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The silkworm (Bombyx mori) microRNAs and their expressions in multiple developmental stages.

Yu X, Zhou Q, Li SC, Luo Q, Cai Y, Lin WC, Chen H, Yang Y, Hu S, Yu J - PLoS ONE (2008)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Genome Sciences and Information, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, China.

ABSTRACT

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.

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Related in: MedlinePlus

Predicted stem-loop structures of precursors and mature forms (highlight in red) of newly-identified miRNAs based on our direct cloning approach from the silkworm.Hairpins longer than 120 nt were truncated; these hairpins are indicated by a double slash preceding the stem. Scaffold numbers and genomic positions are indicated in parentheses.
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pone-0002997-g001: Predicted stem-loop structures of precursors and mature forms (highlight in red) of newly-identified miRNAs based on our direct cloning approach from the silkworm.Hairpins longer than 120 nt were truncated; these hairpins are indicated by a double slash preceding the stem. Scaffold numbers and genomic positions are indicated in parentheses.

Mentions: Our sequence analyses on the sequenced clones have yielded 55 miRNAs, representing 17 unique conserved miRNAs already discovered computationally. We also found several miRNA*s, such as bmo-miR-263a* in bluish egg (BS), bmo-miR-71* in spinning larva (SS) and pre-pupa (PPS), which providing solid evidence for Dicer-like processing [55], [56], [57], as it was reported that miRNA*s can also be functional [43], [58], [59]. Another group of 11 clones representing 11 novel putative miRNAs in our predicted data were confirmed by direct cloning (Table 3). In addition, four highly-conserved miRNAs (bmo-miR-278, bmo-miR-306, bmo-miR-317 and bmo-miR-768) and three less-conserved miRNAs (bmo-miR-1920, bmo-miR-1921 and bmo-miR-1922) were also cloned directly, which were not predicted based on our predicting criteria albeit their canonical precursors (Figure 1). To validate the novel miRNAs identified through direct cloning (bmo-miR-1920—bmo-miR-1926 and bmo-miR-2007—bmo-miR-2010), we deployed a stem-loop RT-PCR to assess the expression of these miRNAs (except bmo-miR-2009*). Expression of all 13 novel miRNAs at the different developmental stages was confirmed (Figure 2).


The silkworm (Bombyx mori) microRNAs and their expressions in multiple developmental stages.

Yu X, Zhou Q, Li SC, Luo Q, Cai Y, Lin WC, Chen H, Yang Y, Hu S, Yu J - PLoS ONE (2008)

Predicted stem-loop structures of precursors and mature forms (highlight in red) of newly-identified miRNAs based on our direct cloning approach from the silkworm.Hairpins longer than 120 nt were truncated; these hairpins are indicated by a double slash preceding the stem. Scaffold numbers and genomic positions are indicated in parentheses.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2500172&req=5

pone-0002997-g001: Predicted stem-loop structures of precursors and mature forms (highlight in red) of newly-identified miRNAs based on our direct cloning approach from the silkworm.Hairpins longer than 120 nt were truncated; these hairpins are indicated by a double slash preceding the stem. Scaffold numbers and genomic positions are indicated in parentheses.
Mentions: Our sequence analyses on the sequenced clones have yielded 55 miRNAs, representing 17 unique conserved miRNAs already discovered computationally. We also found several miRNA*s, such as bmo-miR-263a* in bluish egg (BS), bmo-miR-71* in spinning larva (SS) and pre-pupa (PPS), which providing solid evidence for Dicer-like processing [55], [56], [57], as it was reported that miRNA*s can also be functional [43], [58], [59]. Another group of 11 clones representing 11 novel putative miRNAs in our predicted data were confirmed by direct cloning (Table 3). In addition, four highly-conserved miRNAs (bmo-miR-278, bmo-miR-306, bmo-miR-317 and bmo-miR-768) and three less-conserved miRNAs (bmo-miR-1920, bmo-miR-1921 and bmo-miR-1922) were also cloned directly, which were not predicted based on our predicting criteria albeit their canonical precursors (Figure 1). To validate the novel miRNAs identified through direct cloning (bmo-miR-1920—bmo-miR-1926 and bmo-miR-2007—bmo-miR-2010), we deployed a stem-loop RT-PCR to assess the expression of these miRNAs (except bmo-miR-2009*). Expression of all 13 novel miRNAs at the different developmental stages was confirmed (Figure 2).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Genome Sciences and Information, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, China.

ABSTRACT

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.

Show MeSH
Related in: MedlinePlus