Characterization and expression patterns of let-7 microRNA in the silkworm (Bombyx mori)

Abstract

Background: lin-4 and let-7, the two founding members of heterochronic microRNA genes, are firstly confirmed in Caenorhabditis elegans to control the proper timing of developmental programs in a heterochronic pathway. let-7 has been thought to trigger the onset of adulthood across animal phyla. Ecdysone and Broad-Complex are required for the temporal expression of let-7 in Drosophila melanogaster. For a better understanding of the conservation and functions of let-7, we seek to explore how it is expressed in the silkworm (Bombyx mori).

Results: One member of let-7 family has been identified in silkworm computationally and experimentally. All known members of this family share the same nucleotides at ten positions within the mature sequences. Sequence logo and phylogenetic tree show that they are not only conserved but diversify to some extent among some species. The bmo-let-7 was very lowly expressed in ova harvested from newborn unmated female adult and in individuals from the first molt to the early third instar, highly expressed after the third molt, and the most abundant expression was observed after mounting, particularly after pupation. The expression levels were higher at the end of each instar and at the beginning of each molt than at other periods, coinciding with the pulse of ecdysone and BR-C as a whole. Using cultured ovary cell line, BmN-SWU1, we examined the effect of altered ecdysone levels on bmo-let-7 expression. The expression was also detected in various tissues of day 3 of the fifth instar and of from day 7 of the fifth to pupa, suggesting a wide distributing pattern with various signal intensities.

Conclusion: bmo-let-7 is stage- and tissue-specifically expressed in the silkworm. Although no signals were detected during embryonic development and first larval instar stages, the expression of bmo-let-7 was observed from the first molt, suggesting that it might also function at early larval stage of the silkworm. The detailed expression profiles in the whole life cycle and cultured cell line of silkworm showed a clear association with ecdysone pulse and a variety of biological processes.

Figure 1

Stem-loop structures of alternative forms of bmo-let-7 precursor. The mature sequences are in red, shown in the foldback of the precursors.let-7 small RNA of silkworm is 22 nt or 23 nt long, and the precursor is 87 nt or 72 nt long. The actual size of the stem-loop structure is not known experimentally and may be slightly shorter or longer than represented.

Figure 2

Sequence logo of known let-7 family members. Ninety known let-7 members together with bmo-let-7 were aligned using the CLUSTAL × program, then were submitted to logo analysis by using the WebLogo program available [66]. The height of the letters indicates the relative frequency of the letter at that position and the overall height of the stack indicates the sequence conservation in terms of information content in bits.

Figure 3

Sample clusters of known precursors of let-7 family members based on sequence similarity. Twenty-six of all known let-7 members were selected in terms of sequence- and species-specific and aligned against each other by a Smith-Waterman algorithm.

Figure 4

Phylogenetic tree based on the selected precursors of let-7 family members. In order to be convenient for analysis, we selected 26 representative let-7 precursors. Phylogenetic tree was constructed using MEGA v3.0 with 1000 times bootstrap sampling. Full species names coresponding to the microRNAs are as follows: hsa, Homo sapiens; fru, Fugu rubripes; dre, Danio rerio; xtr, Xenopus tropicalis; bta, Bos taurus; cbr, Caenorhabditis briggsae; cel, Caenorhabditis elegans; bmo, Bombyx mori; dme, Drosophila melanogaster; dps, Drosophila pseudoobscura; aga, Anopheles gambiae; ssc, Sus scrofa; tni, Tetraodon nigroviridis.

Figure 5

Stage durations in the lifespan of B. mori. When reared at 25°C and 85% H.R., the lifespan of silkworm is over 50 days, consisting of ova, larva, pupa and imago. The embyo duration is about 10 days if proper acid-treatment is performed at hour 24 after oviposition. The first instar is 3 days long, starting at the newly-hatched silkworm or named ant silkworm, ending at just stopping eating mulberry leaves for the first molt. After spinning, the silkworm will live through pre-pupa of two days before becoming pupae. The durations vary a lot, ranging from 1 to 10 days.

Figure 6

General profile of bmo-let-7 accumulation during development of silkworm. Both the sense and antisense probes were examined here, but only two antisense probes brought out the same positive results. And lin-4 escaped detection in all samples. All samples were quantified to be 15 μg per well before loading (see Materials and Methods). U6 and 5srRNA hybridizations were used as loading controls and levels of let-7 RNA are quantified relative to 5srRNA. Reproducible and consistent results were confirmed experiments repeated (data not shown).

Figure 7

Detailed profile of bmo-let-7 accumulation from ova to the late fifth instar. (A). Expression profile from pre-laid egg (ova) to to early 3^rd molt. In the first test, the oviduct attached to egg was not discarded. In the second test, the oviduct was completely removed. T-1 stand for the first test (Test 1), T-2 means the second test (Test 2). The mulberry leaf was mainly used as a control to eliminate the potential contamination. U6 and 5srRNA hybridizations were used as loading controls and levels of let-7 RNA are quantified relative to 5srRNA. (B). Expression profile from early 4^th instar to day-7 5^th instar. The female and male silkworms were differentiated on the basis of gonad while harvesting the materials. U6 and 5srRNA hybridizations were used as loading controls and levels of let-7 RNA are quantified relative to U6.

Figure 8

Detailed expression profile of bmo-let-7 during B.mori pupa metamorphosis. (A). Profiling during female pupa metamorphosis under artificial culture conditions. (B). Profiling during male pupa metamorphosis under natural culture conditions. The two groups were incubated under different conditions, group A under 25°C and 85%RH, group B under 18–30°C and 40–100%RH. Spinning stage, as well as adult stage, was simultaneously tested in favor of comparative analysis. Two or three days after mounting, cocoon spinning is finished and the larva becomes prepupa. In the following two or three days, the prepupa becomes pupa after molting. The pupa takes 8~11 days to transform into a moth which is white-grey colored. Under natural conditions(18~30°C), the pupa metamorphosis will take 10~11 days; Under man-made conditions (25°C), however, pupa lasts 8~9 days ended as adult moth. U6 and 5srRNA were used as loading controls. 5srRNA and U6 were used as the loading controls and levels of bmo-let-7 RNA are quantified relative to U6.

Figure 9

bmo-let-7 expression profile in tissues from females and males of fifth-instar day-3 larvae. Eighteen tissues were harvested from females and males of day-3 5^th instar larvae bred at 25°C and 85% H.R. The two antisense probes(anti-bmo-let-7 and anti-bmo-let-7*) were firstly hybridized on different blots, respectively, with equal amount of small RNAs on all loading wells. After stripping, exchanged the probe for the other blot. The candidate mature sequence is 22 nt or 23 nt, and the potential precursor is 72 nt or 87 nt long, but the precursor transcribed by silkworm genome can not be accumulated enough to be seen by Northern blotting. 5srRNA and U6 were used as the loading controls and levels of bmo-let-7 RNA are quantified relative to 5srRNA.

Figure 10

bmo-let-7 expression in male tissues from just mounting to day-3 pupa. At day-3 pupa, it is unlikely to get enough silk gland and midgut, say nothing of small RNAs. Only two tissues were harvested at this period. 5srRNA and U6 were used as the loading controls and levels of bmo-let-7 RNA are quantified relative to U6.

Figure 11

The effect of concentrations of ecdysone on the expression of bmo-let-7 in cultured cells. (A). The effect of ecdysone for 72 h. (B). The effect of ecdysone for 24 h. The cells were cultured in Grace's medium with or without ecdysone for the periods of time shown. The small RNAs were harvested and two probes for bmo-let-7 were used in the detection of Northern blot. 0 h is just before treating with ecdysone or the beginning of treatment with ecdysone, and all cell cultures are synchronous in this assay by transferring from the common vessel. Levels of bmo-let-7 are quantified relative to the loading control, 5srRNA.