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Evidence for the expression of abundant microRNAs in the locust genome.

Wang Y, Jiang F, Wang H, Song T, Wei Y, Yang M, Zhang J, Kang L - Sci Rep (2015)

Bottom Line: We observed that abundant local duplication events of miRNAs, which were unique in locusts compared with those in other insects with small genome sizes, may be responsible for the substantial acquisition of miRNAs in locusts.Together, multiple evidence showed that the locust genome experienced a burst of miRNA acquisition, suggesting that genome size expansion may have considerable influences of miRNA innovation.These results provide new insight into the genomic dynamics of miRNA repertoires under genome size evolution.

View Article: PubMed Central - PubMed

Affiliation: Institute of Applied Biology, Shanxi University, Taiyuan, Shanxi, China.

ABSTRACT
Substantial accumulation of neutral sequences accounts for genome size expansion in animal genomes. Numerous novel microRNAs (miRNAs), which evolve in a birth and death manner, are considered evolutionary neutral sequences. The migratory locust is an ideal model to determine whether large genomes contain abundant neutral miRNAs because of its large genome size. A total of 833 miRNAs were discovered, and several miRNAs were randomly chosen for validation by Northern blot and RIP-qPCR. Three additional verification methods, namely, processing-dependent methods of miRNA biogenesis using RNAi, evolutionary comparison with closely related species, and evidence supported by tissue-specific expression, were applied to provide compelling results that support the authenticity of locust miRNAs. We observed that abundant local duplication events of miRNAs, which were unique in locusts compared with those in other insects with small genome sizes, may be responsible for the substantial acquisition of miRNAs in locusts. Together, multiple evidence showed that the locust genome experienced a burst of miRNA acquisition, suggesting that genome size expansion may have considerable influences of miRNA innovation. These results provide new insight into the genomic dynamics of miRNA repertoires under genome size evolution.

No MeSH data available.


Evidence for the presence of numerous locust miRNAs in an evolutionary view.(A) Box plot of miRNA expression for evolutionarily conserved miRNAs and lineage-specific miRNAs. *indicates P < 0.001 (Mann–Whitney–Wilcoxon tests). (B) Box plot of lineage-specific miRNA expression for lineage-specific miRNAs that showed no variations and those with sequence variations. The variable group represents the lineage-specific miRNAs in locusts that differed from those in band-winged grasshoppers. *indicates P = 0.009 (Mann–Whitney–Wilcoxon tests). (C) Effects of Drosha knockdown using RNAi for miRNAs of the three different categories, namely evolutionarily conserved miRNAs, lineage-specific miRNAs with moderate/high expression and lineage-specific miRNAs with low expression. (D) Inferred MFEs of the lineage-specific miRNAs were similar to those of the evolutionarily conserved miRNAs, and significantly stronger than the binding of shuffled control sequences. (E,F) The miRNA 5′ end and 3′ end processing precision of evolutionarily conserved and lineage-specific miRNAs. The processing precision was calculated as the fraction of mapped reads that corresponded precisely to the consensus sequences of genomic locus. The miRNAs are shown on the x-axis and ordered by the processing precision. The miRNA with the most precision is at percentile 1, and the one with the least precision is at percentile 100.
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f3: Evidence for the presence of numerous locust miRNAs in an evolutionary view.(A) Box plot of miRNA expression for evolutionarily conserved miRNAs and lineage-specific miRNAs. *indicates P < 0.001 (Mann–Whitney–Wilcoxon tests). (B) Box plot of lineage-specific miRNA expression for lineage-specific miRNAs that showed no variations and those with sequence variations. The variable group represents the lineage-specific miRNAs in locusts that differed from those in band-winged grasshoppers. *indicates P = 0.009 (Mann–Whitney–Wilcoxon tests). (C) Effects of Drosha knockdown using RNAi for miRNAs of the three different categories, namely evolutionarily conserved miRNAs, lineage-specific miRNAs with moderate/high expression and lineage-specific miRNAs with low expression. (D) Inferred MFEs of the lineage-specific miRNAs were similar to those of the evolutionarily conserved miRNAs, and significantly stronger than the binding of shuffled control sequences. (E,F) The miRNA 5′ end and 3′ end processing precision of evolutionarily conserved and lineage-specific miRNAs. The processing precision was calculated as the fraction of mapped reads that corresponded precisely to the consensus sequences of genomic locus. The miRNAs are shown on the x-axis and ordered by the processing precision. The miRNA with the most precision is at percentile 1, and the one with the least precision is at percentile 100.

Mentions: Newly emerged miRNAs in an ancestral lineage are integrated into gene regulatory networks and play important roles in expression regulation. Therefore, their mature sequences should be under strong purifying selection, and they are rarely mutated with secondary loss in the descendants8. To check the presence of locust miRNAs in the closely related species sharing common ancestor with L. migratoria, we generated 9,483,626 million reads of 18–30 bp from adults of band-winged grasshopper, Oedaleus asiaticus26. A substantial fraction (46%, 385 in 833) of the locust lineage-specific miRNA precursors (37%, 255 in 689) and evolutionarily conserved miRNA precursors (90%, 130 in 144) were detected in the band-winged grasshopper, which confirmed that they were evolutionarily conserved at least after the emergence of Orthoptera. Similar to a previous report, evolutionarily conserved miRNAs had significantly higher expression levels than the lineage-specific miRNAs (Fig. 3A, P < 0.001, Mann–Whitney–Wilcoxon tests), which implied that the lineage-specific miRNAs were prone to show low expression27. Newly emerged miRNAs have been continuously created in metazoan genomes as lineage-specific miRNAs, and these evolutionarily young miRNAs are generally lowly expressed and appear to have nonsubstantial effects on regulatory networks2829. Pearson’s correlation tests showed that the expression of the lineage-specific miRNAs in adult locusts had a significantly positive correlation with that of their respective homologs in adult band-winged grasshoppers (P < 0.001, Pearson’s correlation tests). Based on the expression cut-off of TPM > 10, miRNAs were classified into the moderate/high and low expression groups. Numerous homologs (34%, 86 in 255) of the lineage-specific miRNAs exhibit moderate/high expression [transcripts per million (TPM) > 10] in the band-winged grasshoppers. Almost all the homologs of the lineage-specific miRNAs (92%, 79 in 86) that displayed moderate/high expression in band-winged grasshoppers were also found with moderate/high expression in locusts. The substantial expression of these miRNAs implied that these lineage-specific miRNAs might not be lowly expressed genomic by-products, and they have considerable effects on the transcriptome and their target genes in terms of regulatory networks29. We examined the strength of natural selection acting on the lineage-specific miRNAs to determine whether the lineage-specific miRNAs with low expression have undergone rapid sequence evolution28. Specifically, we determined the sequence variations in the miRNA mature region between band-winged grasshoppers and locusts, and compared the number of variant miRNAs between the lineage-specific miRNAs with moderate/high expression and those with low expression. We excluded the first and last three bases of sequencing reads in sequence variation detection, because of the frequent un-templated modifications at both 5P and 3P ends of mature miRNAs30. For the 130 evolutionarily conserved miRNAs detected in band-winged grasshoppers, only one miRNA in the moderate/high expression group had sequence variation in their mature region. This characteristic was consistent with the fact that strong purifying selection intensely constrained evolutionarily conserved miRNAs. However, contrary to that observed in evolutionarily conserved miRNAs (1 in 144), we observed a significant signal of sequence variances for the lineage-specific miRNAs (26 in 255, P < 0.001, χ2-tests). We found that the lineage-specific miRNAs that showed no variations were more highly expressed than those with sequence variations (Fig. 3B, P = 0.009, Mann–Whitney–Wilcoxon tests). Based on the expression cut-off of TPM > 10, miRNAs were classified into the moderate/high and low expression groups. Compared with those in the moderate/high expression groups, the lineage-specific miRNAs in the low expression group were subject to a significant signal of sequence variances (3 in 86 of the moderate/high expression group and 23 in 169 of the low expression group; P = 0.025, χ2-tests). We repeated the analysis with different cut-offs of TPM (P = 0.010 for TPM > 5 and P = 0.004 for TPM > 15, χ2-tests), reaching the same significant signals. Therefore, our analytical results suggested that the numerous lineage-specific miRNAs with moderate/high expression might be under high selective pressures, and showed evolutionary conservation at least after the emergence of Orthoptera. This finding was consistent with the fact that highly expressed miRNAs are under strict selective constraints to maintain sequence uniformity and play critical roles in a broad manner31. To determine the silencing effects of Drosha knockdown for miRNAs with different evolutionary origin, we compared the expression changes for the evolutionarily conserved miRNAs, the lineage-specific miRNAs with moderate/high expression and with low expression (Fig. 3C). The fold changes between the lineage-specific miRNAs with moderate/high expression and the lineage-specific miRNAs with low expression were comparable (P = 0.681, Mann–Whitney–Wilcoxon tests). Compared with those of the lineage-specific miRNAs with moderate/high expression (P = 0.9347, Mann–Whitney–Wilcoxon tests) and with those of the lineage-specific miRNAs with low expression (P = 0.9815, Mann–Whitney–Wilcoxon tests), the fold changes of evolutionarily conserved miRNAs was not significantly low. These data suggested that, similar to the evolutionarily conserved miRNAs, the lineage-specific miRNAs dependent on Drosha processing for their biogenesis, implying the authenticity of the lineage-specific miRNAs.


Evidence for the expression of abundant microRNAs in the locust genome.

Wang Y, Jiang F, Wang H, Song T, Wei Y, Yang M, Zhang J, Kang L - Sci Rep (2015)

Evidence for the presence of numerous locust miRNAs in an evolutionary view.(A) Box plot of miRNA expression for evolutionarily conserved miRNAs and lineage-specific miRNAs. *indicates P < 0.001 (Mann–Whitney–Wilcoxon tests). (B) Box plot of lineage-specific miRNA expression for lineage-specific miRNAs that showed no variations and those with sequence variations. The variable group represents the lineage-specific miRNAs in locusts that differed from those in band-winged grasshoppers. *indicates P = 0.009 (Mann–Whitney–Wilcoxon tests). (C) Effects of Drosha knockdown using RNAi for miRNAs of the three different categories, namely evolutionarily conserved miRNAs, lineage-specific miRNAs with moderate/high expression and lineage-specific miRNAs with low expression. (D) Inferred MFEs of the lineage-specific miRNAs were similar to those of the evolutionarily conserved miRNAs, and significantly stronger than the binding of shuffled control sequences. (E,F) The miRNA 5′ end and 3′ end processing precision of evolutionarily conserved and lineage-specific miRNAs. The processing precision was calculated as the fraction of mapped reads that corresponded precisely to the consensus sequences of genomic locus. The miRNAs are shown on the x-axis and ordered by the processing precision. The miRNA with the most precision is at percentile 1, and the one with the least precision is at percentile 100.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4556993&req=5

f3: Evidence for the presence of numerous locust miRNAs in an evolutionary view.(A) Box plot of miRNA expression for evolutionarily conserved miRNAs and lineage-specific miRNAs. *indicates P < 0.001 (Mann–Whitney–Wilcoxon tests). (B) Box plot of lineage-specific miRNA expression for lineage-specific miRNAs that showed no variations and those with sequence variations. The variable group represents the lineage-specific miRNAs in locusts that differed from those in band-winged grasshoppers. *indicates P = 0.009 (Mann–Whitney–Wilcoxon tests). (C) Effects of Drosha knockdown using RNAi for miRNAs of the three different categories, namely evolutionarily conserved miRNAs, lineage-specific miRNAs with moderate/high expression and lineage-specific miRNAs with low expression. (D) Inferred MFEs of the lineage-specific miRNAs were similar to those of the evolutionarily conserved miRNAs, and significantly stronger than the binding of shuffled control sequences. (E,F) The miRNA 5′ end and 3′ end processing precision of evolutionarily conserved and lineage-specific miRNAs. The processing precision was calculated as the fraction of mapped reads that corresponded precisely to the consensus sequences of genomic locus. The miRNAs are shown on the x-axis and ordered by the processing precision. The miRNA with the most precision is at percentile 1, and the one with the least precision is at percentile 100.
Mentions: Newly emerged miRNAs in an ancestral lineage are integrated into gene regulatory networks and play important roles in expression regulation. Therefore, their mature sequences should be under strong purifying selection, and they are rarely mutated with secondary loss in the descendants8. To check the presence of locust miRNAs in the closely related species sharing common ancestor with L. migratoria, we generated 9,483,626 million reads of 18–30 bp from adults of band-winged grasshopper, Oedaleus asiaticus26. A substantial fraction (46%, 385 in 833) of the locust lineage-specific miRNA precursors (37%, 255 in 689) and evolutionarily conserved miRNA precursors (90%, 130 in 144) were detected in the band-winged grasshopper, which confirmed that they were evolutionarily conserved at least after the emergence of Orthoptera. Similar to a previous report, evolutionarily conserved miRNAs had significantly higher expression levels than the lineage-specific miRNAs (Fig. 3A, P < 0.001, Mann–Whitney–Wilcoxon tests), which implied that the lineage-specific miRNAs were prone to show low expression27. Newly emerged miRNAs have been continuously created in metazoan genomes as lineage-specific miRNAs, and these evolutionarily young miRNAs are generally lowly expressed and appear to have nonsubstantial effects on regulatory networks2829. Pearson’s correlation tests showed that the expression of the lineage-specific miRNAs in adult locusts had a significantly positive correlation with that of their respective homologs in adult band-winged grasshoppers (P < 0.001, Pearson’s correlation tests). Based on the expression cut-off of TPM > 10, miRNAs were classified into the moderate/high and low expression groups. Numerous homologs (34%, 86 in 255) of the lineage-specific miRNAs exhibit moderate/high expression [transcripts per million (TPM) > 10] in the band-winged grasshoppers. Almost all the homologs of the lineage-specific miRNAs (92%, 79 in 86) that displayed moderate/high expression in band-winged grasshoppers were also found with moderate/high expression in locusts. The substantial expression of these miRNAs implied that these lineage-specific miRNAs might not be lowly expressed genomic by-products, and they have considerable effects on the transcriptome and their target genes in terms of regulatory networks29. We examined the strength of natural selection acting on the lineage-specific miRNAs to determine whether the lineage-specific miRNAs with low expression have undergone rapid sequence evolution28. Specifically, we determined the sequence variations in the miRNA mature region between band-winged grasshoppers and locusts, and compared the number of variant miRNAs between the lineage-specific miRNAs with moderate/high expression and those with low expression. We excluded the first and last three bases of sequencing reads in sequence variation detection, because of the frequent un-templated modifications at both 5P and 3P ends of mature miRNAs30. For the 130 evolutionarily conserved miRNAs detected in band-winged grasshoppers, only one miRNA in the moderate/high expression group had sequence variation in their mature region. This characteristic was consistent with the fact that strong purifying selection intensely constrained evolutionarily conserved miRNAs. However, contrary to that observed in evolutionarily conserved miRNAs (1 in 144), we observed a significant signal of sequence variances for the lineage-specific miRNAs (26 in 255, P < 0.001, χ2-tests). We found that the lineage-specific miRNAs that showed no variations were more highly expressed than those with sequence variations (Fig. 3B, P = 0.009, Mann–Whitney–Wilcoxon tests). Based on the expression cut-off of TPM > 10, miRNAs were classified into the moderate/high and low expression groups. Compared with those in the moderate/high expression groups, the lineage-specific miRNAs in the low expression group were subject to a significant signal of sequence variances (3 in 86 of the moderate/high expression group and 23 in 169 of the low expression group; P = 0.025, χ2-tests). We repeated the analysis with different cut-offs of TPM (P = 0.010 for TPM > 5 and P = 0.004 for TPM > 15, χ2-tests), reaching the same significant signals. Therefore, our analytical results suggested that the numerous lineage-specific miRNAs with moderate/high expression might be under high selective pressures, and showed evolutionary conservation at least after the emergence of Orthoptera. This finding was consistent with the fact that highly expressed miRNAs are under strict selective constraints to maintain sequence uniformity and play critical roles in a broad manner31. To determine the silencing effects of Drosha knockdown for miRNAs with different evolutionary origin, we compared the expression changes for the evolutionarily conserved miRNAs, the lineage-specific miRNAs with moderate/high expression and with low expression (Fig. 3C). The fold changes between the lineage-specific miRNAs with moderate/high expression and the lineage-specific miRNAs with low expression were comparable (P = 0.681, Mann–Whitney–Wilcoxon tests). Compared with those of the lineage-specific miRNAs with moderate/high expression (P = 0.9347, Mann–Whitney–Wilcoxon tests) and with those of the lineage-specific miRNAs with low expression (P = 0.9815, Mann–Whitney–Wilcoxon tests), the fold changes of evolutionarily conserved miRNAs was not significantly low. These data suggested that, similar to the evolutionarily conserved miRNAs, the lineage-specific miRNAs dependent on Drosha processing for their biogenesis, implying the authenticity of the lineage-specific miRNAs.

Bottom Line: We observed that abundant local duplication events of miRNAs, which were unique in locusts compared with those in other insects with small genome sizes, may be responsible for the substantial acquisition of miRNAs in locusts.Together, multiple evidence showed that the locust genome experienced a burst of miRNA acquisition, suggesting that genome size expansion may have considerable influences of miRNA innovation.These results provide new insight into the genomic dynamics of miRNA repertoires under genome size evolution.

View Article: PubMed Central - PubMed

Affiliation: Institute of Applied Biology, Shanxi University, Taiyuan, Shanxi, China.

ABSTRACT
Substantial accumulation of neutral sequences accounts for genome size expansion in animal genomes. Numerous novel microRNAs (miRNAs), which evolve in a birth and death manner, are considered evolutionary neutral sequences. The migratory locust is an ideal model to determine whether large genomes contain abundant neutral miRNAs because of its large genome size. A total of 833 miRNAs were discovered, and several miRNAs were randomly chosen for validation by Northern blot and RIP-qPCR. Three additional verification methods, namely, processing-dependent methods of miRNA biogenesis using RNAi, evolutionary comparison with closely related species, and evidence supported by tissue-specific expression, were applied to provide compelling results that support the authenticity of locust miRNAs. We observed that abundant local duplication events of miRNAs, which were unique in locusts compared with those in other insects with small genome sizes, may be responsible for the substantial acquisition of miRNAs in locusts. Together, multiple evidence showed that the locust genome experienced a burst of miRNA acquisition, suggesting that genome size expansion may have considerable influences of miRNA innovation. These results provide new insight into the genomic dynamics of miRNA repertoires under genome size evolution.

No MeSH data available.