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Identification and characterization of FGF2-dependent mRNA: microRNA networks during lens fiber cell differentiation.

Wolf L, Gao CS, Gueta K, Xie Q, Chevallier T, Podduturi NR, Sun J, Conte I, Zelenka PS, Ashery-Padan R, Zavadil J, Cvekl A - G3 (Bethesda) (2013)

Bottom Line: Specific miRNA:mRNA interaction networks were predicted for c-Maf, N-Myc, and Nfib (DNA-binding transcription factors); Cnot6, Cpsf6, Dicer1, and Tnrc6b (RNA to miRNA processing); and Ash1l, Med1/PBP, and Kdm5b/Jarid1b/Plu1 (chromatin remodeling).Three miRNAs, including miR-143, miR-155, and miR-301a, down-regulated expression of c-Maf in the 3'-UTR luciferase reporter assays.These present studies demonstrate for the first time global impact of activated FGF signaling in lens cell culture system and predicted novel gene regulatory networks connected by multiple miRNAs that regulate lens differentiation.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Albert Einstein College of Medicine, Bronx, New York 10461.

ABSTRACT
MicroRNAs (miRNAs) and fibroblast growth factor (FGF) signaling regulate a wide range of cellular functions, including cell specification, proliferation, migration, differentiation, and survival. In lens, both these systems control lens fiber cell differentiation; however, a possible link between these processes remains to be examined. Herein, the functional requirement for miRNAs in differentiating lens fiber cells was demonstrated via conditional inactivation of Dicer1 in mouse (Mus musculus) lens. To dissect the miRNA-dependent pathways during lens differentiation, we used a rat (Rattus norvegicus) lens epithelial explant system, induced by FGF2 to differentiate, followed by mRNA and miRNA expression profiling. Transcriptome and miRNome analysis identified extensive FGF2-regulated cellular responses that were both independent and dependent on miRNAs. We identified 131 FGF2-regulated miRNAs. Seventy-six of these miRNAs had at least two in silico predicted and inversely regulated target mRNAs. Genes modulated by the greatest number of FGF-regulated miRNAs include DNA-binding transcription factors Nfib, Nfat5/OREBP, c-Maf, Ets1, and N-Myc. Activated FGF signaling influenced bone morphogenetic factor/transforming growth factor-β, Notch, and Wnt signaling cascades implicated earlier in lens differentiation. Specific miRNA:mRNA interaction networks were predicted for c-Maf, N-Myc, and Nfib (DNA-binding transcription factors); Cnot6, Cpsf6, Dicer1, and Tnrc6b (RNA to miRNA processing); and Ash1l, Med1/PBP, and Kdm5b/Jarid1b/Plu1 (chromatin remodeling). Three miRNAs, including miR-143, miR-155, and miR-301a, down-regulated expression of c-Maf in the 3'-UTR luciferase reporter assays. These present studies demonstrate for the first time global impact of activated FGF signaling in lens cell culture system and predicted novel gene regulatory networks connected by multiple miRNAs that regulate lens differentiation.

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FGF2-regulated miRNAs and posttranscriptional control of c-Maf. (A) Localization of miRNA-binding regions in the mouse c-Maf 3′-UTR (NCBI Reference Sequence NM_001025577.2) and their separation into three shorter regions, WT1 (~0.8 kb), WT2 (~0.2 kb), and WT3 (~0.5 kb). These regions are evolutionary conserved among mammals. (B) Sequence alignment of individual miRNAs:3′-UTR mRNA pairs. Watson-Crick (vertical lines) and wobble (G:U) base pairing are shown. The nucleotides changed in mutated reporter plasmids are highlighted in gray. (C) Luciferase reporter assays using the WT1, WT2, and WT3. (D) Luciferase reporter assays using specific mutants of WT1, WT2, and WT3 (see panel B).
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fig10: FGF2-regulated miRNAs and posttranscriptional control of c-Maf. (A) Localization of miRNA-binding regions in the mouse c-Maf 3′-UTR (NCBI Reference Sequence NM_001025577.2) and their separation into three shorter regions, WT1 (~0.8 kb), WT2 (~0.2 kb), and WT3 (~0.5 kb). These regions are evolutionary conserved among mammals. (B) Sequence alignment of individual miRNAs:3′-UTR mRNA pairs. Watson-Crick (vertical lines) and wobble (G:U) base pairing are shown. The nucleotides changed in mutated reporter plasmids are highlighted in gray. (C) Luciferase reporter assays using the WT1, WT2, and WT3. (D) Luciferase reporter assays using specific mutants of WT1, WT2, and WT3 (see panel B).

Mentions: The DNA-binding transcription factors Pax6 and c-Maf are key regulators of lens-specific GRNs that control the crystallin gene machinery (Chauhan et al. 2004; Yang et al. 2004, 2006; Yang and Cvekl 2005; Xie and Cvekl 2011). Expression of c-Maf, but not of Pax6, is up-regulated in the postmitotic cells of the lens vesicle that give rise to the primary lens fiber cells, raising the possibility that c-Maf is regulated at the level of transcription through FGFs originating from the prospective neuroretina (Lovicu and McAvoy 2005). Subsequent genetic studies supported this model (Zhao et al. 2008; Garcia et al. 2011; Qu et al. 2011). The current data suggest that multiple miRNAs, including miR-9, miR-137, miR-155, miR-301a, miR455, and miR-543 (Figure 7A and Figure 8A), regulate c-Maf expression through its 3′-UTR. In addition, c-Maf 3′-UTR contains a miR-143 target sequence (Figure 10, A and B). Herein, we conducted a series of luciferase reporter gene assays using three wild-type and eight mutated reporters using mouse lens epithelial cell line αTN4-1 (Yang and Cvekl 2005). The c-Maf 3′-UTR was divided into three shorter fragments, WT1 to WT3, as shown in Figure 10A. The predicted 3′-UTR:miRNA pairs for selected six binding sites are shown in Figure 10B. The results of luc reporter assays are summarized in Figure 10, C and D. The data showed that miR-143 and miR-155 down-regulated the WT2 c-Maf 3′-UTR, and miR-301a down-regulated the WT3 c-Maf 3′-UTR (Figure 10C, n = 6, P < 0.05). Site-directed mutagenesis of the predicted target sites yielded no statistically significant changes between the wild-type and mutated reporters (Figure 10D).


Identification and characterization of FGF2-dependent mRNA: microRNA networks during lens fiber cell differentiation.

Wolf L, Gao CS, Gueta K, Xie Q, Chevallier T, Podduturi NR, Sun J, Conte I, Zelenka PS, Ashery-Padan R, Zavadil J, Cvekl A - G3 (Bethesda) (2013)

FGF2-regulated miRNAs and posttranscriptional control of c-Maf. (A) Localization of miRNA-binding regions in the mouse c-Maf 3′-UTR (NCBI Reference Sequence NM_001025577.2) and their separation into three shorter regions, WT1 (~0.8 kb), WT2 (~0.2 kb), and WT3 (~0.5 kb). These regions are evolutionary conserved among mammals. (B) Sequence alignment of individual miRNAs:3′-UTR mRNA pairs. Watson-Crick (vertical lines) and wobble (G:U) base pairing are shown. The nucleotides changed in mutated reporter plasmids are highlighted in gray. (C) Luciferase reporter assays using the WT1, WT2, and WT3. (D) Luciferase reporter assays using specific mutants of WT1, WT2, and WT3 (see panel B).
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fig10: FGF2-regulated miRNAs and posttranscriptional control of c-Maf. (A) Localization of miRNA-binding regions in the mouse c-Maf 3′-UTR (NCBI Reference Sequence NM_001025577.2) and their separation into three shorter regions, WT1 (~0.8 kb), WT2 (~0.2 kb), and WT3 (~0.5 kb). These regions are evolutionary conserved among mammals. (B) Sequence alignment of individual miRNAs:3′-UTR mRNA pairs. Watson-Crick (vertical lines) and wobble (G:U) base pairing are shown. The nucleotides changed in mutated reporter plasmids are highlighted in gray. (C) Luciferase reporter assays using the WT1, WT2, and WT3. (D) Luciferase reporter assays using specific mutants of WT1, WT2, and WT3 (see panel B).
Mentions: The DNA-binding transcription factors Pax6 and c-Maf are key regulators of lens-specific GRNs that control the crystallin gene machinery (Chauhan et al. 2004; Yang et al. 2004, 2006; Yang and Cvekl 2005; Xie and Cvekl 2011). Expression of c-Maf, but not of Pax6, is up-regulated in the postmitotic cells of the lens vesicle that give rise to the primary lens fiber cells, raising the possibility that c-Maf is regulated at the level of transcription through FGFs originating from the prospective neuroretina (Lovicu and McAvoy 2005). Subsequent genetic studies supported this model (Zhao et al. 2008; Garcia et al. 2011; Qu et al. 2011). The current data suggest that multiple miRNAs, including miR-9, miR-137, miR-155, miR-301a, miR455, and miR-543 (Figure 7A and Figure 8A), regulate c-Maf expression through its 3′-UTR. In addition, c-Maf 3′-UTR contains a miR-143 target sequence (Figure 10, A and B). Herein, we conducted a series of luciferase reporter gene assays using three wild-type and eight mutated reporters using mouse lens epithelial cell line αTN4-1 (Yang and Cvekl 2005). The c-Maf 3′-UTR was divided into three shorter fragments, WT1 to WT3, as shown in Figure 10A. The predicted 3′-UTR:miRNA pairs for selected six binding sites are shown in Figure 10B. The results of luc reporter assays are summarized in Figure 10, C and D. The data showed that miR-143 and miR-155 down-regulated the WT2 c-Maf 3′-UTR, and miR-301a down-regulated the WT3 c-Maf 3′-UTR (Figure 10C, n = 6, P < 0.05). Site-directed mutagenesis of the predicted target sites yielded no statistically significant changes between the wild-type and mutated reporters (Figure 10D).

Bottom Line: Specific miRNA:mRNA interaction networks were predicted for c-Maf, N-Myc, and Nfib (DNA-binding transcription factors); Cnot6, Cpsf6, Dicer1, and Tnrc6b (RNA to miRNA processing); and Ash1l, Med1/PBP, and Kdm5b/Jarid1b/Plu1 (chromatin remodeling).Three miRNAs, including miR-143, miR-155, and miR-301a, down-regulated expression of c-Maf in the 3'-UTR luciferase reporter assays.These present studies demonstrate for the first time global impact of activated FGF signaling in lens cell culture system and predicted novel gene regulatory networks connected by multiple miRNAs that regulate lens differentiation.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Albert Einstein College of Medicine, Bronx, New York 10461.

ABSTRACT
MicroRNAs (miRNAs) and fibroblast growth factor (FGF) signaling regulate a wide range of cellular functions, including cell specification, proliferation, migration, differentiation, and survival. In lens, both these systems control lens fiber cell differentiation; however, a possible link between these processes remains to be examined. Herein, the functional requirement for miRNAs in differentiating lens fiber cells was demonstrated via conditional inactivation of Dicer1 in mouse (Mus musculus) lens. To dissect the miRNA-dependent pathways during lens differentiation, we used a rat (Rattus norvegicus) lens epithelial explant system, induced by FGF2 to differentiate, followed by mRNA and miRNA expression profiling. Transcriptome and miRNome analysis identified extensive FGF2-regulated cellular responses that were both independent and dependent on miRNAs. We identified 131 FGF2-regulated miRNAs. Seventy-six of these miRNAs had at least two in silico predicted and inversely regulated target mRNAs. Genes modulated by the greatest number of FGF-regulated miRNAs include DNA-binding transcription factors Nfib, Nfat5/OREBP, c-Maf, Ets1, and N-Myc. Activated FGF signaling influenced bone morphogenetic factor/transforming growth factor-β, Notch, and Wnt signaling cascades implicated earlier in lens differentiation. Specific miRNA:mRNA interaction networks were predicted for c-Maf, N-Myc, and Nfib (DNA-binding transcription factors); Cnot6, Cpsf6, Dicer1, and Tnrc6b (RNA to miRNA processing); and Ash1l, Med1/PBP, and Kdm5b/Jarid1b/Plu1 (chromatin remodeling). Three miRNAs, including miR-143, miR-155, and miR-301a, down-regulated expression of c-Maf in the 3'-UTR luciferase reporter assays. These present studies demonstrate for the first time global impact of activated FGF signaling in lens cell culture system and predicted novel gene regulatory networks connected by multiple miRNAs that regulate lens differentiation.

Show MeSH
Related in: MedlinePlus