 to cancer.
Tan, M.H., Li, Q., Shanmugam, R., Piskol, R., Kohler, J., Young, A.N., Liu, K.I., Zhang, R., Ramaswami, G., Ariyoshi, K., et al. "Dynamic landscape and regulation of RNA editing in mammals." Nature. 2017 Oct 11;550(7675):249-254. doi: 10.1038/nature24041.
Sakurai, M., Shiromoto, Y., Ota, H., Song, C., Kossenkov, A.V., Wickramasinghe, J., Showe, L.C., Skordalakes, E., Tang, H.Y., Speicher, D.W., et al. "ADAR1 controls apoptosis of stressed cells by inhibiting Staufen1-mediated mRNA decay." Nat Struct Mol Biol. 2017 Jun;24(6):534-543. doi: 10.1038/nsmb.3403. Epub 2017 Apr 24.
Song, C., Sakurai, M., Shiromoto, Y., Nishikura, K. "Functions of the RNA Editing Enzyme ADAR1 and Their Relevance to Human Diseases." Genes (Basel). 2016 Dec 17;7(12). pii: E129. doi: 10.3390/genes7120129.
Gumireddy, K., Li, A., Kossenkov, A.V., Sakurai, M., Yan, J., Li, Y., Xu, H., Wang, J., Zhang, P.J., Zhang, L., et al. "The mRNA-edited form of GABRA3 suppresses GABRA3-mediated Akt activation and breast cancer metastasis." Nat Commun. 2016 Feb 12;7:10715. doi: 10.1038/ncomms10715.
Nishikura, K. "A-to-I editing of coding and non-coding RNAs by ADARs." Nat Rev Mol Cell Biol. 2016 Feb;17(2):83-96. doi: 10.1038/nrm.2015.4. Epub 2015 Dec 9.
Yulia Nefedova, M.D., Ph.D.
Associate Professor, Immunology, Microenvironment & Metastasis Program, The Wistar Institute Cancer Center