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.
Associate Staff Scientist
Yusuke Shiromoto, Ph.D.
Moeko Minakuchi, Ph.D.
Visiting Research Associate
YuChen Hwang, Ph.D.
ADAR1 FUNCTION IN REGULATION OF INTERFERON SIGNALING AND ITS RELEVANCE TO AICARDI-GOUTIÈRES SYNDROME AUTOIMMUNE DISEASE
Adenosine deaminase acting on RNA (ADAR) is the enzyme involved in adenosine-to-inosine (A-to-I) RNA editing and three ADAR gene family members (ADAR1, ADAR2, and ADAR3) have been identified in mammals. Research efforts of the laboratory focus on biological functions of ADAR1 and human diseases caused by their deficiency.
There are two ADAR1 isoforms, an interferon-inducible full length p150 and a shorter N-terminus truncated p110. The cytoplasmic ADAR1p150 edits 3’UTR double-stranded RNAs primarily comprising inverted Alu repeats and thereby suppresses activation of the MDA5-MAVS-IFN signaling and induction of interferons. Loss of this ADAR1p150 function underlies the embryonic lethality of Adar1 null mice, pathogenesis of the severe autoimmune disease Aicardi-Goutières syndrome (AGS), and resistance to immune checkpoint blockade in cancer. Mutations of seven genes including ADAR1 (AGS6) have been identified in association with AGS and 10 AGS6 mutations of ADAR1 have been reported so far. Finally, this ADAR1p150-mediated suppression of IFN signaling also represses tumor responsiveness to immune checkpoint blockade, revealing the pro-oncogenic function of ADAR1p150. Analysis of TCGA cancer genome atlas database revealed elevated ADAR1 expression and A-to-I editing levels in almost all types of cancers, indicating that this pro-oncogenic ADAR1p150 function helps cancer cells suppress inflammatory responses and thus avoid host immunosurveillance.
We are currently conducting enzymatic analysis of 10 known ADAR1 AGS6 mutants in order to understand how each mutation affects the ADAR1 function that regulates the dsRNA sensing mechanism.
ADAR1 REGULATES R-LOOP FORMATION AND GENOME STABILITY AT TELOMERES IN CANCER CELLS
In contrast to the recent advance in knowledge of ADAR1p150 functions, the biological functions of the nuclear-localized ADAR1p110 have remained mostly unknown. Newly transcribed RNA usually dissociates from its template DNA strand immediately after transcription, but occasionally it forms a stable RNA:DNA hybrid, which consequently leaves the sense DNA in a single-stranded form. This structure, called an R-loop, causes genome instability. Human diseases including amyotrophic lateral sclerosis type 4 (ALS4), ataxia-ocular apraxia type 2 (AOA2) and Aicardi-Goutières syndrome (AGS), are caused by accumulation of R-loops. We recently discovered that ADAR1p110 regulates R-loop formation and genome stability at telomeres in cancer cells carrying non-canonical variants of telomeric repeats. ADAR1p110 edits the A-C mismatches within RNA:DNA hybrids formed between canonical and non-canonical variant repeats, converting them to I:C matched pairs, which facilitates resolution of telomeric R-loops by RNase H2. This ADAR1p110-dependent control of telomeric R-loops is required for continued proliferation of telomerase-reactivated (non-ALT) cancer cells but not primary fibroblast cells, revealing the pro-oncogenic nature of ADAR1p110 and identifying ADAR1 as a promising therapeutic target of telomerase-positive cancers.
STRESS RESPONSE FUNCTIONS OF ADAR1 AND THEIR RELEVANCE TO CANCER
Although the ADAR1p110 isoform usually localizes in the nucleus, recent studies in our lab revealed that ADAR1p110 moves to the cytoplasm in response to stress such as UV irradiation and heat shock. We 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. Our 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. ADAR1p110 promotes survival of stressed cells, therefore ADAR1 may be considered to be pro-oncogenic. Ongoing studies aim at defining the relevance of the ADAR1 stress response functions to cancer.
ADAR1 STIMULATES DICER FUNCTIONS AND REGULATES RNA INTERFERENCE
The laboratory has shown a new function of ADAR1 in the RNAi mechanism. ADAR1 forms a heterodimer complex with Dicer, facilitates the pre-miRNA dicing reaction and promotes RISC loading of miRNA, revealing the presence of a stimulative interaction of the RNA editing and the RNAi machineries. In addition to miRNA synthesis, DICER is involved in processing of long dsRNAs into small RNAs (endo-siRNAs). Generation of retrotransposon-derived endo-siRNAs by DICER and their functions in regulation of transcripts in mouse oocytes has been previously reported. However, the synthesis and functions of endo-siRNAs in somatic cells remain largely unknown. We recently discovered that ADAR1 together with DICER generates endogenous small RNAs, Alu endo-siRNAs by cleaving long double-stranded regions of inverted Alu repeats. We found that Alu endo-siRNAs derived from AluSz and AluJr family elements target CUB Domain Containing Protein 1 mRNAs containing an antisense copy of AluJb in their 3’UTRs and consequently induce apoptosis in HeLa cells. Our study revealed that ADAR1 could contribute to controlling the potential of long dsRNAs for induction of the MDA5-MAVS-IFN pathway in two ways: introducing extensive A-to-I editing into dsRNAs and thereby suppressing MDA5 binding in differentiated somatic cells; or promoting the DICER activity and processing dsRNAs to siRNAs in oocytes and embryonic stem cells where the IFN pathway is absent.
Shiromoto, Y., Sakurai, M., Qu, H., Kossenkov, A., Nishikura, K. "Processing of Alu small RNAs by DICER/ADAR1 complexes and their RNAi targets." RNA. 2020 Aug 17;rna.076745.120. doi: 10.1261/rna.076745.120.
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.
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.