Deep sequencing of small RNA libraries reveals dynamic regulation of conserved and novel microRNAs and microRNA-stars during silkworm development

Abstract

Background: In eukaryotes, microRNAs (miRNAs) have emerged as critical regulators of gene expression. The Silkworm (Bombyx mori L.) is one of the most suitable lepidopteran insects for studying the molecular aspects of metamorphosis because of its large size, availability of mutants and genome sequence. Besides, this insect also has been amply studied from a physiological and biochemical perspective. Deep sequencing of small RNAs isolated from different stages of silkworm is a powerful tool not only for measuring the changes in miRNA profile but also for discovering novel miRNAs.

Results: We generated small RNA libraries from feeding larvae, spinning larvae, pupae and adults of B. mori and obtained approximately 2.5 million reads of 18-30 nt. Sequence analysis identified 14 novel and 101 conserved miRNAs. Most novel miRNAs are preferentially expressed in pupae, whereas more than 95% of the conserved miRNAs are dynamically regulated during different developmental stages. Remarkably, the miRNA-star (miR*) of four miRNAs are expressed at much higher levels than their corresponding miRNAs, and their expression profiles are distinct from their corresponding miRNA profiles during different developmental stages. Additionally, we detected two antisense miRNA loci (miR-263-S and miR-263-AS; miR-306-S and miR-306-AS) that are expressed in sense and antisense directions. Interestingly, miR-263 and miR-306 are preferentially and abundantly expressed in pupae and adults, respectively.

Conclusions: We identified 101 homologs of conserved miRNAs, 14 species-specific and two antisense miRNAs in the silkworm. Our results provided deeper insights into changes in conserved and novel miRNA and miRNA* accumulation during development.

Figure 1

Length distribution and abundance of small RNAs in silkworm. a) Schematic representation of the silkworm life cycle beginning with the feeding larval stage. Abundance of each size class of small RNAs based on nucleotide (nt) length in feeding larvae, spinning larvae, pupae and moths plotted using total small RNA reads (b) and unique reads (c).

Figure 2

Predicted fold-back structures for the newly identified miRNAs in silkworm. Mature miRNA sequence is shown in red letters.

Figure 3

Identification of a novel miRNA family. (a) sequence alignment of the mature miRNAs, (b), sequence alignment of the miRNA precursor (miRNA and miRNA* are shown in red and blue colored letters, respectively) and c), expression levels of the individual members in different developmental stages.

Figure 4

Temporal regulation of miRNA levels. (a-e), Plots of expression levels of miRNAs. Normalized read counts in feeding larvae, spinning larvae, pupae and moths are used for the graphical presentation.

Figure 5

Temporal regulation of miRNA-star (miR*) abundance (a-e), Plots of expression levels of miRNAs along with their miRNA* reads. Normalized read counts in feeding larvae, spinning larvae, pupae and moth stages in silkworm are used for the graphical representation.

Figure 6

Antisense miRNAs in B. mori. Predicted fold-back of miR-306 (a) and miR-263 (b) from sense (top panel) and antisense strand (bottom panel) and mature miRNA is shown in red. Sequencing reads uniquely aligned to hairpin sequence of reverse complement of miR-306 (c) and miR-263 (d) followed by their cloning frequency and length (nt). Mature sequences of miR-306:miR-306-AS pair (e) and miR-263:miR263-AS (f) aligned for showing identical nucleotide sequences.

Figure 7

Overlapping small RNA reads derived from annotated miR-1920 and miR-1921 loci. Annotated mature miRNAs are shown in red in the predicted fold-back structures for miR-1920 (a) and miR-1921 (b). Overlapping small RNA reads derived from the predicted hairpin structure are shown in the bottom panel and number of reads recovered in the libraries and their length (nt) are shown on the right.