Identification and characterization of microRNAs in white and brown alpaca skin
© Tian et al.; licensee BioMed Central Ltd. 2012
Received: 9 April 2012
Accepted: 8 October 2012
Published: 16 October 2012
MicroRNAs (miRNAs) are small, non-coding 21–25 nt RNA molecules that play an important role in regulating gene expression. Little is known about the expression profiles and functions of miRNAs in skin and their role in pigmentation. Alpacas have more than 22 natural coat colors, more than any other fiber producing species. To better understand the role of miRNAs in control of coat color we performed a comprehensive analysis of miRNA expression profiles in skin of white versus brown alpacas.
Two small RNA libraries from white alpaca (WA) and brown alpaca (BA) skin were sequenced with the aid of Illumina sequencing technology. 272 and 267 conserved miRNAs were obtained from the WA and BA skin libraries, respectively. Of these conserved miRNAs, 35 and 13 were more abundant in WA and BA skin, respectively. The targets of these miRNAs were predicted and grouped based on Gene Ontology and KEGG pathway analysis. Many predicted target genes for these miRNAs are involved in the melanogenesis pathway controlling pigmentation. In addition to the conserved miRNAs, we also obtained 22 potentially novel miRNAs from the WA and BA skin libraries.
This study represents the first comprehensive survey of miRNAs expressed in skin of animals of different coat colors by deep sequencing analysis. We discovered a collection of miRNAs that are differentially expressed in WA and BA skin. The results suggest important potential functions of miRNAs in coat color regulation.
KeywordsAlpaca MicroRNAs Deep sequencing Coat color Skin
Since the first small RNA (sRNA) – lin4 was discovered in C. elegans, identification of small endogenous non-coding RNA (sncRNA) has received considerable attention. SncRNAs include small interfering RNA, miRNA and piwi-interacting RNA, which all regulate genes at the post-transcriptional level. Among these sncRNAs, miRNAs are small, non-coding 21–25 nt RNA molecules that play an important role in regulating gene expression in animals and plants by promoting mRNA degradation and inhibiting mRNA translation. However, other studies revealed that some miRNAs may also function to induce gene expression. Many miRNAs are evolutionary conserved in related species and some even show conservation between invertebrates and vertebrates. Generally, the functions of miRNAs are not limited to the regulation of developmentally timed events. Instead, they have diverse expression patterns and probably regulate many aspects of development and physiology. To date, the miRBase database contains 9072 mature miRNA products from 25 mammalian species. Recently, transcriptome profiling of miRNAs has led to identification of novel miRNAs, provided valuable information on tissue specific expression and evolutionary conservation across species, and served as a cornerstone for subsequent functional analyses.
In adult animals, both hair and skin color depend on pigment produced by melanocytes at the base of the epithelium. Melanocytes in mammals and birds produce two types of melanin, black to brown eumelanin and yellow to reddish brown pheomelanin[9, 10]. The quality and ratio of eumelanin to pheomelanin influence the color of skin, fleece and eyes. At present, a large number of genes have been found to affect hair and skin color in humans and other vertebrate species[11–13], although the molecular and cellular mechanisms regulating coat color in fiber-producing species, such as the alpaca, are not completely understood.
Alpacas have a great variety of natural coat colors. Studies of the melanocortin-1 receptor (MC1R) and agouti signaling protein (ASIP) genes in alpacas of different coat colors revealed several polymorphisms that may be predictive of coat color[14–18]. Powell et al. reported that the M87V and S126G variants of MC1R may affect coat color with brown coat color animals showing homozygosity for the methionine and serine alleles. Tyrosinase related protein 1 (TYRP1) is encoded by the brown locus and is involved in eumelanin synthesis. Mutations in TYRP1 are associated with brown coat color in many species[20, 21], but mutations in TYRP1 associated with brown coat color in alpacas have not been reported to date. Previous studies in our laboratory revealed that the expression of many coat color genes including MC1R, TYRP1, tyrosinase (TYR), tyrosinase related protein 2 (TYRP2), nitric oxide synthase 2 (NOS2), paired box protein 3 (PAX3), and WNT3A was higher in skin of brown versus white alpacas, suggestive of a functional contribution to coat color regulation in this species. However, the potential contribution of miRNAs to differential expression (associated with coat color) of such genes has not been fully elucidated, primarily due to a lack of knowledge of the miRNA transcriptome profile in alpaca skin.
Previous studies have addressed expression and function of miRNAs in mouse, goat and sheep skin[22–25]. For example, miR-203, a skin specific miRNA, was up-regulated in psoriatic plaques and shown to be involved in inflammatory responses and keratinocyte functions. However, studies of the functional role of miRNAs in coat color regulation are limited. Dong et al. over-expressed miR-137 in mice and obtained a series of coat color changes in transgenic animals. Zhu et al. reported differential expression of miR-25 in skin of white versus brown alpacas and a functional role of this miRNA in the regulation of expression of microphthalmia-associated transcription factor (MITF), a transcription factor regulating expression of key genes involved in melanogenesis.
To better understand the potential role of miRNAs in the post-transcriptional regulation of genes linked to pigmentation, we have characterized the miRNA transcriptome in skin of alpaca of different coat colors by deep sequencing analysis. We report here the identification of hundreds of conserved miRNAs and candidate novel miRNAs in WA and BA skin. We also provide evidence of differential expression of miRNAs in WA and BA, foundational to future studies of the potential functional role for such miRNAs in post-transcriptional gene regulation linked to coat color.
Overall complexity of sRNA pools between the WA and BA skin libraries
Identification of conserved miRNAs in alpaca skin
Differential expression of conserved miRNAs in white and brown alpaca skin
Significantly expressed miRNAs in white and brown alpaca skin
NEL in brown
NEL in white
Sequence divergence within known miRNAs
Target gene prediction for miRNAs in alpaca
Targets for top 4 significantly expressed miRNAs in white and brown alpaca involved in melanogenesis
Predicted targets involved in melanogenesis
CPNE8, PRKCG, TMEM135, ARMC8, DCT, PLCB1,WNT3
GNAI2, TCF7, CREB3, FZD7, MYOF, PRKACB, KIT, GNAI1, PLCB1, SYT11, SYT1, MCTP1, FZD1, SYT4, CALM
CPNE8,TCF7, KRAS, WNT7A, PRKCB, KIT, CAMK2D, CABP2, EDNRB, WNT5A, FZD4
CAMK2A, FAM193B, MYOF, ADCY5, ADCY8, PRKCB, PLCB3, RAF1, SYT1, WNT11, PRKACA, WNT16, PLCB2
Identification of novel miRNAs in alpaca skin
Novel miRNAs identified in alpaca skin
Studies on expression and function of miRNAs in evolutionarily diverse agricultural species are limited. The alpaca is a domestic mammal specialized in fiber production and has more than 22 natural coat colors[40–44]. In this report, we explored the miRNA transcriptome in alpaca skin using deep sequencing technology, and provided a foundation for future functional studies on the relationship between miRNA expression and development and function of skin including coat color.
Many miRNAs have been identified by traditional Sanger sequencing and microarray methods. However, the high throughout-sequencing method which yields 400,000 reads per run and identifies low abundance tags is a much more comprehensive method for miRNA profiling. Using this sequencing approach, we found 22 novel, as well as 272 and 267 conserved miRNAs in WA and BA skin libraries. According to the new miRNA family classifications in Rfam (miRBase 17.0), 272 miRNAs from WA and 267 miRNAs from BA were grouped into 155 families. Most of these identified miRNA families are also conserved in other mammalian species, such as mouse[23, 46], human, goat and sheep. Expression of these conserved alpaca miRNA families differed between WA and BA skin, based on both abundance and representation within miRNA families. For example, miR-146 and miR-8 families both had abundant reads in WA and BA skin libraries, but miR-146b (miR-146 family) and miR-141 (miR-8 family) were of greater abundance in WA than BA. This significant variation maybe attributed to the timing and location of miRNA expression during skin development in adult alpacas. In many miRNA families, there was a predominant member whose reads were significantly higher than other members. For example, the Let-7 miRNA family contains 10 members. The reads for let-7a were more than one million, but let-7i only had five thousand reads. It is possible that the predominant member in each miRNA was responsible for the regulatory role at the developmental stage in which skin samples were collected in the present studies.
We compared the expression of conserved miRNAs between WA and BA skin and identified differentially expressed miRNAs from the two libraries. The expression of miRNAs has an established relationship with cell proliferation and differentiation. As such, differences in miRNA expression between animals possibly reflect alterations in growth and differentiation. Heterochrony, or change in the timing of developmental growth, is known to alter the overall shape of developing tissues, leading to differences in physiology. Alternatively, there are pronounced phenotypic differences between WA and BA which include fiber diameter, staple length, hair color and other hair characteristics[48, 49]. Thus, differences in miRNA abundance may also be related to hair color and other fiber characteristics. The functional role of such differentially expressed miRNAs is the focus of future investigation.
In animals, miRNAs target mRNAs with partial sequence complementarity. One miRNA is inferred to influence expression of hundreds of mRNAs via this flexible recognition. There were many differentially expressed miRNAs identified between WA and BA skin, so the next step was to search for putative specific genes targeted by these miRNAs. The major physiological difference of interest between the samples analyzed was coat color, so we focused on potential target genes regulating pigmentation. Based on KEGG pathway analysis, we found 117 potential miRNA target genes involved in melanogenesis. Many miRNAs targeting these genes have been reported. For examples, miR-137 and miR-25 were previously shown to regulate the expression of MITF, which is an important regulator of melanocyte growth, maturation, apoptosis and melanogenesis. In the present study, miR-137 and miR-25 were found in both libraries and their target genes also included MITF. Haflidadóttir et al. showed that miR-148 affects Mitf mRNA expression in melanoma cells through a conserved binding site in the 3’-UTR sequences of mouse and human Mitf. In our studies, miR-148 showed greater expression in WA versus BA. So, it is possible that miR-148 may contribute to coat color differences between WA and BA.
The present studies also revealed that both miR-193a and miR-193b are differentially expressed in WA and BA skin. Gao et al. reported that Kit expression was subject to post-transcriptional regulation by miR-193a and miR-193b in human acute myeloid leukemia[53, 54]. In humans, Kit contributes to the regulation of skin pigmentation and we have previously reported Kit expression is higher in skin of BA versus WA. Thus, miR-193a and miR-193b may play a role in coat color determination in alpacas via regulating the expression of Kit.
Studies reported provide novel information on miRNA expression in alpaca species, including conserved miRNAs and potential novel miRNAs, and insight into differential miRNA expression associated with coat color and respective target genes. The predicted targets of the conserved miRNAs include 117 genes related to melanogenesis. Further validation of the functional role of differentially expressed miRNAs and their targets will provide better understanding of the miRNA regulatory mechanisms influencing coat color.
Sample collection and total RNA extraction
Housing and care of alpacas and collection of skin samples for use in described experiments were conducted in accordance with the International Guiding Principles for Biomedical Research Involving Animals (http://www.cioms.ch/frame 1985 texts of guidelines. html). Three white and three brown healthy adult alpaca (lama paco) were selected from the alpaca farm in Shanxi Agriculture University (Shanxi, China) for sample collection. Three pieces of skin (2cm ×3 cm) from the hindquarter were collected under local anesthesia and immediately stored in liquid nitrogen. Total RNA from the samples was extracted using Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. The RNA integrity was evaluated by gel electrophoresis and the RNA concentration was measured by OD260 reading using a Nanodrop spectrophotometer.
Small RNA library preparation and sequencing
For deep sequencing, total RNA samples were size-fractionated on a 15% PAGE gel, and the 16–30 nt fraction was collected. The 5’RNA adaptor and 3’RNA adaptor were ligated to the RNA pools, and the RNAs of 64–99 nts were isolated through gel elution and ethanol precipitation. PCR products were purified and small RNA libraries were sequenced using Illumina Genome Analyzer. Sequencing was carried out at the ShenZheng Genomics Institute (BGI, China).
Initial processing of reads and identification of conserved miRNAs
Raw sequence reads were produced by the Illumina HiSeq2000 and processed into clean full length reads by the BGI small RNA pipeline. During the procedure, all low quality reads, including 3’ and 5’ adapter contaminants were removed. The remaining high quality sequences larger than 30nt and smaller than 18nt were discarded. Then, all high quality sequences were mapped to Vicugna pacos genome (http://www.ncbi.nlm.nih.gov/nuccore/206581138) and Bos Taurus genome (http://hgdownload.cse.ucsc.edu/goldenPath/bosTau4/bigZips/bosTau4.fa.gz) by SOAP. All clean reads were compared with the sequences of non-coding RNA (rRNA, tRNA, snRNA, snoRNA) available in Rfam (http://www.sanger.ac.uk/software/Rfam) and the GenBank non-coding RNA database (http://www.ncbi.nlm.nih.gov/) to classify degradation fragments of non-coding RNA. We followed the rule: rRNAetc, in which Genbank > Rfam> known miRNA > repeat > exon > intron. Subsequently, the remaining sequences were aligned to the whole animal miRNAs deposited in miRBase 17.0 to obtain the known miRNAs in alpaca as well as base bias at the first position of identified miRNAs and at each position of all identified miRNAs respectively. If a known miRNA in alpaca showed homology with less than two mismatches (or >90% homology), it was considered as evolutionary conserved.
Validation of conserved miRNA expression
Ten conserved miRNAs were selected for validation by quantitative real time PCR analysis. Total RNA was isolated from eight alpaca skin samples (4 brown, 4 white). One μg of DNase-treated RNA was converted to cDNA using specific stem-loop RT primer (Additional file5) and HiFi-MMLV cDNA kit mix (CWBIO, Beijing, China). The cDNA was then used for real time PCR quantification of miRNAs using miRNA specific primers (Additional file5) in combination with the universal primer. U6 snRNA was used as an endogenous control (primers listed in Additional file5). Quantitative real-time PCR was performed in triplicate on the Stratagene Mx3005P system. The 25 μL PCR reaction included 12.5μL SYBR Premix Ex TaqTM II (TaKaRa, Dalian, China), 1.0 μL specific forward primer, 1μL universal primer, 0.5 μL ROX reference dye, 2.5 μL diluted (4 times) cDNA and 7.5 μL water. Cycling parameters were 95°C for 30 sec followed by 40 cycles of 95°C for 5 sec, 56 °C or 58 °C for 20 sec and 72 °C for 15 sec. Melting curve analyses were performed following amplifications. Quantification of selected miRNA transcript abundance was performed using the comparative threshold cycle (CT) method established by Livak et al.. The abundance of selected miRNAs was normalized relative to that of U6 snRNA.
Identification of novel miRNAs
The prediction of alpaca candidate novel miRNAs was conducted using criteria that were previously developed for animal miRNA prediction. The criteria were as follows: the putative mature sequence must reside at the stem region and its size was limited to 20-24nt; the frequency of putative mature sequence should not be below 5; the folding free energy of the stem-loop structure was limited to below −18 kcal mol-1; the maximum tolerance of a bugle size was 4 nucleotides; the maximum size of difference between miRNA and miRNA* was 5 nucleotides and 35 nucleotides; the sequence asymmetry between miRNA and miRNA* could not exceed 5 nucleotides. The selected sequences were then folded into a secondary structure using mFold3.2 (http://mfold.rit.albany.edu). If a perfect stem-loop structure was formed, the small RNA sequence was located at one arm of the stem and met the above criteria, the small RNA was then considered as a candidate novel alpaca miRNA. The formula for calculating MFEI was: MFEI = [(MFE / length of the RNA sequence) ×100] / (G + C )%].
Target gene prediction
Mireap software (https://sourceforge.net/projects/mireap/) was used to predict the target genes for conserved miRNAs identified. The criteria were as follows: no more than four mismatches between miRNA & target (G-U bases count as 0.5 mismatches); no more than two adjacent mismatches in positions 2–12 of the miRNA/target duplex (5′ of miRNA); no mismatches in positions 10–11 of miRNA/target duplex; no more than 2.5 mismatches in positions 1–12 of the miRNA/target duplex (5′ of miRNA); MFE of the miRNA/target duplex should be >75% of the miRNA bound to its perfect complement.
Small endogenous non-coding RNA
Agouti signaling protein
Tyrosinase related protein 1
Tyrosinase related protein 2
Nitric oxide synthase 2
Paired box protein 3
Minimum free energy
Microphthalmia-associated transcription factor
We thank Dr. Xinmin Li (Department of Pathology & Laboratory Medicine, David Geffer School of Medicine, UCLA), professor Lihua Lü, Yun Zhang for useful advice, discussion, and assistance and Mr. Wang Ying (China alpaca farm, Zhuangzi, Yuci, China) for providing experimental animals. The study was sponsored by the National Natural Science Foundation of China (30571070, 2006031060).
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