ADAR1- and ADAR2-mediated regulation of maturation and targeting of miR-376b to modulate GABA neurotransmitter catabolism

Abstract

miRNAs are short noncoding RNA molecules that regulate gene expression by inhibiting translation or inducing degradation of target mRNAs. miRNAs are often expressed as polycistronic transcripts, so-called miRNA clusters, containing several miRNA precursors. The largest mammalian miRNA cluster, the miR-379–410 cluster, is expressed primarily during embryonic development and in the adult brain; however, downstream regulation of this cluster is not well understood. Here, we investigated adenosine deamination to inosine (RNA editing) in the miR-379–410 cluster by adenosine deaminase acting on RNA (ADAR) enzymes as a possible mechanism modulating the expression and activity of these miRNAs in a brain-specific manner. We show that the levels of editing in the majority of mature miRNAs are lower than the editing levels of the corresponding site in primary miRNA precursors. However, for one miRNA, miR-376b-3p, editing was significantly higher in the mature form than in the primary precursor. We found miR-376b-3p maturation is negatively regulated by ADAR2 in an editing activity–independent manner, whereas ADAR1-mediated and ADAR2-mediated editing were observed to be competitive. In addition, the edited miR-376b-3p targets a different set of mRNAs than unedited miR-376b-3p, including 4-aminobutyrate aminotransferase, encoding the enzyme responsible for the catabolism of the neurotransmitter gamma aminobutyric acid (GABA). Expression of edited miR-376b-3p led to increased intracellular GABA levels as well as increased cell surface presentation of GABA type A receptors. Our results indicate that both editing and editing-independent effects modulate the expression of miR-376b-3p, with the potential to regulate GABAergic signaling in the brain. miRNAs are short noncoding RNA molecules that regulate gene expression by inhibiting translation or inducing degradation of target mRNAs. miRNAs are often expressed as polycistronic transcripts, so-called miRNA clusters, containing several miRNA precursors. The largest mammalian miRNA cluster, the miR-379–410 cluster, is expressed primarily during embryonic development and in the adult brain; however, downstream regulation of this cluster is not well understood. Here, we investigated adenosine deamination to inosine (RNA editing) in the miR-379–410 cluster by adenosine deaminase acting on RNA (ADAR) enzymes as a possible mechanism modulating the expression and activity of these miRNAs in a brain-specific manner. We show that the levels of editing in the majority of mature miRNAs are lower than the editing levels of the corresponding site in primary miRNA precursors. However, for one miRNA, miR-376b-3p, editing was significantly higher in the mature form than in the primary precursor. We found miR-376b-3p maturation is negatively regulated by ADAR2 in an editing activity–independent manner, whereas ADAR1-mediated and ADAR2-mediated editing were observed to be competitive. In addition, the edited miR-376b-3p targets a different set of mRNAs than unedited miR-376b-3p, including 4-aminobutyrate aminotransferase, encoding the enzyme responsible for the catabolism of the neurotransmitter gamma aminobutyric acid (GABA). Expression of edited miR-376b-3p led to increased intracellular GABA levels as well as increased cell surface presentation of GABA type A receptors. Our results indicate that both editing and editing-independent effects modulate the expression of miR-376b-3p, with the potential to regulate GABAergic signaling in the brain. miRNAs are short noncoding RNA that act as post-transcriptional regulators of gene expression by inhibiting translation and/or inducing degradation of target mRNAs (1Jonas S. Izaurralde E. Towards a molecular understanding of microRNA-mediated gene silencing.Nat. Rev. Genet. 2015; 16: 421-433Google Scholar). miRNAs are transcribed as primary-miRNAs (pri-miRNAs), which are hairpin structures of dsRNA structure. pri-miRNAs are recognized by a complex consisting of the RNA-binding protein DiGeorge syndrome critical region 8 and the endonuclease Drosha, which cleaves the dsRNA structure, releasing a ∼70 nucleotide (nt) precursor miRNA (pre-miRNA) (2Denli A.M. Tops B.B.J. Plasterk R.H.A. Ketting R.F. Hannon G.J. Processing of primary microRNAs by the microprocessor complex.Nature. 2004; 432: 231-235Google Scholar). The pre-miRNA is then exported to the cytoplasm, where it is recognized by Dicer that cleaves the hairpin structure again, releasing a duplexed pair of mature miRNAs (3Zhang H. Kolb F.A. Jaskiewicz L. Westhof E. Filipowicz W. Single processing center models for human Dicer and bacterial RNase III.Cell. 2004; 118: 57-68Google Scholar). One strand of the duplex is then incorporated into the RNA-induced silencing complex (RISC), whereupon it serves to guide the complex to mRNAs containing complementary sequences. The region of nucleotides 2 to 8 of the miRNA is critical for targeting and is termed the seed region (4Lewis B.P. Burge C.B. Bartel D.P. Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets.Cell. 2005; 120: 15-20Google Scholar). miRNA genes are often found clustered in the genome and are expressed as a single polycistronic transcript, which contains several pri-miRNA hairpins (5Altuvia Y. Landgraf P. Lithwick G. Elefant N. Pfeffer S. Aravin A. Brownstein M.J. Tuschl T. Margalit H. Clustering and conservation patterns of human microRNAs.Nucleic Acids Res. 2005; 33: 2697-2706Google Scholar, 6Baskerville S. Bartel D.P. Microarray profiling of microRNAs reveals frequent coexpression with neighboring miRNAs and host genes.RNA. 2005; 11: 241-247Google Scholar). This enables coordinated transcriptional regulation of miRNAs involved in the same biological function or regulatory network (7Wang E. of clustered microRNAs on regulatory Scholar). regulation of miRNA in miRNA and/or miRNA enables of levels of miRNAs a cluster G. of miRNA Scholar). The largest miRNA cluster in is the miR-379–410 cluster, which contains as as pri-miRNA genes in a region H. H. A microRNA gene cluster the Res. 2004; Scholar, S. G. of the gene cluster the Genet. Scholar). This cluster is expressed in the whereas expression to the in the adult S. P. The cluster the 33: Scholar). the miR-379–410 cluster in with a function of the cluster in development and in the adult as the miR-379–410 of as well as and of the adult S. G. of the gene cluster the Genet. Scholar, S. P. The cluster the 33: Scholar, S. L. T. G. A microRNA cluster as a for in Scholar). were to an increased and of S. L. T. G. A microRNA cluster as a for in Scholar). In the of the miR-379–410 cluster led to increased expression of and expression of genes to gamma aminobutyric acid signaling S. L. T. G. A microRNA cluster as a for in Scholar). of the signaling were found to be of the miR-379–410 cluster in cell microRNAs gene in Scholar). of the miRNA cluster in a with miRNAs to regulate as and in of the in embryonic and regulators of 2015; Expression patterns of of the cluster in different and different indicate that post-transcriptional modulate the expression of miRNAs the cluster microRNAs gene in Scholar). of the miR-379–410 cluster found to be to RNA editing Y. P. H. of silencing targets by editing of Scholar, S. E. A. N. E. of edited microRNAs in the human Res. Scholar). This biological of the deamination of dsRNA structures by the adenosine deaminase acting on RNA (ADAR) of The inosine to of which that editing of a miRNA a form that a different set of target mRNAs Y. P. H. of silencing targets by editing of Scholar, S. G. Y. Y. H. A for of the silencing of and edited Scholar, H. editing in the miRNA seed region target and silencing Acids Res. Scholar). of pri-miRNA miRNA often pri-miRNA processing Y. E. H. L. and of microRNA editing in human Acids Res. where RNA editing miRNA processing S. A. ADAR2 in and of mature microRNAs in the Acids Res. Scholar). In addition, the enzymes dsRNA structures as found in pri-miRNA hairpins and miRNA in an editing-independent L. G. effects of on the Scholar). The and different editing site S. of RNA editing in and different effects on miRNA L. G. effects of on the Scholar). In addition, is expressed as different a in the and a that the and the of the region of the human adenosine deaminase gene and of an Scholar, A. L. The human not the enzyme an and the of a Scholar). expression of the enzymes in different and different to miRNA regulation G. A. A. N. and regulation of RNA editing in Scholar, L. Y. S. W. N. The of miRNA editing in and on miRNA and Res. Scholar). In expression and miRNA editing are in the and editing of miRNAs found to miRNAs to mRNAs involved in and L. Y. S. W. N. The of miRNA editing in and on miRNA and Res. Scholar). miRNA editing in the mammalian is with miRNA editing during development Y. editing of microRNAs in the mammalian during Res. Scholar). The editing of the miR-379–410 cluster in the adult which is in the expressed miRNA cluster S. A. that act on RNA in and of embryonic Res. is to be a mechanism for modulating miRNA expression and targeting in a manner. Here, we show that expression and editing of miR-376b-3p is by and with increased levels of edited miR-376b-3p in of edited miR-376b-3p the levels of genes involved in genes 4-aminobutyrate a miR-376b-3p editing and regulation of GABAergic signaling in the brain. The miR-379–410 cluster contains miRNAs that are to RNA editing Y. P. H. of silencing targets by editing of Scholar, Y. E. H. L. and of microRNA editing in human Acids Res. Scholar, Y. editing of microRNAs in the mammalian during Res. Scholar). RNA editing the maturation of these miRNAs in the we the editing levels the primary and corresponding site in the mature miRNA in of the different editing levels the of miRNA these significantly lower editing in the mature miRNA with the the editing site in where the pri-miRNA was edited to whereas the editing of the corresponding site in the mature form was This that RNA editing of the miR-379–410 cluster miRNA However, the miRNAs of the cluster, miR-376b-3p editing was significantly higher in the mature miRNA with the pri-miRNA one of the edited mature In to this and enzymes miRNAs of the cluster, we this miRNA for RNA editing of miRNA found to be regulated Y. editing of microRNAs in the mammalian during Res. we a of miR-376b-3p in different of development in to editing is to expression levels during We acid to unedited and edited miR-376b-3p in We the expression of the of miR-376b-3p in different embryonic and adult The levels of the edited miR-376b-3p increased significantly during whereas the unedited in with increased editing of miR-376b-3p Y. editing of microRNAs in the mammalian during Res. Scholar). we investigated the of unedited and edited mature with the editing activity in the primary The contains edited in the mature the site in the edited by ADAR2 and the site in the that is edited by Y. P. H. of silencing targets by editing of Scholar, S. A. ADAR2 in and of mature microRNAs in the Acids Res. Scholar, S. A. that act on RNA in and of embryonic Res. Scholar). editing these during we of of the for the editing of the site to and we not and as be the levels of edited of the site was and which it increased to the in ADAR2 activity H. A. of ADAR2 RNA editing during Scholar). The that levels of edited miR-376b-3p a in pri-miRNA editing the corresponding site the that editing the mechanism of editing and the of and we a expression containing the pri-miRNA hairpin and of The pri-miRNA expression was in human embryonic with expression for the or containing a to the enzymes A.M. G. RNA editing by of dsRNA as 2015; Scholar, is in the ADAR2 and for RNA 2005; Scholar). was on to editing activity the of Y. E. H. L. and of microRNA editing in human Acids Res. Scholar, S. A. ADAR2 in and of mature microRNAs in the Acids Res. Scholar, S. A. that act on RNA in and of embryonic Res. we observed a for the with editing the site and ADAR2 editing the site a was to the site on the site to whereas ADAR2 was to editing of the In ADAR2 in a of lower editing the site than observed as a of the editing in This indicate that ADAR2 is to ADAR1-mediated editing of the of both and ADAR2 the editing by the ADAR2 was to significantly and ADAR2 not ADAR2-mediated editing of the that and are both to ADAR2-mediated ADAR2 the same on ADAR1-mediated editing of the that the is editing results the of the enzymes for of the editing of and that ADAR2 ADAR1-mediated editing of in an editing-independent the of ADAR1-mediated editing in to the of ADAR2-mediated editing in the mature of in we investigated the of the different enzymes to the activity of miR-376b-3p, we miR-376b-3p in in the of in a This was with the expression and the expression with not the silencing whereas led to a ADAR2 in in an editing-independent manner, that ADAR2 an on miR-376b-3p the enzymes the maturation of the strand miRNA, we a with a for The different of were not found to However, as for the miRNA, ADAR2 an editing-independent In to that the effects are editing we miRNA expression in which the editing in the or the miRNAs were by a the of In to the targeting of the the complementary in the were for Here, we that of the or the miRNA not silencing of the miR-376b-3p of the site not silencing of the However, led to a of the on expression with results the editing-independent of the observed effects of and ADAR2 on miR-376b-3p that maturation of is by editing the In the a silencing activity be the of an in the this and the results of the we of miR-376b-3p of miRNA expression and enzymes and of unedited miR-376b-3p not with whereas led to an of in a of edited miR-376b-3p, corresponding to a in miRNA whereas not levels of edited miRNA In with results the in A and ADAR2 miRNA for both unedited and edited of the In these results indicate that a on the levels of miR-376b-3p, whereas ADAR2 levels of both miR-376b-3p and The enzymes to the processing of different often by the of S. A. ADAR2 in and of mature microRNAs in the Acids Res. Scholar, L. G. effects of on the Scholar, W. of microRNA processing and expression RNA editing by and the of to form mature miRNAs Y. RNA editing of the precursor by the Scholar, H. L. H. a complex with Dicer to microRNA processing and RNA-induced gene Scholar). the enzymes were we the of pre-miRNA and mature miRNA with This to of pre-miRNA as well as mature The for the of an of unedited and edited of the miRNA, in for editing to not of of and mature miR-376b-3p was not significantly different with or or and ADAR2 on the led to of both pre-miRNA and mature miRNA in an editing-independent manner. of the site not significantly of pre-miRNA or mature of the site pre-miRNA and with the pre-miRNA a lower molecular and whereas mature miRNA In these results that ADAR2 of both and mature miR-376b-3p in an editing-independent manner. The of ADAR2 on the levels of pre-miRNA and mature miRNA be to of pri-miRNA or processing of these we in the of the pre-miRNA hairpin to pri-miRNA with the different of for led to in pri-miRNA whereas ADAR2 led to a than in This indicates that ADAR2 to an of pri-miRNA transcripts, in the in miR-376b-3p and ADAR2 to for to and ADAR2 pri-miRNA mature miR-376b-3p be for that with This is by the increased ADAR1-mediated editing of mature miR-376b-3p in to the lower editing observed in the pri-miRNA and the ADAR2-mediated editing of mature In ADAR2 a in and mature miR-376b-3p by inhibiting processing of the of the enzymes for to it is possible that this to the higher ADAR1-mediated editing observed in the mature we investigated the of the regulatory that the of editing of miR-376b-3p is in the this miRNA is expressed in S. A. that act on RNA in and of embryonic Res. and that is regulated by we that edited miR-376b-3p the levels of genes with We an in of targets of edited miR-376b-3p with the of Y. for of microRNA Acids Res. the edited adenosine of miR-376b-3p with in to the effects of an of miR-376b-3p we the for unedited miR-376b-3p to targets of a of and genes were targets of the unedited and edited with one gene the and were on the to of the target genes The for the targets of edited miR-376b-3p several to as and regulation of neurotransmitter whereas the for the unedited miR-376b-3p not to the of targets of edited miR-376b-3p, we genes with a higher than and expression in the cell for The genes 4-aminobutyrate and we with the miR-376b-3p miRNA with or we observed significantly levels of target genes with miR-376b-3p, whereas with the unedited miRNA not the targeting of these genes by the edited miR-376b-3p, and to we where a of the of target gene containing one or target was into the of of the with the expression of the not the led to activity for genes This was of the nucleotides in the complementary to the seed of the edited miR-376b-3p In these results show that not miR-376b-3p targets these genes in a manner. the genes are to this the that editing of miR-376b-3p and ADAR2-mediated of miR-376b-3p maturation are to function in the brain. In to targets of edited miR-376b-3p with we targeting conservation and this we the of to targeting of the miR-376b-3p and a of targets the edited adenosine for in we observed and the with of and we were to the targeting of miR-376b-3p to by the human cell with miR-376b-3p miRNA and a of with the and not the miR-376b-3p is the enzyme responsible for the catabolism of the neurotransmitter GABA and be involved in the regulation of GABAergic we to on this target an of with the miR-376b-3p expression that the of by edited miR-376b-3p was with a significantly protein Y. T. Kolb A for in of 16: a of where of GABA by the protein the of a this with an miRNA of miR-376b-3p, we observed an increased of as by cell This indicates that the of by miR-376b-3p to increased intracellular levels of GABA of GABA levels of intracellular GABA the levels of GABA in of GABAergic In addition, it observed that intracellular GABA as a in the for the cell surface expression of where increased levels of intracellular GABA in increased of on the cell surface GABA as a in the to cell surface expression of Res. Scholar, P. acid type A in the by 2015; Scholar). This that increased levels of intracellular GABA as a expression of edited miR-376b-3p the of on the cell surface by this this we with the and and of with We observed a with miRNA for edited miR-376b-3p, we observed significantly increased surface In addition, we a cell surface protein and in to for on the cell an on the we observed increased levels of with miRNA for edited miR-376b-3p This was not observed for the protein levels of increased surface presentation of not increased protein In these results that the of levels by edited miR-376b-3p to increased intracellular GABA and increased cell surface genes one transcript, as the genes of polycistronic mRNAs and the miRNA clusters, that transcriptional regulation is and enables and of gene This is for genes with biological of the same or genes involved with a for In the of the miR-379–410 cluster, expressed in both embryonic development and in the adult an is the same miRNA genes function in different biological The is to be post-transcriptional of miRNA the that the miRNAs of the cluster transcriptional the expression patterns of the miRNAs embryonic and adult microRNAs gene in Scholar). levels of editing of of the cluster, to of miRNAs in a manner. Our and S. A. ADAR2 in and of mature microRNAs in the Acids Res. Scholar, L. G. effects of on the Scholar, S. A. that act on RNA in and of embryonic Res. show that the enzymes are to the expression of of the miR-379–410 cluster in to that expression and activity in different and G. A. A. N. and regulation of RNA editing in Scholar, Y. editing of microRNAs in the mammalian during Res. Scholar, H. regulation of RNA editing during Res. this a mechanism for modulating the expression of of the miR-379–410 cluster as for biological In addition, expression and activity different and cell G. A. A. N. and regulation of RNA editing in Scholar, E. A. of RNA editing in the in reveals and Scholar, T. Y. A. regulation of RNA editing in human development and and of miRNA expression a regulatory mechanism for the complex transcriptional of the brain. In this we that levels of edited miR-376b-3p during a corresponding in the expression of unedited miR-376b-3p or in the editing of with expression during development H. A. of ADAR2 RNA editing during Scholar, H. regulation of RNA editing during Res. Scholar, T. Y. A. regulation of RNA editing in human development and Scholar). One for this be of miR-376b-3p during with a mechanism that the of edited and unedited miRNA Here, we that ADAR2 the processing of indicate by and ADAR2 to in with that miR-376b-3p editing is increased in the Y. editing of microRNAs in the mammalian during Res. Scholar). this results in the of miR-376b-3p with the editing observed in the ADAR2-mediated of processing then be the for the of the edited form of miR-376b-3p during increased of ADAR2 in H. A. of ADAR2 RNA editing during to an increased expression of to an in the edited form the miRNA with not the unedited form the miRNA was to with inhibiting In of this the in levels of edited miR-376b-3p was found to be to an in the editing of the ADAR2-mediated the of ADAR2 is and in this a possible of on levels is with a whereas not show a for be the observed increased pre-miRNA processing by Dicer in complex with H. L. H. a complex with Dicer to microRNA processing and RNA-induced gene for the A possible of on maturation to the of that for pri-miRNA hairpin and editing by the enzymes to on a Y. T. T. Y. A of reveals regulation of RNA Scholar). The of ADAR2 to editing by the of and that it editing of in the cytoplasm, whereas ADAR2 is and not with this ADAR2 both pre-miRNA and mature miRNA an of the results indicate that ADAR2 is to processing by Drosha, as for miRNAs S. A. ADAR2 in and of mature microRNAs in the Acids Res. Scholar, L. G. effects of on the Scholar, W. of microRNA processing and expression RNA editing by Scholar). that the of ADAR2 to both editing and processing of are and the of the ADAR2 and the ADAR2 a and with the miRNA hairpin and by and to the of ADAR2 for to this the be higher than the of the of ADAR2 is editing we a in activity of the ADAR2-mediated which the that a to modulate this site to the of an the of the miRNA This strand the mechanism by which one of the mature miRNAs is incorporated into the whereas the is as the strand with the is to incorporated into the A. A. and miRNAs strand Scholar, G. T. N. in the of the enzyme Scholar). However, the site not miR-376b-3p which be the of a in strand is that the increased the site to of the strand Y. Bartel D.P. of into enzyme 2005; Scholar, S. cleaves the strand of during 2005; miRNA editing observed to miRNAs to target genes with L. Y. S. W. N. The of miRNA editing in and on miRNA and Res. Scholar). miRNA editing is in whereas in miRNA editing is found in as the L. E. editing of microRNAs and function in where miRNA editing miRNAs to involved in development L. Y. S. W. N. The of miRNA editing in and on miRNA and Res. Scholar). This a development of the of miRNA to the higher of the human brain. miR-376b-3p is not and seed which is in is with miRNAs in and A. S. microRNA to Acids Res. Scholar). This the of this miRNA, which not is in is a target and it is possible that regulation of GABAergic signaling in the human a edited miR-376b-3p and GABAergic signaling is the that a of is by edited miR-376b-3p In is the gene in the of targets for the A in regulation of GABAergic signaling by of the miR-379–410 cluster is by the of the miRNA cluster, as of GABAergic signaling to the observed and S. G. of the gene cluster the Genet. Scholar, S. L. T. G. A microRNA cluster as a for in Scholar). this on the of genes involved in genes involved in GABAergic signaling were found to be S. L. T. G. A microRNA cluster as a for in which the that of is of a regulatory network that GABAergic the of the results in this is to be we a regulation of an miRNA and of GABAergic were and were by the of the and were in containing and were in an with RNA was to with to the to complementary the RNA was with was and to downstream of the pri-miRNA were with A in to by and to of the pri-miRNA were then with in to by and The was and then to single and the The were by The were and with to and The was the and the RNA were as was by the the RNA and on an the of RNA and RNA was by and were the of the and were to was the for short were to genome were to edited were and to for with RNA editing for was E. G. RNA editing were and and of short to the human were and to the the to A and on to are the for A of was and into The A to editing site in and was by to the and miR-376b-3p, with and to miRNA, were by into the site of miRNA Expression The of complementary to miRNA targeting of and the containing one or were by or and into the as miRNA target were the or to the Expression for and ADAR2 were by of the of the genes The were into the was to the and ADAR2 A.M. G. RNA editing by of dsRNA as 2015; Scholar, is in the ADAR2 and for RNA 2005; Scholar). 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A to gene and gene targets edited or not edited miRNA were to the biological were with a A was expression of were with containing protein was with and were on to and The protein were with the primary and protein were with the primary and of cell surface and was on to the was on the protein as as well as protein with and were a of protein was with the and were with In to in intracellular were with a of a were in with GABA well a were in by a of and on the with a and a was with a was by and was for for single with and were on in and in with were with The were then with to and with containing or were with an The were then with the function with a of a were with the and the function for the cell was for and for were as and was by or of were and the are the are the for This contains The that of with the of this We for on the and critical of the for with cell and for We the in by for the and and the and for for for with and to the A. E. A. and A. and E. A. S. A. and A. W. and A. W. and E. 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