processes Article ADAR Mediated RNA Editing Modulates MicroRNA Targeting in Human Breast Cancer Justin T. Roberts 1 ID , Dillon G. Patterson 1, Valeria M. King 1, Shivam V. Amin 1, Caroline J. Polska 1, Dominika Houserova 2, Aline Crucello 1, Emmaline C. Barnhill 1, Molly M. Miller 1, Timothy D. Sherman 1 and Glen M. Borchert 1,2,* ID 1 Department of Biology, University of South Alabama, Mobile, AL 36688-0002, USA; [email protected] (J.T.R.); [email protected] (D.G.P.); [email protected] (V.M.K.); [email protected] (S.V.A.); [email protected] (C.J.P.); [email protected] (A.C.); [email protected] (E.C.B.); [email protected] (M.M.M.); [email protected] (T.D.S.) 2 Department of Pharmacology, USA College of Medicine, Mobile, AL 36688-0002, USA; [email protected] * Correspondence: [email protected]; Tel.: +1-251-460-7310 Received: 6 April 2018; Accepted: 21 April 2018; Published: 25 April 2018 Abstract: RNA editing by RNA specific adenosine deaminase acting on RNA (ADAR) is increasingly being found to alter microRNA (miRNA) regulation. Editing of miRNA transcripts can affect their processing, as well as which messenger RNAs (mRNAs) they target. Further, editing of target mRNAs can also affect their complementarity to miRNAs. Notably, ADAR editing is often increased in malignancy with the effect of these RNA changes being largely unclear. In addition, numerous reports have now identified an array of miRNAs that directly contribute to various malignancies although the majority of their targets remain largely undefined. Here we propose that modulating the targets of miRNAs via mRNA editing is a frequent occurrence in cancer and an underappreciated participant in pathology. In order to more accurately characterize the relationship between these two regulatory processes, this study examined RNA editing events within mRNA sequences of two breast cancer cell lines (MCF-7 and MDA-MB-231) and determined whether or not these edits could modulate miRNA associations. Computational analyses of RNA-Seq data from these two cell lines identified over 50,000 recurrent editing sites within human mRNAs, and many of these were located in 30 untranslated regions (UTRs). When these locations were screened against the list of currently-annotated miRNAs we discovered that editing caused a subset (~9%) to have significant alterations to mRNA complementarity. One miRNA in particular, miR-140-3p, is known to be misexpressed in many breast cancers, and we found that mRNA editing allowed this miRNA to directly target the apoptosis inducing gene DFFA in MCF-7, but not in MDA-MB-231 cells. As these two cell lines are known to have distinct characteristics in terms of morphology, invasiveness and physiological responses, we hypothesized that the differential RNA editing of DFFA in these two cell lines could contribute to their phenotypic differences. Indeed, we confirmed through western blotting that inhibiting miR-140-3p increases expression of the DFFA protein product in MCF-7, but not MDA-MB-231, and further that inhibition of miR-140-3p also increases cellular growth in MCF-7, but not MDA-MB-231. Broadly, these results suggest that the creation of miRNA targets may be an underappreciated function of ADAR and may help further elucidate the role of RNA editing in tumor pathogenicity. Keywords: ADAR; breast; cancer; inosine; microRNA; microRNA targeting; RNA editing Processes 2018, 6, 42; doi:10.3390/pr6050042 www.mdpi.com/journal/processes Processes 2018, 6, 42 2 of 14 Processes 2018, 6, x FOR PEER REVIEW 2 of 14 1.1. Introduction TranscriptTranscript variationvariation atat thethe singlesingle nucleotidenucleotide levellevel isis increasingly being foundfound to have widespread occurrencesoccurrences withinwithin thethe transcriptometranscriptome withwith fundamentalfundamental roles inin numerousnumerous biologicalbiological processesprocesses includingincluding developmentdevelopment andand disease.disease. Specifically, Specifically, several independent studies have reported thatthat therethere areare hundredshundreds ofof thousandsthousands ofof RNARNA editingediting sitessites catalyzedcatalyzed byby thethe enzymeenzyme ADARADAR (adenosine(adenosine deaminasedeaminase actingacting onon RNA)RNA) withinwithin humanhuman mRNAsmRNAs [[11––66].]. EditingEditing viavia ADARADAR isis characterizedcharacterized byby thethe conversionconversion ofof thethe nucleic acid adenosine to inosine via deamination at the C6 position [7] [7] (Fig (Figureure 11).). SinceSince inosinesinosines havehave been shown to preferentially bind to cytosines, functionally the ADAR-catalyzedADAR-catalyzed editingediting changeschanges an ‘A’‘A’ toto aa ‘G’‘G’ inin thethe transcripttranscript sequencesequence [[7]7].. Interestingly,Interestingly, thethe vastvast majoritymajority (>99%)(>99%) ofof editingediting sitessites occuroccur inin thethe UTRsUTRs ofof primate-specificprimate-specific AluAlu elementselements [[88––1010],], likelylikely duedue toto thethe commoncommon occurrenceoccurrence ofof two two oppositely oppositely oriented oriented Alus Alus located located in the in same the pre-mRNAsame pre-mRNA pairing pairing together together to produce to theproduce long andthe stablelong and double-stranded stable double- RNAstranded structure RNA thatstructure is required that is for required ADAR for to bind.ADAR As to the bind. ability As tothe convert ability nucleotidesto convert nucleotides adds a great adds deal a ofgreat functionality deal of functionality to the transcriptome, to the transcriptome, it is not surprising it is not thatsurprising it has fundamentalthat it has fundamental roles in many roles cellular in many activities. cellular Editing activities. events Editing within events mRNA within coding mRNA for variouscoding for neuroreceptors, various neuroreceptors such as serotonin, such as and serotonin glutamate, and have glutamate been intensively, have been detailed intensively in an detailed array of organismsin an array from of organisms flatworms from to primates flatworms and to found primates to be criticaland found for routine to be criti neuralcal for activity routine [11 ].neural With regardsactivity to[11] gene. With regulation, regards to sequence gene regulation, editing ofsequence RNA has editing widespread of RNA implications, has widespread including implications splice, siteincluding alteration, splice localization site alteration, and nuclear localization retention, and andnuclear modification retention, to and the RNAmodification secondary to structurethe RNA itselfsecondary [12]. Further, structure given itself these [12] abundant. Further, roles given for these ADAR abundant editing inroles routine for cellularADAR function,editing in it routine should alsocellular not befunction, surprising it should that dysfunction also not be ofsurprising this important that dysfunction mechanism of can this have important detrimental mechanism effects and,can indeed,have detrimental an increasing effects number and, ofindeed reports, an indicate increasing a strong number correlation of reports between indicate altered a strong ADAR correlation activity andbetween a variety altered of pathologies. ADAR activity Specifically, and a variety because of ADARpathologies. has been Specifically, shown to bebecause such an ADAR integral has player been inshown apoptotic to be regulationsuch an integral and cellular player differentiation, in apoptotic regulation cancer is ofand especially cellular heighteneddifferentiation, interest cancer and, is inof fact,especially more andheightened more evidence interest isand pointing, in fact to, more dysregulation and more of evidence the editing is pointing process beingto dysregul a majoration factor of inthe tumorigenesis editing process [13 being–18]. a major factor in tumorigenesis [13–18]. FigureFigure 1.1. ADARs deaminate deaminate adenosine adenosine to to inosine, inosine, potentially potentially altering altering miRNA miRNA complementarities complementarities.. A Acartoon cartoon depicting depicting adenosine adenosine (left (left), ),deaminated deaminated adenosine adenosine (inosine, (inosine, in in centercenter), ),and and guanine guanine (right (right). ). EditingEditing eventsevents can can also also have have widespread widespread effects effects on theon genethe gene regulatory regulatory ability ability of noncoding of noncoding RNAs, suchRNAs as, miRNAssuch as miRNAs [17,19]. MiRNAs [17,19]. areMiRNAs small regulatoryare small RNAregulatory molecules RNA roughly molecules 20 to roughly 23 nucleotides 20 to 23 in lengthnucleotides that regulatein length cell that processes regulate by cell binding processes to their by binding target mRNAs to their andtarget inhibiting mRNAs translation and inhibiting [20]. MiRNAstranslation are [20]. initially MiRNAs transcribed are initially as primary transcribed miRNAs as primary (pri-miRNAs) miRNAs consisting (pri-miRNAs) of several consisting thousand of nucleotidesseveral thousand in length nucleotides that are thenin length processed that are by intothen matureprocessed miRNAs by into by mature the enzymes miRNAs Dicer by andthe Droshaenzymes before Dicer enteringand Dros theha RNAibefore gene entering silencing the RNAi complex gene where silencing they complex regulate where gene expressionthey regulate by bindinggene expression to the 30 byUTR binding of their to mRNAthe 3′ UTR targets of their via complimentarymRNA targets via base complimentary