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  • 1
    Publication Date: 2016-08-23
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
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  • 2
    Publication Date: 2020-03-23
    Description: In eukaryotic cells, with the exception of the specialized genomes of mitochondria and plastids, all genetic information is sequestered within the nucleus. This arrangement imposes constraints on how the information can be tailored for different cellular regions, particularly in cells with complex morphologies like neurons. Although messenger RNAs (mRNAs), and the proteins that they encode, can be differentially sorted between cellular regions, the information itself does not change. RNA editing by adenosine deamination can alter the genome’s blueprint by recoding mRNAs; however, this process too is thought to be restricted to the nucleus. In this work, we show that ADAR2 (adenosine deaminase that acts on RNA), an RNA editing enzyme, is expressed outside of the nucleus in squid neurons. Furthermore, purified axoplasm exhibits adenosine-to-inosine activity and can specifically edit adenosines in a known substrate. Finally, a transcriptome-wide analysis of RNA editing reveals that tens of thousands of editing sites (〉70% of all sites) are edited more extensively in the squid giant axon than in its cell bodies. These results indicate that within a neuron RNA editing can recode genetic information in a region-specific manner.
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
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  • 3
    Publication Date: 2022-05-25
    Description: © The Author(s), 2016. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Nucleic Acids Research 44 (2016): e157, doi:10.1093/nar/gkw738.
    Description: Site-directed RNA editing (SDRE) is a strategy to precisely alter genetic information within mRNAs. By linking the catalytic domain of the RNA editing enzyme ADAR to an antisense guide RNA, specific adenosines can be converted to inosines, biological mimics for guanosine. Previously, we showed that a genetically encoded iteration of SDRE could target adenosines expressed in human cells, but not efficiently. Here we developed a reporter assay to quantify editing, and used it to improve our strategy. By enhancing the linkage between ADAR's catalytic domain and the guide RNA, and by introducing a mutation in the catalytic domain, the efficiency of converting a UAG premature termination codon (PTC) to tryptophan (UGG) was improved from ∼11% to ∼70%. Other PTCs were edited, but less efficiently. Numerous off-target edits were identified in the targeted mRNA, but not in randomly selected endogenous messages. Off-target edits could be eliminated by reducing the amount of guide RNA with a reduction in on-target editing. The catalytic rate of SDRE was compared with those for human ADARs on various substrates and found to be within an order of magnitude of most. These data underscore the promise of site-directed RNA editing as a therapeutic or experimental tool.
    Description: National Institutes of Health [1R0111223855, 1R01NS64259]; Cystic Fibrosis Foundation Therapeutics [Rosent14XXO]; Infrastructural support was provided by the National Institutes of Health [NIGMS 1P20GM103642, NIMHD 8G12-MD007600]; National Science Foundation [DBI 0115825, DBI 1337284]; Department of Defense [52680-RT-ISP].
    Repository Name: Woods Hole Open Access Server
    Type: Article
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  • 4
    Publication Date: 2022-05-26
    Description: © The Author(s), 2020. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Vallecillo-Viejo, I. C., Liscovitch-Brauer, N., Diaz Quiroz, J. F., Montiel-Gonzalez, Maria F., Nemes, Sonya E., Rangan, K. J., Levinson, S. R., Eisenberg, E., & Rosenthal, J. J. C. Spatially regulated editing of genetic information within a neuron. Nucleic Acids Research, (2020): gkaa172, doi: 10.1093/nar/gkaa172.
    Description: In eukaryotic cells, with the exception of the specialized genomes of mitochondria and plastids, all genetic information is sequestered within the nucleus. This arrangement imposes constraints on how the information can be tailored for different cellular regions, particularly in cells with complex morphologies like neurons. Although messenger RNAs (mRNAs), and the proteins that they encode, can be differentially sorted between cellular regions, the information itself does not change. RNA editing by adenosine deamination can alter the genome’s blueprint by recoding mRNAs; however, this process too is thought to be restricted to the nucleus. In this work, we show that ADAR2 (adenosine deaminase that acts on RNA), an RNA editing enzyme, is expressed outside of the nucleus in squid neurons. Furthermore, purified axoplasm exhibits adenosine-to-inosine activity and can specifically edit adenosines in a known substrate. Finally, a transcriptome-wide analysis of RNA editing reveals that tens of thousands of editing sites (〉70% of all sites) are edited more extensively in the squid giant axon than in its cell bodies. These results indicate that within a neuron RNA editing can recode genetic information in a region-specific manner.
    Description: National Science Foundation (NSF) [IOS1557748 to J.R.]; United States–Israel Binational Science Foundation [BSF2013094 to J.R. and E.E.]; The Grass Foundation grant in support of the Doryteuthis pealeii Genome Project, and a gift by Mr. Edward Owens. Funding for open access charge: United States–Israel Binational Science Foundation [BSF2013094].
    Repository Name: Woods Hole Open Access Server
    Type: Article
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