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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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Synteny conservation and duplication dating of MIR168 paralogs. A) Synteny conservation of the genomic regions encompassing MIR168a and MIR168b in A. thaliana and P. trichocarpa. Arrows represent MIR168a and MIR168b; the squares represent coding genes with at least one homolog in both genomes; the black lines represent RBMs and the gray lines connect BLASTP hits with lower homology within the same syntenic regions. Dashed lines connect At4g19410 homologs; diagonal lines on Ptc_LG_III represent a 7 Mbp long region not syntenic to Arabidopsis. B) Phylogenetic reconstruction of At4g19410 homologs in the Arabidopsis and poplar genomes. The portion of the linearized tree representing the homologs of At4g19410 located in the same genomic regions as MIR168a and MIR168b is highlighted in black. Values at the branch roots correspond to majority rule consensus bootstrap values ≥ 50%. Ath: A. thaliana; Ptc: P. trichocarpa; Ks: number of synonymous nucleotide substitutions per synonymous site.
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Figure 1: Synteny conservation and duplication dating of MIR168 paralogs. A) Synteny conservation of the genomic regions encompassing MIR168a and MIR168b in A. thaliana and P. trichocarpa. Arrows represent MIR168a and MIR168b; the squares represent coding genes with at least one homolog in both genomes; the black lines represent RBMs and the gray lines connect BLASTP hits with lower homology within the same syntenic regions. Dashed lines connect At4g19410 homologs; diagonal lines on Ptc_LG_III represent a 7 Mbp long region not syntenic to Arabidopsis. B) Phylogenetic reconstruction of At4g19410 homologs in the Arabidopsis and poplar genomes. The portion of the linearized tree representing the homologs of At4g19410 located in the same genomic regions as MIR168a and MIR168b is highlighted in black. Values at the branch roots correspond to majority rule consensus bootstrap values ≥ 50%. Ath: A. thaliana; Ptc: P. trichocarpa; Ks: number of synonymous nucleotide substitutions per synonymous site.

Mentions: Analyses of synteny conservation were carried out by searching in poplar for the putative orthologs of the 20 Arabidopsis genes flanking MIR168a and MIR168b by screening for Reciprocal Best Matches (RBM) in BLASTP searches [28] (see Methods; Fig. 1A and Additional File 1). The queries from the former analyses were then used to identify recent segmental duplications (see Methods). Assuming orthology among the Arabidopsis and poplar genomic regions encompassing the MIR168 loci, the surrounding RBM pairs should be found mainly among the same pair of chromosomes. The uneven distribution of loci forming RBM pairs, however, indicated that the MIR168 loci may have been the result of independent duplication events.


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)

Synteny conservation and duplication dating of MIR168 paralogs. A) Synteny conservation of the genomic regions encompassing MIR168a and MIR168b in A. thaliana and P. trichocarpa. Arrows represent MIR168a and MIR168b; the squares represent coding genes with at least one homolog in both genomes; the black lines represent RBMs and the gray lines connect BLASTP hits with lower homology within the same syntenic regions. Dashed lines connect At4g19410 homologs; diagonal lines on Ptc_LG_III represent a 7 Mbp long region not syntenic to Arabidopsis. B) Phylogenetic reconstruction of At4g19410 homologs in the Arabidopsis and poplar genomes. The portion of the linearized tree representing the homologs of At4g19410 located in the same genomic regions as MIR168a and MIR168b is highlighted in black. Values at the branch roots correspond to majority rule consensus bootstrap values ≥ 50%. Ath: A. thaliana; Ptc: P. trichocarpa; Ks: number of synonymous nucleotide substitutions per synonymous site.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Synteny conservation and duplication dating of MIR168 paralogs. A) Synteny conservation of the genomic regions encompassing MIR168a and MIR168b in A. thaliana and P. trichocarpa. Arrows represent MIR168a and MIR168b; the squares represent coding genes with at least one homolog in both genomes; the black lines represent RBMs and the gray lines connect BLASTP hits with lower homology within the same syntenic regions. Dashed lines connect At4g19410 homologs; diagonal lines on Ptc_LG_III represent a 7 Mbp long region not syntenic to Arabidopsis. B) Phylogenetic reconstruction of At4g19410 homologs in the Arabidopsis and poplar genomes. The portion of the linearized tree representing the homologs of At4g19410 located in the same genomic regions as MIR168a and MIR168b is highlighted in black. Values at the branch roots correspond to majority rule consensus bootstrap values ≥ 50%. Ath: A. thaliana; Ptc: P. trichocarpa; Ks: number of synonymous nucleotide substitutions per synonymous site.
Mentions: Analyses of synteny conservation were carried out by searching in poplar for the putative orthologs of the 20 Arabidopsis genes flanking MIR168a and MIR168b by screening for Reciprocal Best Matches (RBM) in BLASTP searches [28] (see Methods; Fig. 1A and Additional File 1). The queries from the former analyses were then used to identify recent segmental duplications (see Methods). Assuming orthology among the Arabidopsis and poplar genomic regions encompassing the MIR168 loci, the surrounding RBM pairs should be found mainly among the same pair of chromosomes. The uneven distribution of loci forming RBM pairs, however, indicated that the MIR168 loci may have been the result of independent duplication events.

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