Abstract
BackgroundMicroRNAs (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 FindingsWe 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/SignificanceTaking 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.
Highlights
As key regulators for gene expression at post-transcriptional levels, microRNAs are a class of endogenous noncoding RNAs transcribed by RNA polymerase II with a size range of,22 nt; they are processed from larger hairpin structures—known as pri- and pre-miRNAs—by two specialized proteins, namely Drosha and Dicer [1,2]
We first surveyed the B. mori genome assembly to predict candidate pre-miRNAs using Srnaloop program and customdesigned filters, we searched Rfam database [54], an insect ncRNA dataset, and a CDS dataset assembled from the silkworm genome for removing those overlapping with these specialized sequences, yielding 7,241,352 candidate pre-miRNAs
We discovered six pairs that are organized as clusters; bmo-miR-1/bmo-miR-133, bmo-let-7/bmo-miR-100, bmo-miR-12/bmo-miR-283, and bmo-miR-275/bmo-miR-305 are separated by less than 20 kb apart and in the same orientation; bmo-miR-9b overlaps with bmo-miR-79 on the opposite strand; and bmo-miR-2 is adjacent to bmo-miR-13 but on the reverse strand in a tail-to-tail orientation about some twenty basepairs away
Summary
As key regulators for gene expression at post-transcriptional levels, microRNAs (miRNAs) are a class of endogenous noncoding RNAs transcribed by RNA polymerase II with a size range of ,22 nt (nucleotides); they are processed from larger hairpin structures—known as pri- and pre-miRNAs—by two specialized proteins, namely Drosha and Dicer (or Dicer-like proteins, DCLs) [1,2]. Similar to lin-4 and let-7 of Caenorhabditis elegans, the majority of miRNA genes are transcribed as independent transcriptional units [4,8,9] albeit a few of them were found in introns of premRNAs and co-expressed with their host genes [5,10,11,12,13]. More than a third of miRNAs found in C. elegans have homologs in humans [5], suggesting that they have important functions that are evolutionarily-conserved [14]. 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
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
