MicroRNA (miRNA) Transcriptome of Mouse Retina and Identification of a Sensory Organ-specific miRNA Cluster
2007; Elsevier BV; Volume: 282; Issue: 34 Linguagem: Inglês
10.1074/jbc.m700501200
ISSN1083-351X
AutoresShunbin Xu, P. Dane Witmer, Stephen Lumayag, Beatrix Kovacs, David Valle,
Tópico(s)interferon and immune responses
ResumoAlthough microRNAs (miRNAs) provide a newly recognized level of regulation of gene expression, the miRNA transcriptome of the retina and the contributions of miRNAs to retinal development and function are largely unknown. To begin to understand the functions of miRNAs in retina, we compared miRNA expression profiles in adult mouse retina, brain, and heart by microarray analysis. Our results show that at least 78 miRNAs are expressed in adult mouse retina, 21 of which are potentially retina-specific. Among these, we identified a polycistronic, sensory organ-specific paralogous miRNA cluster that includes miR-96, miR-182, and miR-183 on mouse chromosome 6qA3 with conservation of synteny to human chromosome 7q32.2. In situ hybridization showed that members of this cluster are expressed in photoreceptors, retinal bipolar and amacrine cells. Consistent with their genomic organization, these miRNAs have a similar expression pattern during development with abundance increasing postnatally and peaking in adult retina. Target prediction and in vitro functional studies showed that MITF, a transcription factor required for the establishment and maintenance of retinal pigmented epithelium, is a direct target of miR-96 and miR-182. Additionally, to identify miRNAs potentially involved in circadian rhythm regulation of the retina, we performed miRNA expression profiling with retinal RNA harvested at noon (Zeitgeber time 5) and midnight (Zeitgeber time 17) and identified a subgroup of 12 miRNAs, including members of the miR-183/96/182 cluster with diurnal variation in expression pattern. Our results suggest that miR-96 and miR-182 are involved in circadian rhythm regulation, perhaps by modulating the expression of adenylyl cyclase VI (ADCY6). Although microRNAs (miRNAs) provide a newly recognized level of regulation of gene expression, the miRNA transcriptome of the retina and the contributions of miRNAs to retinal development and function are largely unknown. To begin to understand the functions of miRNAs in retina, we compared miRNA expression profiles in adult mouse retina, brain, and heart by microarray analysis. Our results show that at least 78 miRNAs are expressed in adult mouse retina, 21 of which are potentially retina-specific. Among these, we identified a polycistronic, sensory organ-specific paralogous miRNA cluster that includes miR-96, miR-182, and miR-183 on mouse chromosome 6qA3 with conservation of synteny to human chromosome 7q32.2. In situ hybridization showed that members of this cluster are expressed in photoreceptors, retinal bipolar and amacrine cells. Consistent with their genomic organization, these miRNAs have a similar expression pattern during development with abundance increasing postnatally and peaking in adult retina. Target prediction and in vitro functional studies showed that MITF, a transcription factor required for the establishment and maintenance of retinal pigmented epithelium, is a direct target of miR-96 and miR-182. Additionally, to identify miRNAs potentially involved in circadian rhythm regulation of the retina, we performed miRNA expression profiling with retinal RNA harvested at noon (Zeitgeber time 5) and midnight (Zeitgeber time 17) and identified a subgroup of 12 miRNAs, including members of the miR-183/96/182 cluster with diurnal variation in expression pattern. Our results suggest that miR-96 and miR-182 are involved in circadian rhythm regulation, perhaps by modulating the expression of adenylyl cyclase VI (ADCY6). MicroRNAs (miRNAs) 3The abbreviations used are: miRNAmicroRNARPEretinal pigmented epitheliumpri-miRNAprimary miRNAZTZeitgeber timeqRTquantitative reverse transcriptionEembryonic dayPpostnatal dayUTRuntranslated regionntnucleotide 3The abbreviations used are: miRNAmicroRNARPEretinal pigmented epitheliumpri-miRNAprimary miRNAZTZeitgeber timeqRTquantitative reverse transcriptionEembryonic dayPpostnatal dayUTRuntranslated regionntnucleotide are small, noncoding, regulatory RNAs of 18–24 nucleotides in length found in all metazoans. Since their discovery in 1993, at least 100 different miRNA genes have been documented in the genomes of Drosophila and Caenorhabditis elegans and more than 250 in vertebrate genomes (1Bartel D.P. Cell. 2004; 116: 281-297Abstract Full Text Full Text PDF PubMed Scopus (26540) Google Scholar) with recent estimates as high as 800 (2Bentwich I. Avniel A. Karov Y. Aharonov R. Gilad S. Barad O. Barzilai A. Einat P. Einav U. Meiri E. Sharon E. Spector Y. Bentwich Z. Nat. Genet. 2005; 37: 766-770Crossref PubMed Scopus (1487) Google Scholar). By influencing translation and stability of mRNAs, miRNAs contribute a newly recognized level of regulation of gene expression affecting a variety of biological processes. miRNAs are transcribed by RNA polymerase II as transcripts (pri-miRNAs) that are capped, polyadenylated, and spliced (3Lee Y. Kim M. Han J. Yeom K.H. Lee S. Baek S.H. Kim V.N. EMBO J. 2004; 23: 4051-4060Crossref PubMed Scopus (2932) Google Scholar). pri-miRNAs fold into hairpin structures that are cleaved by an RNase III endonuclease, the Drosha-DGCR8 complex, to form 60–70-nt stem loop intermediates known as pre-miRNA (1Bartel D.P. Cell. 2004; 116: 281-297Abstract Full Text Full Text PDF PubMed Scopus (26540) Google Scholar, 4Lee Y. Jeon K. Lee J.T. Kim S. Kim V.N. EMBO J. 2002; 21: 4663-4670Crossref PubMed Scopus (1550) Google Scholar, 5Zeng Y. Yi R. Cullen B.R. Proc. Natl. Acad. Sci. U. S. 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Despite this progress in understanding miRNA functions and disease associations, little is known about the miRNA complement of mammalian retina. One recent report focused on miRNA expression of the anterior segment of the mouse eye but does list 10 miRNAs abundant in retina (35Ryan D.G. Oliveira-Fernandes M. Lavker R.M. Mol. Vis. 2006; 12: 1175-1184PubMed Google Scholar), another reports cloning of 9 miRNAs from the eye of the newt (36Makarev E. Spence J.R. Del Rio-Tsonis K. Tsonis P.A. Mol. Vis. 2006; 12: 1386-1391PubMed Google Scholar), and a third described the spatiotemporal expression of 7 miRNAs in embryonic and postnatal mouse eye (37Karali M. Peluso I. Marigo V. Banfi S. Investig. Ophthalmol. Vis. Sci. 2007; 48: 509-515Crossref PubMed Scopus (0) Google Scholar). Retina is a derivative of the forebrain ectoderm, and regulation of gene expression through intrinsic and extrinsic factors ensures a tightly controlled temporal and spatial developmental sequence (38Cepko C.L. Austin C.P. Yang X. Alexiades M. Ezzeddine D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 589-595Crossref PubMed Scopus (790) Google Scholar). It is likely that, as in other tissues, miRNAs play important roles in the normal development and function of the retina. To better understand these functions, we used microarray analysis to determine the expression pattern of miRNAs in mouse retina as compared with brain and heart. We find at least 78 miRNAs are expressed in adult mouse retina, 21 of which are expressed preferentially or specifically in retina, including a cluster of three paralogous miRNAs that are also expressed in other neurosensory tissues. We also present evidence for circadian cycling in the expression of certain of these miRNAs. Mouse RNA Samples—Wild type SVJ129 mice and retinal degeneration mice, rd1, were purchased from The Jackson Laboratories. Mice were kept in 12-h light/12-h dark cycles with lights on at 7:00 a.m. (ZT0) and the lights off at 7:00 p.m. (ZT12). Total retinal RNA was prepared from adult SVJ129 mice (∼3 months old) at 12:00 p.m. (ZT5) or 12:00 a.m. (ZT17) using mirVana miRNA isolation system (Ambion). Mice were anesthetized with pentobarbital (75 mg/kg intraperitoneally) and sacrificed by cervical dislocation. The eyes were enucleated, and the retina was quickly removed and submerged in 300 μl of lysis/binding buffer, homogenized with a plastic pestle homogenizer. Thereafter, the manufacturer's protocol for isolation of small RNA-enriched total RNA was followed (Ambion). Adult mouse total RNA samples from brain, heart, thymus, lung, liver, spleen, testicle, ovary, kidney, and embryonic day 10 embryos of Swiss Webster mice were purchased from Ambion. For the circadian rhythm study, total retinal RNA was isolated at ZT1, ZT5, ZT9, ZT13, ZT17, and ZT21. At each time point, three mice were sacrificed, and total retinal RNA from each mouse was prepared separately. Adult rd1 mouse (∼4 months old) retinal RNA was prepared as described above. Mouse olfactory epithelium total RNA was prepared as described previously (39Xu S. Wang Y. Zhao H. Zhang L. Xiong W. Yau K.W. Hiel H. Glowatzki E. Ryugo D.K. Valle D. Mol. Cell. Biol. 2004; 24: 9137-9151Crossref PubMed Scopus (13) Google Scholar) except that we used the mirVana miRNA isolation system. For mouse tongue epithelium total RNA preparation, we removed adult SVJ129 mouse tongues and soaked them in enzyme mixture (1 mg/ml collagenase A (Sigma), dispase (Invitrogen), and 1 mg/ml trypsin inhibitor (Roche Applied Science)) (40Huang Y.J. Maruyama Y. Lu K.S. Pereira E. Plonsky I. Baur J.E. Wu D. Roper S.D. J. Neurosci. 2005; 25: 843-847Crossref PubMed Scopus (150) Google Scholar) for 10 min. We also injected the mixture under the epithelium of the tongue. We peeled the epithelium off and soaked it in the lysis buffer of mirVana miRNA isolation system (Ambion) and prepared total RNA as described above. miRNA Labeling and Microarray Hybridization—We purified small RNA (<40 nt) from the total RNA samples using a FlashPAGE fractionator (Ambion) according to the manufacturer's protocol. We used the mirVana miRNA labeling kit (Ambion) and followed the Ambion protocol to label the miRNA with Cy3. We performed three independent microarray hybridizations with independent probes labeled from independent mouse retinal RNA preparations at ZT5 or ZT17. For the brain and heart profiling, we performed three independent microarray hybridizations with independently prepared probes from the total RNA purchased from Ambion. We used the mirVana miRNA bioarray (Ambion) for the miRNA profiling. This array contains 385 independent miRNAs each as a duplicate feature, representing a comprehensive panel of all human, mouse, and rat microRNAs in the miRNA Registry. We followed the manufacturers' protocol for hybridization. Microarray Scanning and Data Analysis—We used a Packard Biochip scanner at the Research Resource Center of the University of Illinois, Chicago, and ScanArray software to scan the arrays at 90–95% power and a photomultiplier of 75–80 and 5 μm resolution. The spot intensity was determined in QuantArray. The average intensity of the “empty” spots (negative controls) was calculated for each array as the background. We calculated the background-corrected signal intensity (BgCor Signal) according to the formula: BgCor Signal = spot intensity - (average of the empty spots). We reported values less than zero as zero. We normalized the BgCor Signal as a function of the average of the BgCor Signal of all noncontrol spots: normalized signal = BgCor Signal × 100/average of the BgCor Signal of all noncontrol spots. We calculated the average of the normalized intensity of the six spots for each miRNA gene (duplicate spots/array × three independent arrays) as the specific signal for each miRNA. We calculated significance of the specific signal as a two-tailed p value of Z-test (X = 0, σ = standard deviation of all noncontrol feature signals on array) for normalized signals. We identify a signal with p < 0.01 as a “positive” or as “expressed miRNA.” We removed the following features from analysis due to observation of apparent hybridization artifacts on the array images: BA10458, BA10440, BA10339, BA10333, BA10283, BA10442, BA10376, BA10245, BA10479, and BA10351. In the differential expression between day (ZT5) and night (ZT17) RNA samples, we calculated the fold of change for each miRNA as specific signal-ZT17/specific signal-ZT5. We evaluated the significance of differential expression using a two-tailed pairwise Student's t test between the specific signal intensity at ZT5 and the one at ZT17. The miRNA genes with p < 0.1 are identified as differentially expressed. RT-PCR—We purchased mirVana qRT-PCR primer sets of mmu-miR-7, mmu-miR-9, mmu-miR-31, mmu-miR-96, mmu-miR-181c, mmu-miR-182, mmu-miR-185, mmu-miR-194, mmu-miR-210, mmu-miR-219, mmu-miR-320, mmu-miR-335, mmu-miR-361, and mirVana qRT-PCR miRNA detection kit from Ambion and used 5 S rRNA as a normalizing control. For real time quantitative PCR (qPCR), we used 10 ng of total RNA and an Opticon 2 real time detector in a DNA engine 2 (Bio-Rad) with SYBR Green I for detection. For end product PCR, we used a DNA Engine Dyad Peltier thermal cycler and separated the product on 3.5% high resolution agarose electro-phoresis gel. We used NCod SYBR Green miRNA first-strand synthesis and qRT-PCR system (Invitrogen) to amplify mmu-miR-9-AS, mmu-miR-25, mmu-miR-92, mmu-miR-106b, mmu-miR-183, mmu-miR-184, mmu-miR-211, mmu-miR-140-AS, and rno-miR151-AS with the forward primer (Sigma Genosys) sequences as the corresponding mature miRNA sequences. For the circadian rhythm studies on miR-182 and miR-96, we amplified 10 ng of total mouse retinal RNA prepared at ZT1, ZT5, ZT9, ZT13, ZT17, and ZT21. Three RNA samples from each of three mice were amplified at each time point. The relative quantity of the miR-182 and miR-96 was normalized to the 5 S RNA quantitation. We used a nonparametric one-way analysis of variance test to determine the significance of the differences. For the circadian rhythm study on Adcy6 expression, we employed a QuantiFast SYBR Green RT-PCR system (Qiagen) with Mm_Adcy6_1_SG QuantiTect primer set (Qiagen, QT00136850) and used 18 S rRNA amplified with primer set (Qiagen, QT01036875) to normalize. We followed the manufacturer's protocol for the qRT-PCRs. In Situ Hybridization—We purchased 5′-digoxigenin-labeled miRCURY LNA detection probes for mmu-miR-182, mmu-miR-183, and mmu-miR-96 from Exiqon and used ∼6 pmol of probe on each section. We prepared fresh-frozen adult mouse retinal sections (10 μm) and followed the manufacturer's protocol with minor modifications. We prehybridized the sections in hybridization buffer (50% formamide, 0.3 m NaCl, 20 mm Tris-HCl, pH 8.0, 5 mm EDTA, 10 mm NaPO4, pH 8.0, 10% dextran sulfate, 1× Denhardt's, 0.5 mg/ml baker yeast RNA) for 1 h at room temperature. We diluted ∼6 pmol of the probe in 300 μl of hybridization buffer for hybridization. The hybridizations were carried out at 52 °C overnight in a humidified chamber. Luciferase Reporter Assays of miRNA Targeting—We utilized RT-PCR to amplify and subclone fragments (SpeI/HindIII) of the 3′-UTR of ADCY6 (nt 4206–6520 of GenBank™ accession number NM_015270, containing four potential target sites for miR-96 and miR-182) or MITF (nt 3219–4654 of GenBank™ accession number NM_198159, containing four potential target sites for miR-96 and for miR-182) into the luciferase reporter vector, pMIR-REPORT (Ambion), 3′ to the firefly luciferase cassette. We plated HEK293 cells at a density of 5 × 105 cells/well in 24-well plates coated with poly-d-lysine (Sigma) and transfected them with 150 ng of pMIR reporter construct (pMIR-REPORT-3′-UTR/Adcy6 or pMIR-REPORT-3′-UTR/Mitf), 15 ng of hpRL-SV40 (Promega), and 5–10 pmol of the specified miRNA mimics or control oligonucleotide with a scrambled sequence (Dharmacon) using Lipofectamine 2000 (Invitrogen). Using the dual luciferase assay kit (Promega), we measured firefly luciferase activity 48 h later and normalized to Renilla luciferase activity. We performed at least three independent experiments for each assay. The miRNA Transcriptomes of Mouse Retina, Brain, and Heart—Using a microarray (Ambion), we profiled the expression of miRNAs in small RNA-enriched total RNA from mouse retina, brain, and heart. We identified at least 78 miRNAs expressed in retina (Fig. 1) (supplemental Table 1). Of these, 40 (∼51%) are expressed in all three tissues (supplemental Table 8). These widely expressed miRNAs may represent a set of “housekeeping” miRNAs important for regulation of the basic cellular functions in all tissues. Of the remaining 38 retinal miRNAs, 12 were also expressed in brain but not in heart (supplemental Table 2A); five were expressed in heart but not brain (supplemental Table 2B); and 21 were detected only in retina making them candidate retina-specific miRNAs (Fig. 1 and Table 1). We also identified two miRNAs (miR-143 and Ambi_miR_7029) that were expressed in brain and heart but not retina (supplemental Table 7). Overall, the miRNA expression pattern of retina more closely resembled that of brain as compared with heart, in both the number of shared miRNAs and in the levels of their expression (Fig. 1, supplemental Tables 2 and 8–10).TABLE 1miRNAs expressed in retina but not in brain or heartNo.Namems chr locationhs chr locationAverage retina (ZT5)Average retina (ZT17)1hsa_miR_1826qA37q32.2693.9869.62hsa_miR_1836qA37q32.2491.7583.03hsa_miR_1849qE3.315q25.1401.5380.24mmu_miR_2117qC15q13.3391.4445.65hsa_miR_2107F511q15.5368.4443.06hsa_miR_966qA37q32.2368.3416.97hsa_miR_314qC49p21.3325.6303.58hsa_miR_181c8C319p13.12325.0233.69hsa_miR_3356qA37q32.2301.9181.010rno_miR_151_AS15qE1/10?8q24.3281.3254.611mmu_miR_140_AS8qD116q22.1273.2208.112hsa_miR_255qG17q22.1263.3116.013hsa_miR_361XqDXq21.2260.0255.814hsa_miR_32014qD18q21.3249.4305.515hsa_miR_21917qB2/2qA36p21.32/9q34.11193.2194.016hsa_miR_9214/XqA513q31.3/Xq26.2192.7188.317hsa_miR_1941qH4/19qA1q41/11q13.1189.1155.618hsa_miR_9_AS9qF1/13qC3/7qD21q23.1167.1109.319hsa_miR_713qB2/7qD2/17qD9q21.33140.9161.020hsa_miR_18516qB122q11.21119.0147.121hsa_miR_106b5qG17q22.157.4188.2 Open table in a new tab Confirmation of the Tissue Specificity of the Retinal miRNAs—We performed quantitative RT-PCR assays for all apparently retina-specific miRNAs in RNA isolated from retina, brain, and eight other tissues (heart, thymus, lung, liver, spleen, kidney, testis, and ovary) and embryonic day (E) 10 whole mouse embryos. We confirmed that all except miR-361 are expressed in retina (Fig. 2 and supplemental Fig. 1). Interestingly, we found that miR-96, miR-182, miR-183, miR-184, miR-210, and miR-140-AS are all highly expressed in adult retina but are not detectable in RNA from brain or the other tissues. This high level of expression with apparent specificity suggests that these miRNAs may play important roles in the normal function of retina. One of the miRNAs, miR-210, is also expressed in E10 embryos (Fig. 2 and supplemental Fig. 1), but not in the other adult tissues and may have a role in early retinal development. We also found nine miRNAs (miR-7, miR-9, miR-9-AS, miR-31, miR-181c, miR-211, miR-219, miR-320, and miR-335) that are preferentially expressed in retina or retina/brain with low levels of expression in various other tissues (Fig. 2 and supplemental Fig. 1). miR-25, miR-92, miR-194, and miR-151-AS are expressed in several other tissues in addition to retina. miR-106b and miR-185 are expressed at low level in most of the tissues. Developmental Patterns of Expression—To study the developmental time course of the 21 miRNAs shown to be specifically or preferentially expressed in retina, we isolated mouse total retinal RNA from eye cups of embryos at E10, and the developing retinas of embryos at E14 and E18, as well as from retinas of postnatal days 1 (P1) and 10 (P10) mice and performed qRT-PCR (Fig. 3 and supplemental Table 13). In the E10 samples, all of the assayed miRNAs have little or no expression. Most (17/21) showed a pattern of increasing expression with development peaking at P10 or adult (Fig. 3, A–C). Of these, 14 showed at least a 10-fold increase of expression at the peak time (adult or P10) (supplemental Table 13), compared with E10. Eight (miR-183, miR-182, mir-96, miR-9_AS, miR-184, miR-211, miR-151_AS, and miR-140_AS) of the 12 miRNAs with peak expression in adult retina showed at least a 10-fold increase from P1 to adult, suggesting that these miRNAs correspond to the time when the late retinal progenitor cells are differentiating into the mature retinal cell types and functioning as mature retinal neurons and/or Muller glia. Four of the miRNAs (miR-335, miR-219, miR-194, and miR-185) showed peak expression at E14 and E18.5 (Fig. 3D and supplemental Table 13), whereas other miRNAs (miR-151_AS, miR-211, miR-184, miR-25, and miR-140_AS) showed a moderate increase at E14 or E18 (Fig. 3, B and C, and supplemental Table 13), suggesting that these miRNAs may have stage-specific functions. Identification of a Sensory Tissue-specific, Paralogous miRNA Cluster—The genes for three of the highly expressed retinal miRNAs, miR-183, miR-96, and miR-182, are clustered within 4 kb on mouse chr6qA3, transcribed in the same direction (telomere → centromere), and located in an ∼60-kb gap between the genes encoding nuclear respiratory factor-1 (Nrf1) and ubiquitin-conjugating enzyme E2H (Ube2h). The orthologous region in humans is 7q32.2, where hsa-miR-183, miR-96, and miR-182 have similar arrangement (Fig. 4A). The sequences of these three miRNAs are similar to one another as follow: in mouse, miR-96 has 52 and 56% identity with miR-182 and miR-183, respectively; miR-182 and miR-183 are 78% identical (Fig. 4B). All three of these miRNAs have a similar developmental expression pattern (Fig. 4D). The sequence similarity, clustered location, common orientation, and shared developmental pattern suggest that these three miRNAs comprise a paralogous cluster transcr
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