MicroRNAs Of Bombyx Mori And Their Expression Rhythms

MicroRNAs are small endogenous RNAs that silence gene expression post-transcriptionally via base-pair interactions with the 3′untranslated regions of target genes.Most miRNAs are evolutionarily conserved in related species and some even show conservation between invertebrates and vertebrates.In this report,we predicted 120 miRNAs genes in all on the 6X and 9X genome assembly of Bombyx mor based on sequence conservation and structural similarity to known miRNAs. They can be grouped into at least 60 families.MiRNAs intersperse on the silkworm genome,over 2/3 residing in intergenic regions and the rest inhabiting within the introns or exons of predicted protein-coding genes.Some microRNAs have more than one paralogs in the silkworm(e.g.let-7, miR-2,miR-9,miR-10,miR-92,miR-228).Three paralogous miRNAs,bmo-mir-2a-1,bmo-mir-2a-2 and bmo-mir-13a locate on nscaf1690 in close proximity to each other,thus forming a cluster.It was shown that 23 homolog-hunted and experiment-detected silkworm miRNAs are strongly conserved in a wide range of bilaterian animals across phyla,and 19 conserved between closely related species of insecta.We characterized the temporal and spacial expression patterns for 92 unique miRNAs by microarray and Northern blotting.The results show that 43 out of the 46 homolog-predicted miRNAs and 35 out of the other half from other organisms can be detected in the silkworm.Approximately two thirds of the detected microRNAs are differentially expressed during the whole process of metamorphism development.21 microRNAs are detected in embryo at the stage of head pigmentation, and 16 of them persist high expression level to adult moth,albeit with some oscillation.Stage-by-stage investigations demonstrate that microRNAs come forth groups by groups during embryogenesis, exhibit distinct rhythms in abundance responding to stage transitions.Seen from the whole life cyle of silkworm,bmo-miR-275 is expressed in a changing level opposite to that of bmo-miR-34b;over two thirds of the expressed miRNAs are marginally pulsing in step with the changing rate of ecdysone titer. During the 9 days of pupa metamorphosis,about 30 miRNAs are highly expressed in both sexes from beginning to the end.Whereas,over 2/3 miRNAs including the highly expresed ones share the sharp expression trough at day 3 pupa of females and males.Strikingly,an opposite changing profile is observable between bmo-miR-275 and bmo-miR-29 as the pupa metamorphosis advances.Over 60 microRNAs in all are expressed responding to different biological behaviours,such as hatching, feeding,molting,cocoon spinning,pupation and eclosion.Only 10 miRNAs are widely distributed in the tissues at day 3 fifth instar larvae,whereas most miRNAs present striking tissue-specificity,and one third even show sexual differences in diverse tissues.Further investigation of the tissues undergoing metamorphosis shows that different miRNAs are upregulted or downregulated in the process of apoptosis and histolysis.The independly repeated cell culture experiments indicates that bmo-let-7a,bmo-let-7b,bmo-bantam and dme-miR-184-3p respond to ecdysone in a dose-/time-dependent manner.Subsequently,347 unique miRNAs have been revealed by solexa sequencing of the silk gland and whole-mount of day 3 the fifth instar,51 of which have homologs in orther organisms.Taken together,our data will lay important foundations to the future study on miRNA-involved regulations in the development and metamorphosis of insects and other species.