Deep Sequencing of Organ- and Stage-Specific microRNAs in the Evolutionarily Basal Insect Blattella germanica (L.) (Dictyoptera, Blattellidae)

Alexandre S Cristino,Erica D Tanaka,Mercedes Rubio,Maria-Dolors Piulachs,Xavier Belles,Baohong Zhang

doi:10.1371/journal.pone.0019350

Alexandre S Cristino, Erica D Tanaka + Show 4 more

Open Access

https://doi.org/10.1371/journal.pone.0019350

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Journal: PloS one	Publication Date: Apr 28, 2011
Citations: 145	License type: CC BY 4.0

Affiliation: University of Queensland, Institut de Biologia Evolutiva

Abstract

BackgroundmicroRNAs (miRNAs) have been reported as key regulators at post-transcriptional level in eukaryotic cells. In insects, most of the studies have focused in holometabolans while only recently two hemimetabolans (Locusta migratoria and Acyrthosiphon pisum) have had their miRNAs identified. Therefore, the study of the miRNAs of the evolutionarily basal hemimetabolan Blattella germanica may provide valuable insights on the structural and functional evolution of miRNAs.Methodology/Principal FindingsSmall RNA libraries of the cockroach B. germanica were built from the whole body of the last instar nymph, and the adult ovaries. The high throughput Solexa sequencing resulted in approximately 11 and 8 million reads for the whole-body and ovaries, respectively. Bioinformatic analyses identified 38 known miRNAs as well as 11 known miRNA*s. We also found 70 miRNA candidates conserved in other insects and 170 candidates specific to B. germanica. The positive correlation between Solexa data and real-time quantitative PCR showed that number of reads can be used as a quantitative approach. Five novel miRNA precursors were identified and validated by PCR and sequencing. Known miRNAs and novel candidates were also validated by decreasing levels of their expression in dicer-1 RNAi knockdown individuals. The comparison of the two libraries indicates that whole-body nymph contain more known miRNAs than ovaries, whereas the adult ovaries are enriched with novel miRNA candidates.Conclusions/SignificanceOur study has identified many known miRNAs and novel miRNA candidates in the basal hemimetabolan insect B. germanica, and most of the specific sequences were found in ovaries. Deep sequencing data reflect miRNA abundance and dicer-1 RNAi assay is shown to be a reliable method for validation of novel miRNAs.

Highlights

In recent years, the discovery of short non-coding RNAs has changed the way molecular biologists understand the regulation of gene expression in almost all biological processes
Concerning insects, repertoires of small RNAs have been mainly established for species with their whole genome sequenced, such as 12 Drosophila species [12], three mosquitoes (Anopheles gambiae, Aedes albopictus and Culex quinquefasciatus) [13,14], two hymenopterans (Apis mellifera and Nasonia vitripennis) [15], the flour beetle (Tribolium castaneum) [16,17] and the silkworm (Bombyx mori) [18,19]
A peak at 22-nt size was observed in small RNA libraries of L. migratoria [20], A. albopictus and C. quinquefasciatus [13]

Summary

Introduction

The discovery of short non-coding RNAs has changed the way molecular biologists understand the regulation of gene expression in almost all biological processes. Identification of novel miRNA candidates Many other small RNA sequences were found highly represented in the WB-6 and Ov-A libraries and arguably they are likely to be functional small RNAs. A total of 1,428 different sequences with at least 100 reads were identified in our Solexa data

Results

Conclusion