Identification of Schistosoma mansoni microRNAs
1 Graduate Program in Bioinformatics, Universidade Federal de Minas Gerais, Av. Antonio Carlos 6627, Belo Horizonte, MG, Brazil
2 Department of Statistics, Peter Medawar Building, South Parks Road, Oxford, UK
3 J Craig Venter Institute (JCVI), 9704 Medical Center Drive, Rockville, MD 20850, USA
4 Department of Cell Biology and Molecular Genetics and Center for Bioinformatics and Computational Biology, University of Maryland College Park, MD 20742, USA
5 Institute for Genome Sciences, University of Maryland School of Medicine, Baltimore, MD, USA
6 CEBio, Instituto Nacional de Ciência e Tecnologia em Doenças Tropicais, Laboratory of Cellular and Molecular Parasitology, Centro de Pesquisas René Rachou, Fundação Oswaldo Cruz, Av. Augusto de Lima 1715, Belo Horizonte, 30190-002, Brazil
7 University of Texas Health Science Center, 7703 Floyd Curl Dr. Mail Code 7760, San Antonio, Texas 78229-3900, USA
8 Biosciences eastern and central Africa - International Livestock Research Institute (BecA-ILRI) Hub, P.O. Box 30709 Nairobi, Kenya
BMC Genomics 2011, 12:47 doi:10.1186/1471-2164-12-47Published: 19 January 2011
MicroRNAs (miRNAs) constitute a class of single-stranded RNAs which play a crucial role in regulating development and controlling gene expression by targeting mRNAs and triggering either translation repression or messenger RNA (mRNA) degradation. miRNAs are widespread in eukaryotes and to date over 14,000 miRNAs have been identified by computational and experimental approaches. Several miRNAs are highly conserved across species. In Schistosoma, the full set of miRNAs and their expression patterns during development remain poorly understood. Here we report on the development and implementation of a homology-based detection strategy to search for miRNA genes in Schistosoma mansoni. In addition, we report results on the experimental detection of miRNAs by means of cDNA cloning and sequencing of size-fractionated RNA samples.
Homology search using the high-throughput pipeline was performed with all known miRNAs in miRBase. A total of 6,211 mature miRNAs were used as reference sequences and 110 unique S. mansoni sequences were returned by BLASTn analysis. The existing mature miRNAs that produced these hits are reported, as well as the locations of the homologous sequences in the S. mansoni genome. All BLAST hits aligned with at least 95% of the miRNA sequence, resulting in alignment lengths of 19-24 nt. Following several filtering steps, 15 potential miRNA candidates were identified using this approach. By sequencing small RNA cDNA libraries from adult worm pairs, we identified 211 novel miRNA candidates in the S. mansoni genome. Northern blot analysis was used to detect the expression of the 30 most frequent sequenced miRNAs and to compare the expression level of these miRNAs between the lung stage schistosomula and adult worm stages. Expression of 11 novel miRNAs was confirmed by northern blot analysis and some presented a stage-regulated expression pattern. Three miRNAs previously identified from S. japonicum were also present in S. mansoni.
Evidence for the presence of miRNAs in S. mansoni is presented. The number of miRNAs detected by homology-based computational methods in S. mansoni is limited due to the lack of close relatives in the miRNA repository. In spite of this, the computational approach described here can likely be applied to the identification of pre-miRNA hairpins in other organisms. Construction and analysis of a small RNA library led to the experimental identification of 14 novel miRNAs from S. mansoni through a combination of molecular cloning, DNA sequencing and expression studies. Our results significantly expand the set of known miRNAs in multicellular parasites and provide a basis for understanding the structural and functional evolution of miRNAs in these metazoan parasites.