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The Silkworm (Bombyx mori) microRNAs and Their Expressions in Multiple Developmental Stages
Xiaomin Yu1,2#, Qing Zhou1,2#, Sung-Chou Li4,5#, Qibin Luo1,3, Yimei Cai1,2, Wen-chang Lin4,6, Huan Chen1,3, Yue Yang1,2, Songnian Hu1, Jun Yu1*
1 Key Laboratory of Genome Sciences and Information, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, China,
2 Graduate University of Chinese Academy of Sciences, Beijing, China,
3 James D. Watson Institute of Genome Sciences, Zhejiang University, Hangzhou, China,
4 Institute of BioMedical Informatics, National Yang-Ming University, Taipei, Taiwan,
5 Bioinformatics Program, Taiwan International Graduate Program, Academia Sinica, Taipei, Taiwan,
6 Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan
Background
MicroRNAs (miRNAs) play crucial roles in various physiological processes through post-transcriptional regulation of gene expressions and are involved in development, metabolism, and many other important molecular mechanisms and cellular processes. The Bombyx mori genome sequence provides opportunities for a thorough survey for miRNAs as well as comparative analyses with other sequenced insect species.
Methodology/Principal Findings
We identified 114 non-redundant conserved miRNAs and 148 novel putative miRNAs from the B. mori genome with an elaborate computational protocol. We also sequenced 6,720 clones from 14 developmental stage-specific small RNA libraries in which we identified 35 unique miRNAs containing 21 conserved miRNAs (including 17 predicted miRNAs) and 14 novel miRNAs (including 11 predicted novel miRNAs). Among the 114 conserved miRNAs, we found six pairs of clusters evolutionarily conserved cross insect lineages. Our observations on length heterogeneity at 5′ and/or 3′ ends of nine miRNAs between cloned and predicted sequences, and three mature forms deriving from the same arm of putative pre-miRNAs suggest a mechanism by which miRNAs gain new functions. Analyzing development-related miRNAs expression at 14 developmental stages based on clone-sampling and stem-loop RT PCR, we discovered an unusual abundance of 33 sequences representing 12 different miRNAs and sharply fluctuated expression of miRNAs at larva-molting stage. The potential functions of several stage-biased miRNAs were also analyzed in combination with predicted target genes and silkworm's phenotypic traits; our results indicated that miRNAs may play key regulatory roles in specific developmental stages in the silkworm, such as ecdysis.
Conclusions/Significance
Taking a combined approach, we identified 118 conserved miRNAs and 151 novel miRNA candidates from the B. mori genome sequence. Our expression analyses by sampling miRNAs and real-time PCR over multiple developmental stages allowed us to pinpoint molting stages as hotspots of miRNA expression both in sorts and quantities. Based on the analysis of target genes, we hypothesized that miRNAs regulate development through a particular emphasis on complex stages rather than general regulatory mechanisms.
