Comparative functional RNA editomes of neural differentiation from human PSCs

Abstract

RNA editing is a fundamental mechanism that constitutes the epitranscriptomic complexity. A-to-G editing is the predominant type catalyzed by ADAR1 and ADAR2 in human. Using a CRISPR/Cas9 approach to knockout ADAR1/2, we identified a regulatory role of RNA editing in directed differentiation of human embryonic stem cells (hESCs) toward neural progenitor cells (NPCs). Genome-wide landscapes of A-to-G editing in hESCs and four derivative cell lineages representing all three germ layers and the extraembryonic cell fate were profiled, with a particular focus on neural differentiation. Furthermore, a bioinformatics-guided case study identified a potential functional editing event in ZYG11B 3’UTR that might play a role in regulation of NPC differentiation through gain of miR6089 targeting. Collectively, our study established the functional role of A-to-G RNA editing in neural lineage differentiation; illustrated the RNA editing landscapes of hESCs and NPC differentiation; and shed new light on molecular insights thereof.

Keywords

RNA editing / CRISPR/Cas9 / human pluripotent stem cell / neural differentiation / ZYG11b

References

[1] GottJM, EmesonRB. Functions and mechanisms of RNA editing. Annu Rev Genet 2000;34:499–531.

[2] WalkleyCR, LiJB. Rewriting the transcriptome: adenosine-to-inosine RNA editing by ADARs. Genome Biol 2017;18:205.

[3] FryeM, Jaffrey SR, PanT, et al. RNA modifications: what have we learned and where are we headed? Nat Rev Genet 2016;17:365–72.

[4] LiS, MasonCE. The pivotal regulatory landscape of RNA modifications. Annu Rev Genomics Hum Genet 2014;15:127–50.

[5] NishikuraK. Functions and regulation of RNA editing by ADAR deaminases. Annu Rev Biochem 2010;79:321–49.

[6] SunWJ, LiJH, LiuS, et al. RMBase: a resource for decoding the landscape of RNA modifications from high-throughput sequencing data. Nucleic Acids Res 2016;44:D259–65.

[7] LiJB, ChurchGM. Deciphering the functions and regulation of brain-enriched A-to-I RNA editing. Nat Neurosci 2013;16:1518–22.

[8] TanMH, LiQ, ShanmugamR, et al. Dynamic landscape and regulation of RNA editing in mammals. Nature 2017;550:249–54.

[9] WedekindJE, DanceGS, SowdenMP, et al. Messenger RNA editing in mammals: new members of the APOBEC family seeking roles in the family business. Trends Genet 2003;19:207–16.

[10] SongB, Shiromoto Y, MinakuchiM, et al. The role of RNA editing enzyme ADAR1 in human disease. Wiley Interdiscip Rev RNA 2022;13:e1665.

[11] YangY, OkadaS, SakuraiM. Adenosine-to-inosine RNA editing in neurological development and disease. RNA Biol 2021;18:999–1013.

[12] TakahashiK, TanabeK, OhnukiM, et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 2007;131:861–72.

[13] ThomsonJA, Itskovitz-Eldor J, ShapiroSS, et al. Embryonic stem cell lines derived from human blastocysts. Science 1998;282:1145–7.

[14] YuJ, Vodyanik MA, Smuga-OttoK, et al. Induced pluripotent stem cell lines derived from human somatic cells. Science 2007;318:1917–20.

[15] HartnerJC, Schmittwolf C, KispertA, et al. Liver disintegration in the mouse embryo caused by deficiency in the RNA-editing enzyme ADAR1. J Biol Chem 2004;279:4894–902.

[16] HiguchiM, MaasS, SingleFN, et al. Point mutation in an AMPA receptor gene rescues lethality in mice deficient in the RNA-editing enzyme ADAR2. Nature 2000;406:78–81.

[17] WangQ, Khillan J, GadueP, et al. Requirement of the RNA editing deaminase ADAR1 gene for embryonic erythropoiesis. Science 2000;290:1765–8.

[18] WangQ, Miyakoda M, YangW, et al. Stress-induced apoptosis associated with null mutation of ADAR1 RNA editing deaminase gene. J Biol Chem 2004;279:4952–61.

[19] ShtrichmanR, Germanguz I, MandelR, et al. Altered A-to-I RNA editing in human embryogenesis. PLoS One 2012;7:e41576.

[20] OsenbergS, Paz Yaacov N, SafranM, et al. Alu sequences in undifferentiated human embryonic stem cells display high levels of A-to-I RNA editing. PLoS One 2010;5:e11173.

[21] BurnsCM, ChuH, RueterSM, et al. Regulation of serotonin-2C receptor G-protein coupling by RNA editing. Nature 1997;387:303–8.

[22] EranA, LiJB, VatalaroK, et al. Comparative RNA editing in autistic and neurotypical cerebella. Mol Psychiatry 2013;18:1041–8.

[23] KawaharaY, Grimberg A, TeegardenS, et al. Dysregulated editing of serotonin 2C receptor mRNAs results in energy dissipation and loss of fat mass. J Neurosci 2008;28:12834–44.

[24] SilberbergG, LundinD, NavonR, et al. Deregulation of the A-to-I RNA editing mechanism in psychiatric disorders. Hum Mol Genet 2012;21:311–21.

[25] KonenLM, WrightAL, RoyleGA, et al. A new mouse line with reduced GluA2 Q/R site RNA editing exhibits loss of dendritic spines, hippocampal CA1-neuron loss, learning and memory impairments and NMDA receptor-independent seizure vulnerability. Mol Brain 2020;13:27.

[26] SchwarzAP, Kovalenko AA, ZakharovaMV, et al. The application of the self-probing primer PCR for quantitative expression analysis of R607Q (un)edited GluA2 AMPA receptor mRNA. Biochem Biophys Res Commun 2021;569:174–8.

[27] IwamotoK, KatoT. RNA editing of serotonin 2C receptor in human postmortem brains of major mental disorders. Neurosci Lett 2003;346:169–72.

[28] KaranovicJ, Svikovic S, PantovicM, et al. Joint effect of ADARB1 gene, HTR2C gene and stressful life events on suicide attempt risk in patients with major psychiatric disorders. World J Biol Psychiatry 2015;16:261–71.

[29] AnsellBRE, ThomasSN, BonelliR, et al. A survey of RNA editing at single-cell resolution links interneurons to schizophrenia and autism. RNA 2021;27:1482–96.

[30] Tsivion-VisbordH, Kopel E, FeiglinA, et al. Increased RNA editing in maternal immune activation model of neurodevelopmental disease. Nat Commun 2020;11:5236.

[31] ChenT, XiangJF, ZhuS, et al. ADAR1 is required for differentiation and neural induction by regulating microRNA processing in a catalytically independent manner. Cell Res 2015;25:459–76.

[32] Heraud-FarlowJE, ChalkAM, LinderSE, et al. Protein recoding by ADAR1-mediated RNA editing is not essential for normal development and homeostasis. Genome Biol 2017;18:166.

[33] LiddicoatBJ, Hartner JC, PiskolR, et al. Adenosine-to-inosine RNA editing by ADAR1 is essential for normal murine erythropoiesis. Exp Hematol 2016;44:947–63.

[34] LiddicoatBJ, PiskolR, ChalkAM, et al. RNA editing by ADAR1 prevents MDA5 sensing of endogenous dsRNA as nonself. Science (New York, NY) 2015;349:1115–20.

[35] XieW, Schultz MD, ListerR, et al. Epigenomic analysis of multilineage differentiation of human embryonic stem cells. Cell 2013;153:1134–48.

[36] ChambersSM, FasanoCA, PapapetrouEP, et al. Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling. Nat Biotechnol 2009;27:275–80.

[37] KiranA, Baranov PV. DARNED: a DAtabase of RNa EDiting in humans. Bioinformatics 2010;26:1772–6.

[38] ParkE, Williams B, WoldBJ, et al. RNA editing in the human ENCODE RNA-seq data. Genome Res 2012;22:1626–33.

[39] PengZ, ChengY, TanBC, et al. Comprehensive analysis of RNA-Seq data reveals extensive RNA editing in a human transcriptome. Nat Biotechnol 2012;30:253–60.

[40] RamaswamiG, LinW, PiskolR, et al. Accurate identification of human Alu and non-Alu RNA editing sites. Nat Methods 2012;9:579–81.

[41] XiangJF, YangQ, LiuCX, et al. N(6)-methyladenosines modulate A-to-I RNA editing. Mol Cell 2018;69:126–35.

[42] PiskolR, PengZ, WangJ, et al. Lack of evidence for existence of noncanonical RNA editing. Nat Biotechnol 2013;31:19–20.

[43] WangK, LiM, HakonarsonH. ANNOVAR: functional annotation of genetic variants from high-throughput sequencing data. Nucleic Acids Res 2010;38:e164.

[44] SinghM. Dysregulated A to I RNA editing and non-coding RNAs in neurodegeneration. Front Genet 2012;3:326.

[45] CouzensAL, KnightJD, KeanMJ, et al. Protein interaction network of the mammalian Hippo pathway reveals mechanisms of kinase-phosphatase interactions. Sci Signal 2013;6:rs15.

[46] Das ThakurM, FengY, JagannathanR, et al. Ajuba LIM proteins are negative regulators of the Hippo signaling pathway. Curr Biol 2010;20:657–62.

[47] FoxlerDE, BridgeKS, JamesV, et al. The LIMD1 protein bridges an association between the prolyl hydroxylases and VHL to repress HIF-1 activity. Nat Cell Biol 2012;14:201–8.

[48] BorchertGM, Gilmore BL, SpenglerRM, et al. Adenosine deamination in human transcripts generates novel microRNA binding sites. Hum Mol Genet 2009;18:4801–7.

[49] MaschiettoM, TahiraAC, PugaR, et al. Co-expression network of neural- differentiation genes shows specific pattern in schizophrenia. BMC Med Genomics 2015;8:23.

[50] McCarthyMJ, Barrett TB, NissenS, et al. Allele specific analysis of the ADRBK2 gene in lymphoblastoid cells from bipolar disorder patients. J Psychiatr Res 2010;44:201–8.

[51] FumagalliD, Gacquer D, RotheF, et al. Principles governing A-to-I RNA editing in the breast cancer transcriptome. Cell Rep 2015;13:277–89.

[52] HanL, DiaoL, YuS, et al. The genomic landscape and clinical relevance of A-to-I RNA editing in human cancers. Cancer Cell 2015;28:515–28.

[53] HwangT, ParkCK, LeungAK, et al. Dynamic regulation of RNA editing in human brain development and disease. Nat Neurosci 2016;19:1093–9.

[54] Paz-YaacovN, BazakL, BuchumenskiI, et al. Elevated RNA editing activity is a major contributor to transcriptomic diversity in tumors. Cell Rep 2015;13:267–76.

[55] SlotkinW, Nishikura K. Adenosine-to-inosine RNA editing and human disease. Genome Med 2013;5:105.

[56] XuX, WangY, LiangH. The role of A-to-I RNA editing in cancer development. Curr Opin Genet Dev 2018;48:51–6.

[57] WangQ, HuiH, GuoZ, et al. ADAR1 regulates ARHGAP26 gene expression through RNA editing by disrupting miR-30b-3p and miR- 573 binding. RNA 2013;19:1525–36.

[58] CoxDBT, Gootenberg JS, AbudayyehOO, et al. RNA editing with CRISPR-Cas13. Science 2017;358:1019–27.

[59] YiZ, QuL, TangH, et al. Engineered circular ADAR-recruiting RNAs increase the efficiency and fidelity of RNA editing in vitro and in vivo. Nat Biotechnol 2022;40:946–55.

[60] LuJ, ZhaoC, ZhaoY, et al. Multimode drug inducible CRISPR/Cas9 devices for transcriptional activation and genome editing. Nucleic Acids Res 2018;46:e25.

[61] SauvageauM, GoffLA, LodatoS, et al. Multiple knockout mouse models reveal lincRNAs are required for life and brain development. Elife 2013;2:e01749.

[62] LiH, DurbinR. Fast and accurate long-read alignment with Burrows- Wheeler transform. Bioinformatics 2010;26:589–95.

[63] LiH, Handsaker B, WysokerA, et al. The sequence alignment/map format and SAMtools. Bioinformatics 2009;25:2078–9.

[64] ChenL. Characterization and comparison of human nuclear and cytosolic editomes. Proc Natl Acad Sci USA 2013;110:E2741–7.

[65] LangfelderP, Horvath S. WGCNA: an R package for weighted correlation network analysis. BMC Bioinf 2008;9:559.

[66] ZhangB, Horvath S. A general framework for weighted gene co-expression network analysis. Stat Appl Genet Mol Biol 2005;4:Article17.

[67] LangfelderP, ZhangB, HorvathS. Defining clusters from a hierarchical cluster tree: the Dynamic Tree Cut package for R. Bioinformatics 2008;24:719–20.

[68] HorvathS, DongJ. Geometric interpretation of gene coexpression network analysis. PLoS Comput Biol 2008;4:e1000117.

[69] XueZ, HuangK, CaiC, et al. Genetic programs in human and mouse early embryos revealed by single-cell RNA sequencing. Nature 2013;500:593–7.

[70] Jiang,JJ, Liu,CM, Zhang,BY, et al. MicroRNA-26a supports mammalian axon regeneration in vivo by suppressing GSK3beta expression. Cell Death Dis 2015;6:e1865.

[71] Liu,CM, Wang,RY, Saijilafu,J, et al. MicroRNA-138 and SIRT1 form a mutual negative feedback loop to regulate mammalian axon regeneration. Genes Dev 2013;27:1473–83.

[72] The R Development Core Team. R: A Language And Environment For Statistical Computing. 2019. R Foundation for Statistical Computing, Vienna.

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The Author(s) 2022. Published by Oxford University Press on behalf of Higher Education Press.