Insect Piwi Proteins And Micrornas Bioinformatics And Evolutionary Analysis

As a subfamily of Argonaute proteins, Piwi is poorly understood compared with AGO subfamily until recent discovery of Piwi protein interacting with piRNA. Moreover, identification of Piwi subfamily in invertebrate is not only important for elucidating function and evolution of Piwi proteins but also helpful for piRNA research. We did a large scale screening of insect genomes to identify piwi-like genes. Full or partial cDNA sequences were obtained by EST elongation and GENSCAN. We found that the exon numbers were totally different between vertebrates and invertebrates, approximately 20 exons in mammals but only 6-9 exons in insects. This infers either intron insertion or loss occurred during evolution. Characterized PAZ, c-terminal PIWI domains exist in almost all predicted Piwi-like proteins. We found six conserved motifs, which contain active catalytic triad "Asp-Asp-His/Lys" required for slicer activity. The expression of siwil and siwi2 in Bombyx mori were verified with RT-PCR. Phylogenetic tree inferred by Bayesian algorithm indicates invertebrate Piwi-like proteins are classified into three clades, of which Ago3 clade is closer to mammalian Piwi proteins.The complete genome sequences of 11 Drosophila species provide an opportunity to further investigate the Piwi subfamily and the gene family evolution in closely related species. Drosophila Piwi subfamily, including three members, piwi, Aub and Ago3, has attracted increasing attentions as it participates in the biogenesis of piRNA. Here, following the former work, we identified 33 Piwi homologs from the genome of 11 Drosophila species, in which piwi and Aub genes are within average 20kb in genome. The full-length cDNA sequences of piwi and Aub genes were obtained by using New GENSCAN Web Server. The Ago3 homologs were difficult to get full-length information because they had long introns. The genomic structure of Piwi subfamily genes are highly conserved among diverse Drosophila species. Insect piwi and Aub genes have long first intron. The average length of the first intron is l,284bp for piwi and 840bp for Aub, which is much larger than those of other introns (93bp for piwi and 54bp for Aub). However, this phenomenon is not observed in mammalian piwi genes. We also found that there were abundant repeat sequences in both exons and introns of insect Ago3 genes. Due to recent insertions of LTR elements in four Drosophila species, part of the third introns exhibit higher conservation than adjacent exons and other introns. An evolutional tree created by Minimum Evolution method indicates that mammalian piwi genes are more closely related to insect Ago3 Piwi subfamily. MicroRNAs (miRNAs) are endogenous non-coding genes that interact with AGO protein to regulate gene expression by either degrading mRNA or blocking its translation. We conducted large-scale screening for miRNA genes in the silkworm Bombyx mori using sequence-by-synthesis (SBS) deep sequencing of mixed RNAs from egg, larval, pupal, and adult stages. Of 2,227,930 SBS tags, 1,144,485 ranged from 17 to 25 nt, corresponding to 256,604 unique tags. Among these non-redundant tags, 95,184 were matched to the silkworm genome. We identified 3,750 miRNA candidate genes using a computational pipeline combining RNAfold and TripletSVM algorithms. We confirmed 354 miRNA genes using miRNA microarrays and then performed expression profile analysis on these miRNAs for all developmental stages. While 106 miRNAs were expressed in all stages, 248 miRNAs were egg- and pupa-specific, suggesting that insect miRNAs play a significant role in embryogenesis and metamorphosis. We selected eight miRNAs for quantitative RT-PCR analysis; six of these were consistent with our microarray results. In addition, we searched for orthologous miRNA genes in mammals, a nematode, and other insects and found that most silkworm miRNAs are conserved in insects, whereas only a small number of silkworm miRNAs have orthologs in mammals and the nematode. These results suggest that there are several miRNAs unique to insects.