Abstract
Channel catfish (Ictalurus spp.) is an economically important species in freshwater aquaculture around the world and occupies a prominent position in the aquaculture industry of the United States. MicroRNAs (miRNAs) play important roles in the regulation of almost every biological process in eukaryotes; however, there is little information available concerning miRNAs in channel catfish. In this study, a small-RNA cDNA library was constructed from 10 tissues of channel catfish, and Solexa sequencing technology was used to perform high-throughput sequencing of the library. A total of 14,919,026 raw reads, representing 161,288 unique sequences, were obtained from the small-cDNA library. After comparing the small RNA sequences with the RFam database, 4,542,396 reads that represent 25,538 unique sequences were mapped to the genome sequence of zebrafish to perform distribution analysis and to screen for candidate miRNA genes. Subsequent bioinformatic analysis identified 237 conserved miRNAs and 45 novel miRNAs in the channel catfish. Stem-loop RT-PCR was applied to validate and profile the expression of the novel miRNAs in 10 tissues. Some novel miRNAs, such as ipu-miR-129b, ipu-miR-7562 and ipu-miR-7553, were expressed in all tissues examined. However, some novel miRNAs appear to be tissue specific. Ipu-miR-7575 is predominantly expressed in stomach. Ipu-miR-7147 and ipu-miR-203c are highly expressed in heart, but are relatively weakly expressed in other tissues. Based on sequence complementarity between miRNAs and mRNA targets, potential target sequences for the 45 novel miRNAs were identified by searching for antisense hits in the reference RNA sequences of the channel catfish. These potential target sequences are involved in immune regulation, transcriptional regulation, metabolism and many other biological functions. The discovery of miRNAs in the channel catfish genome by this study contributes to a better understanding of the role miRNAs play in regulating diverse biological processes in fish and vertebrates.
Highlights
Channel catfish (Ictalurus spp.) is an economically important species in freshwater aquaculture around the world and occupies a prominent position in the aquaculture industry of the United States [1]
We have identified 237 conserved miRNAs and 45 novel miRNAs in channel catfish, most of which have more than one isomiR
Solexa Sequencing of Small RNAs To increase the coverage of channel catfish miRNAs, a library of small cDNAs was generated with pooled total RNAs and subjected to Solexa high-throughput sequencing. 14,919,026 raw reads, representing 161,288 unique sequences, were obtained
Summary
Channel catfish (Ictalurus spp.) is an economically important species in freshwater aquaculture around the world and occupies a prominent position in the aquaculture industry of the United States [1]. Little information is available concerning channel catfish microRNAs (miRNAs), which play important roles in the regulation of almost every biological process in eukaryotes. Since the discovery of the first miRNAs in Caenorhabditis elegans in 1993, thousands of mature miRNAs have been discovered in a wide variety of organisms, including animals, plants and even viruses [6,7,8]. This suggests that miRNAs appeared early in eukaryotic evolution and play fundamental roles in the regulation of gene expression. A multitude of studies have discovered critical regulatory functions of miRNAs in many physiological activities, including cell growth, differentiation and apoptosis [9,10]
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