Identification and Profiling of MicroRNAs from Skeletal Muscle of the Common Carp

Citation: Yan X, Ding L, Li Y, Zhang X, Liang Y, et al. ( Identification and Profiling of MicroRNAs from Skeletal Muscle of the Common Carp Xuechun Yan 0 Lei Ding 0 Yunchao Li 0 Xiaofeng Zhang 0 Yang Liang 0 Xiaowen Sun 0 Chun-Bo Teng 0 Zhanjiang Liu, Auburn University, United States of America 0 1 Key Laboratory of Aquatic Biotechnology, Heilongjiang River Fisheries Research Institute, Chinese Academy of Fishery Sciences , Harbin , China , 2 College of Life Sciences, Northeast Forestry University , Harbin , China The common carp is one of the most important cultivated species in the world of freshwater aquaculture. The cultivation of this species is particularly productive due to its high skeletal muscle mass; however, the molecular mechanisms of skeletal muscle development in the common carp remain unknown. It has been shown that a class of non-coding ,22 nucleotide RNAs called microRNAs (miRNAs) play important roles in vertebrate development. They regulate gene expression through sequence-specific interactions with the 39 untranslated regions (UTRs) of target mRNAs and thereby cause translational repression or mRNA destabilization. Intriguingly, the role of miRNAs in the skeletal muscle development of the common carp remains unknown. In this study, a small-RNA cDNA library was constructed from the skeletal muscle of the common carp, and Solexa sequencing technology was used to perform high throughput sequencing of the library. Subsequent bioinformatics analysis identified 188 conserved miRNAs and 7 novel miRNAs in the carp skeletal muscle. The miRNA expression profiling showed that, miR-1, miR-133a-3p, and miR-206 were specifically expressed in muscle-containing organs, and that miR-1, miR-21, miR-26a, miR-27a, miR-133a-3p, miR-206, miR-214 and miR-222 were differentially expressed in the process of skeletal muscle development of the common carp. This study provides a first identification and profiling of miRNAs related to the muscle biology of the common carp. Their identification could provide clues leading towards a better understanding of the molecular mechanisms of carp skeletal muscle development. - Funding: This work was supported by the grants from the National High Technology Research and Development Program of China (863 Program) (No. 2009AA10Z105, 2011AA100400, 2011AA100404GRAM09), and the Fundamental Research Funds for the Central Universities (No. DL09CA16). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. . These authors contributed equally to this work. The common carp (Cyprinus Carpio L.) occupies a prominent position in the world of freshwater aquaculture and serves as a major source of animal protein for millions of people [1]. In spite of its aquacultural importance and a production increase in carp breeding over the last decade, the current productive species are mainly selected through traditional breeding methods, including the use of mutual hybrids and backcrosses to domesticated ancestors [2]. Molecular techniques are still not widely used in the breeding of the common carp due to a lack of genetic and genomic information. In recent years, several carp traits, including cold tolerance, body weight and plasma glucose contents, have been studied through development of molecular markers and construction of genetic linkage maps [35]. Although it is an important economic trait, the skeletal muscle mass of the common carp has not been well defined by molecular markers or control mechanisms to date. Several lines of evidence in mouse and zebrafish models have suggested that the development of vertebrate skeletal muscle is orchestrated by evolutionarily conserved gene expression networks in which transcription factors play important roles in regulating muscle growth and differentiation [6]. An essential myogenic transcription factor is the MADS box transcription factor myocyte enhancer factor-2 (MEF2), which activates the myogenic differentiation program in conjunction with the basic-helix-loop-helix (bHLH) transcription factors MyoD and myogenin in skeletal muscle [7]. Recent studies have shown that, in addition to transcriptional factors involved in muscle proliferation and differentiation, a set of microRNAs (miRNAs) also play important roles in skeletal muscle development in vertebrate animals [8,9]. miRNAs are a class of non-coding RNAs of approximately 22 nucleotides in length, which are processed from stem-loop RNA precursors (pre-miRNAs, ,65 nucleotides) by the ribonuclease III (RNase III) enzyme Dicer [10]. The pre-miRNAs are excised from the longer primary transcripts (pri-miRNAs) in the nucleus by the RNAse III enzyme Drosha [11]. The mature miRNAs exert their functions by incorporating one of the duplex strands into the RNA-induced silencing complexes (RISCs) and then binding the 39 untranslated region (39UTR) of the target mRNAs to degrade or repress their translation [12]. Many studies have demonstrated that miRNAs exist in a wide range of invertebrates and vertebrates, and that they are involved in the regulation of animal development, physiological functions and pathological processes [1316]. miRNAs can be identified through manual cloning and sequencing, miRNA array screening, and bioinformatic approaches [17,18]. Recently, high throughput sequencing technology has Figure 1. Anaylsis of small RNAs derived from the skeletal muscle of the common carp. A: Length distribution of sequenced small RNAs; B: The clean reads were blasted against the GenBank database to annotate rRNA, RNA, snRNA, or snoRNA; C: The clean reads were blasted against the Rfam database noncoding RNA to annotate rRNA, RNA, snRNA, or snoRNA. doi:10.1371/journal.pone.0030925.g001 been widely used to facilitate the identification and detection of miRNAs in multiple species [1923]. For this study, we constructed a small-RNA cDNA library from the skeletal muscle of the common carp. Through high throughput sequencing of the small RNA library and subsequent bioinformatic analysis, miRNAs in skeletal muscle tissue of the common carp were identified. Finally, using expression profiling analysis of the miRNAs from different tissues and multiple development stages, miRNAs likely to play a role in carp muscle development were identified. Construction of cDNA library for sequencing and small RNA discovery The cDNA library of small RNAs was constructed with pooled total RNAs from skeletal muscle tissues collected from three different 1-year-old common carps. Through high throughput Solexa sequencing, 21335605 total reads were obtained; 93.49% of the reads were 2123 nucleotides in length (Figure 1A). After removal of the 59 and 39 adapters, pollution reads and reads smaller than 18 nucleotides, 20842912 high-quality clean reads were extracted, and 25966 of the resulting unique small RNAs were annotated as either r (...truncated)