Limits...
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.

Show MeSH
Expression pattern of MIR168 paralogs in Arabidopsis. A) Genomic region encompassing MIR168a; B) genomic region encompassing MIR168b. Black box: mature miR168; dashed box: miR168*; white boxes: 20 bp sequences forming the basal stem; light gray box: miR168 loop region; dark gray boxes: nearest exons in the genes upstream and downstream of MIR168, arrows indicate gene orientation. Distances are drawn to scale, with the exception of pre-miR168 (to a larger scale for clarity); +1 is the first nucleotide of the mature miR168. The pMIR168a::GFP-GUS, pMIR168b1::GFP-GUS and pMIR168b2::GFP-GUS constructs are represented underneath the genomic regions. C) GUS-staining of Arabidopsis transformant lines carrying the pMIR168a::GFP-GUS and pMIR168b1::GFP-GUS constructs.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2664809&req=5

Figure 5: Expression pattern of MIR168 paralogs in Arabidopsis. A) Genomic region encompassing MIR168a; B) genomic region encompassing MIR168b. Black box: mature miR168; dashed box: miR168*; white boxes: 20 bp sequences forming the basal stem; light gray box: miR168 loop region; dark gray boxes: nearest exons in the genes upstream and downstream of MIR168, arrows indicate gene orientation. Distances are drawn to scale, with the exception of pre-miR168 (to a larger scale for clarity); +1 is the first nucleotide of the mature miR168. The pMIR168a::GFP-GUS, pMIR168b1::GFP-GUS and pMIR168b2::GFP-GUS constructs are represented underneath the genomic regions. C) GUS-staining of Arabidopsis transformant lines carrying the pMIR168a::GFP-GUS and pMIR168b1::GFP-GUS constructs.

Mentions: The high conservation of MIR168b suggests that it could be expressed and functional, even though, up to now, no experimental evidence has been reported. The Arabidopsis intergenic region upstream of the mature miR168b is only approximately 500 bp long. Therefore, we used two genomic regions including the whole intergenic region plus 255 or 1038 bp upstream to functionally characterize the MIR168b promoter and ascertain if some regulatory elements may be present in the upstream gene. These two regions were used to drive the expression of a reporter eGFP-uidA fusion gene (pMIR168b1::GFP-GUS and pMIR168b2::GFP-GUS; Fig. 5B. See Methods) in stably transformed Arabidopsis transgenic lines. A construct encompassing the MIR168a promoter was used as a control (Fig. 5A). Both pMIR168b1::GFP-GUS and pMIR168b2::GFP-GUS constructs produced the same expression pattern (data not shown). This result indicates that the intergenic region used in the shortest construct contains all the regulatory information to drive MIR168b expression. Similarly to what was observed for MIR168a, the expression of MIR168b was localized in emerging leaves and in a region underneath the shoot apical meristem corresponding to leaf primordia (Fig. 5C). None of the MIR168b transgenic lines, in contrast to MIR168a, displayed expression in correspondence with vascular tissues.


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)

Expression pattern of MIR168 paralogs in Arabidopsis. A) Genomic region encompassing MIR168a; B) genomic region encompassing MIR168b. Black box: mature miR168; dashed box: miR168*; white boxes: 20 bp sequences forming the basal stem; light gray box: miR168 loop region; dark gray boxes: nearest exons in the genes upstream and downstream of MIR168, arrows indicate gene orientation. Distances are drawn to scale, with the exception of pre-miR168 (to a larger scale for clarity); +1 is the first nucleotide of the mature miR168. The pMIR168a::GFP-GUS, pMIR168b1::GFP-GUS and pMIR168b2::GFP-GUS constructs are represented underneath the genomic regions. C) GUS-staining of Arabidopsis transformant lines carrying the pMIR168a::GFP-GUS and pMIR168b1::GFP-GUS constructs.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Expression pattern of MIR168 paralogs in Arabidopsis. A) Genomic region encompassing MIR168a; B) genomic region encompassing MIR168b. Black box: mature miR168; dashed box: miR168*; white boxes: 20 bp sequences forming the basal stem; light gray box: miR168 loop region; dark gray boxes: nearest exons in the genes upstream and downstream of MIR168, arrows indicate gene orientation. Distances are drawn to scale, with the exception of pre-miR168 (to a larger scale for clarity); +1 is the first nucleotide of the mature miR168. The pMIR168a::GFP-GUS, pMIR168b1::GFP-GUS and pMIR168b2::GFP-GUS constructs are represented underneath the genomic regions. C) GUS-staining of Arabidopsis transformant lines carrying the pMIR168a::GFP-GUS and pMIR168b1::GFP-GUS constructs.
Mentions: The high conservation of MIR168b suggests that it could be expressed and functional, even though, up to now, no experimental evidence has been reported. The Arabidopsis intergenic region upstream of the mature miR168b is only approximately 500 bp long. Therefore, we used two genomic regions including the whole intergenic region plus 255 or 1038 bp upstream to functionally characterize the MIR168b promoter and ascertain if some regulatory elements may be present in the upstream gene. These two regions were used to drive the expression of a reporter eGFP-uidA fusion gene (pMIR168b1::GFP-GUS and pMIR168b2::GFP-GUS; Fig. 5B. See Methods) in stably transformed Arabidopsis transgenic lines. A construct encompassing the MIR168a promoter was used as a control (Fig. 5A). Both pMIR168b1::GFP-GUS and pMIR168b2::GFP-GUS constructs produced the same expression pattern (data not shown). This result indicates that the intergenic region used in the shortest construct contains all the regulatory information to drive MIR168b expression. Similarly to what was observed for MIR168a, the expression of MIR168b was localized in emerging leaves and in a region underneath the shoot apical meristem corresponding to leaf primordia (Fig. 5C). None of the MIR168b transgenic lines, in contrast to MIR168a, displayed expression in correspondence with vascular tissues.

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