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Evolution of MIR168 paralogs in Brassicaceae.

Gazzani S, Li M, Maistri S, Scarponi E, Graziola M, Barbaro E, Wunder J, Furini A, Saedler H, Varotto C - BMC Evol. Biol. (2009)

Bottom Line: Different phylogenetic footprints, corresponding to known functionally relevant regions (transcription starting site and double-stranded structures responsible for microRNA biogenesis and function) or for which functions could be proposed, were found to be highly conserved among MIR168 homologs.Although their duplication happened at least 40 million years ago, we found evidence that both MIR168 paralogs have been maintained throughout the evolution of Brassicaceae, most likely functionally as indicated by the extremely high conservation of functionally relevant regions, predicted secondary structure and thermodynamic profile.We found further evolutionary evidence that pre-miR168 lower stem (the RNA-duplex structure adjacent to the miR-miR* stem) is significantly longer than animal lower stems and probably plays a relevant role in multi-step miR168 biogenesis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Environment and Natural Resources Area, Fondazione Edmund Mach, via Mach 1, 38010 San Michele all'Adige (TN), Italy. silvia.gazzani@iasma.it

ABSTRACT

Background: In plants, expression of ARGONAUTE1 (AGO1), the catalytic subunit of the RNA-Induced Silencing Complex responsible for post-transcriptional gene silencing, is controlled through a feedback loop involving the miR168 microRNA. This complex auto-regulatory loop, composed of miR168-guided AGO1-catalyzed cleavage of AGO1 mRNA and AGO1-mediated stabilization of miR168, was shown to ensure the maintenance of AGO1 homeostasis that is pivotal for the correct functioning of the miRNA pathway.

Results: We applied different approaches to studying the genomic organization and the structural and functional evolution of MIR168 homologs in Brassicaeae. A whole genome comparison of Arabidopsis and poplar, phylogenetic footprinting and phylogenetic reconstruction were used to date the duplication events originating MIR168 homologs in these genomes. While orthology was lacking between Arabidopsis and poplar MIR168 genes, we successfully isolated orthologs of both loci present in Arabidopsis (MIR168a and MIR168b) from all the Brassicaceae species analyzed, including the basal species Aethionema grandiflora, thus indicating that (1) independent duplication events took place in Arabidopsis and poplar lineages and (2) the origin of MIR168 paralogs predates both the Brassicaceae radiation and the Arabidopsis alpha polyploidization. Different phylogenetic footprints, corresponding to known functionally relevant regions (transcription starting site and double-stranded structures responsible for microRNA biogenesis and function) or for which functions could be proposed, were found to be highly conserved among MIR168 homologs. Comparative predictions of the identified microRNAs also indicate extreme conservation of secondary structure and thermodynamic stability.

Conclusion: We used a comparative phylogenetic footprinting approach to identify the structural and functional constraints that shaped MIR168 evolution in Brassicaceae. Although their duplication happened at least 40 million years ago, we found evidence that both MIR168 paralogs have been maintained throughout the evolution of Brassicaceae, most likely functionally as indicated by the extremely high conservation of functionally relevant regions, predicted secondary structure and thermodynamic profile. Interestingly, the expression patterns observed in Arabidopsis indicate that MIR168b underwent partial subfunctionalization as determined by the experimental characterization of its expression pattern provided in this study. We found further evolutionary evidence that pre-miR168 lower stem (the RNA-duplex structure adjacent to the miR-miR* stem) is significantly longer than animal lower stems and probably plays a relevant role in multi-step miR168 biogenesis.

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Thermodynamic stability and nucleotide substitution profiles of pre-miR168a and pre-miR168b. A) Thermodynamic stability profile of pre-miR168a and pre-miR168b in the Brassicaceae family. Free energy values are given in kcal/mole. Vertical bars: between-species variability calculated as double standard error. B) Distribution of nucleotide substitutions with respect to base pairing in the pre-miR168a and pre-miR168b secondary structures. Yellow: structurally conservative base substitution; ochre: base substitution comporting a change in length of a bulge loop; blue: base substitution comporting a change from unpaired to paired bases; red: base substitution comporting a change from paired to unpaired bases. The rate of nucleotide substitution is given in percentages.
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Figure 4: Thermodynamic stability and nucleotide substitution profiles of pre-miR168a and pre-miR168b. A) Thermodynamic stability profile of pre-miR168a and pre-miR168b in the Brassicaceae family. Free energy values are given in kcal/mole. Vertical bars: between-species variability calculated as double standard error. B) Distribution of nucleotide substitutions with respect to base pairing in the pre-miR168a and pre-miR168b secondary structures. Yellow: structurally conservative base substitution; ochre: base substitution comporting a change in length of a bulge loop; blue: base substitution comporting a change from unpaired to paired bases; red: base substitution comporting a change from paired to unpaired bases. The rate of nucleotide substitution is given in percentages.

Mentions: The average thermodynamic profile calculated from the predicted minimum free energy (MFE) structure of each species was nearly identical at the level of the upper stem and more variable for the lower stem of both microRNAs (Fig. 4A). A common feature of both the upper and lower stem was that the secondary structure was less stable (higher free energy value, dG) at the 5' side with an increase in stability in the central part and at the 3' side. The level of nucleotidic conservation across species, however, did not correlate with the dG values, indicating that the observed footprints could not be explained by a simple increase in the stability of the corresponding secondary structure (see e.g., MIR168a; Fig. 4A). On the contrary, the comparison of MIR168a and MIR168b thermodynamic profiles and the classification of their nucleotide substitutions with respect to base pairing indicated a clear positional effect concerning the lower stem: the central region was more variable than the 3–4 bp close to each end of both stems. In particular the nucleotidic stretch of 5–6 bp connecting upper and lower stems of both microRNAs (position -3, +3) were extremely conserved despite having an average free energy of -1.6 Kcal/mole, which is the average free energy of both stems.


Evolution of MIR168 paralogs in Brassicaceae.

Gazzani S, Li M, Maistri S, Scarponi E, Graziola M, Barbaro E, Wunder J, Furini A, Saedler H, Varotto C - BMC Evol. Biol. (2009)

Thermodynamic stability and nucleotide substitution profiles of pre-miR168a and pre-miR168b. A) Thermodynamic stability profile of pre-miR168a and pre-miR168b in the Brassicaceae family. Free energy values are given in kcal/mole. Vertical bars: between-species variability calculated as double standard error. B) Distribution of nucleotide substitutions with respect to base pairing in the pre-miR168a and pre-miR168b secondary structures. Yellow: structurally conservative base substitution; ochre: base substitution comporting a change in length of a bulge loop; blue: base substitution comporting a change from unpaired to paired bases; red: base substitution comporting a change from paired to unpaired bases. The rate of nucleotide substitution is given in percentages.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2664809&req=5

Figure 4: Thermodynamic stability and nucleotide substitution profiles of pre-miR168a and pre-miR168b. A) Thermodynamic stability profile of pre-miR168a and pre-miR168b in the Brassicaceae family. Free energy values are given in kcal/mole. Vertical bars: between-species variability calculated as double standard error. B) Distribution of nucleotide substitutions with respect to base pairing in the pre-miR168a and pre-miR168b secondary structures. Yellow: structurally conservative base substitution; ochre: base substitution comporting a change in length of a bulge loop; blue: base substitution comporting a change from unpaired to paired bases; red: base substitution comporting a change from paired to unpaired bases. The rate of nucleotide substitution is given in percentages.
Mentions: The average thermodynamic profile calculated from the predicted minimum free energy (MFE) structure of each species was nearly identical at the level of the upper stem and more variable for the lower stem of both microRNAs (Fig. 4A). A common feature of both the upper and lower stem was that the secondary structure was less stable (higher free energy value, dG) at the 5' side with an increase in stability in the central part and at the 3' side. The level of nucleotidic conservation across species, however, did not correlate with the dG values, indicating that the observed footprints could not be explained by a simple increase in the stability of the corresponding secondary structure (see e.g., MIR168a; Fig. 4A). On the contrary, the comparison of MIR168a and MIR168b thermodynamic profiles and the classification of their nucleotide substitutions with respect to base pairing indicated a clear positional effect concerning the lower stem: the central region was more variable than the 3–4 bp close to each end of both stems. In particular the nucleotidic stretch of 5–6 bp connecting upper and lower stems of both microRNAs (position -3, +3) were extremely conserved despite having an average free energy of -1.6 Kcal/mole, which is the average free energy of both stems.

Bottom Line: Different phylogenetic footprints, corresponding to known functionally relevant regions (transcription starting site and double-stranded structures responsible for microRNA biogenesis and function) or for which functions could be proposed, were found to be highly conserved among MIR168 homologs.Although their duplication happened at least 40 million years ago, we found evidence that both MIR168 paralogs have been maintained throughout the evolution of Brassicaceae, most likely functionally as indicated by the extremely high conservation of functionally relevant regions, predicted secondary structure and thermodynamic profile.We found further evolutionary evidence that pre-miR168 lower stem (the RNA-duplex structure adjacent to the miR-miR* stem) is significantly longer than animal lower stems and probably plays a relevant role in multi-step miR168 biogenesis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Environment and Natural Resources Area, Fondazione Edmund Mach, via Mach 1, 38010 San Michele all'Adige (TN), Italy. silvia.gazzani@iasma.it

ABSTRACT

Background: In plants, expression of ARGONAUTE1 (AGO1), the catalytic subunit of the RNA-Induced Silencing Complex responsible for post-transcriptional gene silencing, is controlled through a feedback loop involving the miR168 microRNA. This complex auto-regulatory loop, composed of miR168-guided AGO1-catalyzed cleavage of AGO1 mRNA and AGO1-mediated stabilization of miR168, was shown to ensure the maintenance of AGO1 homeostasis that is pivotal for the correct functioning of the miRNA pathway.

Results: We applied different approaches to studying the genomic organization and the structural and functional evolution of MIR168 homologs in Brassicaeae. A whole genome comparison of Arabidopsis and poplar, phylogenetic footprinting and phylogenetic reconstruction were used to date the duplication events originating MIR168 homologs in these genomes. While orthology was lacking between Arabidopsis and poplar MIR168 genes, we successfully isolated orthologs of both loci present in Arabidopsis (MIR168a and MIR168b) from all the Brassicaceae species analyzed, including the basal species Aethionema grandiflora, thus indicating that (1) independent duplication events took place in Arabidopsis and poplar lineages and (2) the origin of MIR168 paralogs predates both the Brassicaceae radiation and the Arabidopsis alpha polyploidization. Different phylogenetic footprints, corresponding to known functionally relevant regions (transcription starting site and double-stranded structures responsible for microRNA biogenesis and function) or for which functions could be proposed, were found to be highly conserved among MIR168 homologs. Comparative predictions of the identified microRNAs also indicate extreme conservation of secondary structure and thermodynamic stability.

Conclusion: We used a comparative phylogenetic footprinting approach to identify the structural and functional constraints that shaped MIR168 evolution in Brassicaceae. Although their duplication happened at least 40 million years ago, we found evidence that both MIR168 paralogs have been maintained throughout the evolution of Brassicaceae, most likely functionally as indicated by the extremely high conservation of functionally relevant regions, predicted secondary structure and thermodynamic profile. Interestingly, the expression patterns observed in Arabidopsis indicate that MIR168b underwent partial subfunctionalization as determined by the experimental characterization of its expression pattern provided in this study. We found further evolutionary evidence that pre-miR168 lower stem (the RNA-duplex structure adjacent to the miR-miR* stem) is significantly longer than animal lower stems and probably plays a relevant role in multi-step miR168 biogenesis.

Show MeSH
Related in: MedlinePlus