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Regulation of Human PAX6 Expression by miR-7.

Needhamsen M, White RB, Giles KM, Dunlop SA, Thomas MG - Evol. Bioinform. Online (2014)

Bottom Line: Furthermore, transient transfection of cells with synthetic miR-7 inhibits PAX6 protein expression but does not alter levels of PAX6 mRNA, suggesting that miR-7 induces translational repression of PAX6.Finally, a comparison of PAX6 3'-UTRs across species reveals that one of the functional miR-7 target sites is conserved, whereas the second functional target site is found only in primates.Thus, the interaction between PAX6 and miR-7 appears to be highly conserved; however, the precise number of sites through which this interaction occurs may have expanded throughout evolution.

View Article: PubMed Central - PubMed

Affiliation: Parkinson's Centre (ParkC), School of Medical Sciences, Edith Cowan University, Joondalup, Western Australia, Australia. ; Experimental and Regenerative Neurosciences (EaRN), School of Animal Biology, University of Western Australia, Crawley, Western Australia, Australia.

ABSTRACT
The paired box gene 6 (PAX6) is a powerful mediator of eye and brain organogenesis whose spatiotemporal expression is exquisitely controlled by multiple mechanisms, including post-transcriptional regulation by microRNAs (miRNAs). In the present study, we use bioinformatic predictions to identify three candidate microRNA-7 (miR-7) target sites in the human PAX6 3' untranslated region (3'-UTR) and demonstrate that two of them are functionally active in a human cell line. Furthermore, transient transfection of cells with synthetic miR-7 inhibits PAX6 protein expression but does not alter levels of PAX6 mRNA, suggesting that miR-7 induces translational repression of PAX6. Finally, a comparison of PAX6 3'-UTRs across species reveals that one of the functional miR-7 target sites is conserved, whereas the second functional target site is found only in primates. Thus, the interaction between PAX6 and miR-7 appears to be highly conserved; however, the precise number of sites through which this interaction occurs may have expanded throughout evolution.

No MeSH data available.


Diversification of the PAX6 3′-UTR and prediction of 7mer-m8 target sites. (A) Schematic representation of validated PAX6 mRNAs from the following species: H. sapiens (human), M. mulatta (rhesus monkey), S. scrofa (pig), M. musculus (mouse), R. norvegicus (rat), G. gallus (chicken), X. tropicalis (frog), D. rerio (zebrafish): PAX6a and PAX6b, and D. melanogaster (fruit fly), and eyeless (Ey), twin of eyeless (Toy) isoforms a and c, eyegone (Eyg), and twin of eyegone (Toe) variants a and b. The 5′-UTRs and coding DNA sequences (CDSs) are represented in dark gray, and 3′-UTRs in light gray. Percentages illustrate the size of individual 3′-UTRs compared to full-length transcripts, and asterisks illustrate predicted miR-7 target sites. (B) Alignment of the conserved predicted 7mer-m8 miR-7 target sites of H. sapiens, M. mulatta, S. scrofa, M. musculus, R. norvegicus, and X. tropicalis. The seed region is in bold, and identical nucleotides are illustrated with an asterisk. The consensus sequence is given and base pairing to miR-7 is illustrated.
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f3-ebo-10-2014-107: Diversification of the PAX6 3′-UTR and prediction of 7mer-m8 target sites. (A) Schematic representation of validated PAX6 mRNAs from the following species: H. sapiens (human), M. mulatta (rhesus monkey), S. scrofa (pig), M. musculus (mouse), R. norvegicus (rat), G. gallus (chicken), X. tropicalis (frog), D. rerio (zebrafish): PAX6a and PAX6b, and D. melanogaster (fruit fly), and eyeless (Ey), twin of eyeless (Toy) isoforms a and c, eyegone (Eyg), and twin of eyegone (Toe) variants a and b. The 5′-UTRs and coding DNA sequences (CDSs) are represented in dark gray, and 3′-UTRs in light gray. Percentages illustrate the size of individual 3′-UTRs compared to full-length transcripts, and asterisks illustrate predicted miR-7 target sites. (B) Alignment of the conserved predicted 7mer-m8 miR-7 target sites of H. sapiens, M. mulatta, S. scrofa, M. musculus, R. norvegicus, and X. tropicalis. The seed region is in bold, and identical nucleotides are illustrated with an asterisk. The consensus sequence is given and base pairing to miR-7 is illustrated.

Mentions: Recent experiments in mice have demonstrated that miR-7 directly represses the expression of PAX6 through a single miR-7 target site in the mouse PAX6 3′-UTR.8,9 Given our identification of a second functional miR-7 target site (7mer-m8) in human PAX6 3′-UTR compared to mouse, we examined the evolutionary conservation of PAX6 3′-UTRs and their miR-7 target sites across species, and found that the length of PAX6 3′-UTRs varied considerably and that this length seemed to be associated with the number of predicted miR-7 target sites (Fig. 3A). For example, in primates, human (Homo sapiens) and rhesus monkey (Macaca mulatta), PAX6 has relatively long 3′-UTRs (accounting for more than 70% of the total mRNA sequences), and we demonstrated the existence of two functional 7mer-m8 miR-7 target sites within these regions. In comparison, the 3′-UTRs of pig (Sus scrofa), rodents (Mus musculus and Rattus norvegicus), frog (Xenopus tropicalis) and fish (Danio rerio) account for between 30 and 39% of total PAX6 mRNA length and contain only one predicted miR-7 target site (Fig. 3A). In other species such as chicken (Gallus gallus) and fruit fly (Drosophila melanogaster), where the 3′-UTR accounts for less than 20% of the total PAX6 mRNA sequence, no miR-7 target sites were predicted (Fig. 3A). Notably, in zebrafish, there are two duplicated PAX6 gene variants, PAX6a and PAX6b, with distinct 3′-UTR lengths, accounting for 34%–39% of the total mRNA lengths, respectively (Fig. 3A). PAX6a contains a single predicted miR-7 target site, whereas no site was predicted in the shorter 3′-UTR of the PAX6b variant, suggesting that perhaps only PAX6a is post-transcription-ally regulated by miR-7. Also, the fruit fly has multiple PAX6 gene loci, from which six different PAX6 3′-UTRs are transcribed; none have a predicted miR-7 target site in the 3′-UTR despite one transcript having a 3′-UTR that accounts for more than 45% of the total mRNA sequence (Fig. 3A), suggesting that miR-7 might not regulate PAX6 expression levels in fruit flies. These results are in agreement with previous work that suggested the expansion of 3′-UTR sequences increases with organism complexity throughout evolution.29


Regulation of Human PAX6 Expression by miR-7.

Needhamsen M, White RB, Giles KM, Dunlop SA, Thomas MG - Evol. Bioinform. Online (2014)

Diversification of the PAX6 3′-UTR and prediction of 7mer-m8 target sites. (A) Schematic representation of validated PAX6 mRNAs from the following species: H. sapiens (human), M. mulatta (rhesus monkey), S. scrofa (pig), M. musculus (mouse), R. norvegicus (rat), G. gallus (chicken), X. tropicalis (frog), D. rerio (zebrafish): PAX6a and PAX6b, and D. melanogaster (fruit fly), and eyeless (Ey), twin of eyeless (Toy) isoforms a and c, eyegone (Eyg), and twin of eyegone (Toe) variants a and b. The 5′-UTRs and coding DNA sequences (CDSs) are represented in dark gray, and 3′-UTRs in light gray. Percentages illustrate the size of individual 3′-UTRs compared to full-length transcripts, and asterisks illustrate predicted miR-7 target sites. (B) Alignment of the conserved predicted 7mer-m8 miR-7 target sites of H. sapiens, M. mulatta, S. scrofa, M. musculus, R. norvegicus, and X. tropicalis. The seed region is in bold, and identical nucleotides are illustrated with an asterisk. The consensus sequence is given and base pairing to miR-7 is illustrated.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4116382&req=5

f3-ebo-10-2014-107: Diversification of the PAX6 3′-UTR and prediction of 7mer-m8 target sites. (A) Schematic representation of validated PAX6 mRNAs from the following species: H. sapiens (human), M. mulatta (rhesus monkey), S. scrofa (pig), M. musculus (mouse), R. norvegicus (rat), G. gallus (chicken), X. tropicalis (frog), D. rerio (zebrafish): PAX6a and PAX6b, and D. melanogaster (fruit fly), and eyeless (Ey), twin of eyeless (Toy) isoforms a and c, eyegone (Eyg), and twin of eyegone (Toe) variants a and b. The 5′-UTRs and coding DNA sequences (CDSs) are represented in dark gray, and 3′-UTRs in light gray. Percentages illustrate the size of individual 3′-UTRs compared to full-length transcripts, and asterisks illustrate predicted miR-7 target sites. (B) Alignment of the conserved predicted 7mer-m8 miR-7 target sites of H. sapiens, M. mulatta, S. scrofa, M. musculus, R. norvegicus, and X. tropicalis. The seed region is in bold, and identical nucleotides are illustrated with an asterisk. The consensus sequence is given and base pairing to miR-7 is illustrated.
Mentions: Recent experiments in mice have demonstrated that miR-7 directly represses the expression of PAX6 through a single miR-7 target site in the mouse PAX6 3′-UTR.8,9 Given our identification of a second functional miR-7 target site (7mer-m8) in human PAX6 3′-UTR compared to mouse, we examined the evolutionary conservation of PAX6 3′-UTRs and their miR-7 target sites across species, and found that the length of PAX6 3′-UTRs varied considerably and that this length seemed to be associated with the number of predicted miR-7 target sites (Fig. 3A). For example, in primates, human (Homo sapiens) and rhesus monkey (Macaca mulatta), PAX6 has relatively long 3′-UTRs (accounting for more than 70% of the total mRNA sequences), and we demonstrated the existence of two functional 7mer-m8 miR-7 target sites within these regions. In comparison, the 3′-UTRs of pig (Sus scrofa), rodents (Mus musculus and Rattus norvegicus), frog (Xenopus tropicalis) and fish (Danio rerio) account for between 30 and 39% of total PAX6 mRNA length and contain only one predicted miR-7 target site (Fig. 3A). In other species such as chicken (Gallus gallus) and fruit fly (Drosophila melanogaster), where the 3′-UTR accounts for less than 20% of the total PAX6 mRNA sequence, no miR-7 target sites were predicted (Fig. 3A). Notably, in zebrafish, there are two duplicated PAX6 gene variants, PAX6a and PAX6b, with distinct 3′-UTR lengths, accounting for 34%–39% of the total mRNA lengths, respectively (Fig. 3A). PAX6a contains a single predicted miR-7 target site, whereas no site was predicted in the shorter 3′-UTR of the PAX6b variant, suggesting that perhaps only PAX6a is post-transcription-ally regulated by miR-7. Also, the fruit fly has multiple PAX6 gene loci, from which six different PAX6 3′-UTRs are transcribed; none have a predicted miR-7 target site in the 3′-UTR despite one transcript having a 3′-UTR that accounts for more than 45% of the total mRNA sequence (Fig. 3A), suggesting that miR-7 might not regulate PAX6 expression levels in fruit flies. These results are in agreement with previous work that suggested the expansion of 3′-UTR sequences increases with organism complexity throughout evolution.29

Bottom Line: Furthermore, transient transfection of cells with synthetic miR-7 inhibits PAX6 protein expression but does not alter levels of PAX6 mRNA, suggesting that miR-7 induces translational repression of PAX6.Finally, a comparison of PAX6 3'-UTRs across species reveals that one of the functional miR-7 target sites is conserved, whereas the second functional target site is found only in primates.Thus, the interaction between PAX6 and miR-7 appears to be highly conserved; however, the precise number of sites through which this interaction occurs may have expanded throughout evolution.

View Article: PubMed Central - PubMed

Affiliation: Parkinson's Centre (ParkC), School of Medical Sciences, Edith Cowan University, Joondalup, Western Australia, Australia. ; Experimental and Regenerative Neurosciences (EaRN), School of Animal Biology, University of Western Australia, Crawley, Western Australia, Australia.

ABSTRACT
The paired box gene 6 (PAX6) is a powerful mediator of eye and brain organogenesis whose spatiotemporal expression is exquisitely controlled by multiple mechanisms, including post-transcriptional regulation by microRNAs (miRNAs). In the present study, we use bioinformatic predictions to identify three candidate microRNA-7 (miR-7) target sites in the human PAX6 3' untranslated region (3'-UTR) and demonstrate that two of them are functionally active in a human cell line. Furthermore, transient transfection of cells with synthetic miR-7 inhibits PAX6 protein expression but does not alter levels of PAX6 mRNA, suggesting that miR-7 induces translational repression of PAX6. Finally, a comparison of PAX6 3'-UTRs across species reveals that one of the functional miR-7 target sites is conserved, whereas the second functional target site is found only in primates. Thus, the interaction between PAX6 and miR-7 appears to be highly conserved; however, the precise number of sites through which this interaction occurs may have expanded throughout evolution.

No MeSH data available.