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Nmnat1-Rbp7 Is a Conserved Fusion-Protein That Combines NAD+ Catalysis of Nmnat1 with Subcellular Localization of Rbp7.

Chen H, Babino D, Schoenbichler SA, Arkhipova V, Töchterle S, Martin F, Huck CW, von Lintig J, Meyer D - PLoS ONE (2015)

Bottom Line: We find that early embryonic rbp7a expression is negatively regulated by the Nodal/FoxH1-signaling pathway and we show that Nodal/FoxH1 activity has the opposite effect on aldh1a2, which encodes the major enzyme for early embryonic retinoic acid production.Injection experiments in zebrafish further revealed that Nmnat1-Rbp7a and Nmnat1 have similar NAD+ catalyzing activities but a different subcellular localization.HPLC measurements and protein localization analysis highlight Nmnat1-Rbp7a as the only known cytoplasmic and presumably endoplasmic reticulum (ER) specific NAD+ catalyzing enzyme.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Biology/CMBI, University of Innsbruck, Technikerstrasse 25, 6020, Innsbruck, Austria.

ABSTRACT
Retinol binding proteins (Rbps) are known as carriers for transport and targeting of retinoids to their metabolizing enzymes. Rbps are also reported to function in regulating the homeostatic balance of retinoid metabolism, as their level of retinoid occupancy impacts the activities of retinoid metabolizing enzymes. Here we used zebrafish as a model to study rbp7a function and regulation. We find that early embryonic rbp7a expression is negatively regulated by the Nodal/FoxH1-signaling pathway and we show that Nodal/FoxH1 activity has the opposite effect on aldh1a2, which encodes the major enzyme for early embryonic retinoic acid production. The data are consistent with a Nodal-dependent coordination of the allocation of retinoid precursors to processing enzymes with the catalysis of retinoic acid formation. Further, we describe a novel nmnat1-rbp7 transcript encoding a fusion of Rbp7 and the NAD+ (Nicotinamide adenine dinucleotide) synthesizing enzyme Nmnat1. We show that nmnat1-rbp7 is conserved in fish, mouse and chicken, and that in zebrafish regulation of nmnat1-rbp7a is distinct from that of rbp7a and nmnat1. Injection experiments in zebrafish further revealed that Nmnat1-Rbp7a and Nmnat1 have similar NAD+ catalyzing activities but a different subcellular localization. HPLC measurements and protein localization analysis highlight Nmnat1-Rbp7a as the only known cytoplasmic and presumably endoplasmic reticulum (ER) specific NAD+ catalyzing enzyme. These studies, taken together with previously documented NAD+ dependent interaction of RBPs with ER-associated enzymes of retinal catalysis, implicate functions of this newly described NMNAT1-Rbp7 fusion protein in retinol oxidation.

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rbp7a forms a conserved fusion gene with nmnat1.[A] Schematic representation of the exon usage in nmnat1, rbp7a and nmnat1-rbp7a transcripts. Size of exons (boxes) and distances corresponds to the position in the genome. [B] Semi-quantitative RT-PCR analyses of rbp7a, nmnat1, and fusion isoform; mRNA generated from indicated embryonic stages and adult tissues. β-actin was used as a control for cDNA input levels. [C-D] Competitive PCRs reveal the ratios of different isoforms expression level during developmental stages. [C] Schematic indicating primers used for competitive amplification of rbp7a (Pr1+Pr3) versus nmnat1-rbp7a (Pn2+Pr3) in the upper panel and nmnat1 (Pn2+Pn5) versus nmnat1-rbp7a in the lower panel. The different isoform exons are colored respectively; purple: nmnat1, green: rbp7a; the unique exon which belongs to nmnat1 or rbp7a is shown in red and brown. [D] Competition RT-PCR in different embryonic stages, note the temporal changes in ratios of long (nmnat1-rbp7a) versus short (rbp7a and nmnat1) PCR products, which were indicated the different dominated expression time windows for isoforms. [E, F] Schematic representation of exon usage of Rbp7, Nmnat1, and the Nmnat1-rbp7 transcriptions in mouse [E] and chicken [F]. [G] RT-PCR analyses of embryonic mRNA form mouse (E14) and chicken (d9) confirming expression of Nmnat1, Rbp7 and Nmnat1-rbp7 transcripts.
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pone.0143825.g003: rbp7a forms a conserved fusion gene with nmnat1.[A] Schematic representation of the exon usage in nmnat1, rbp7a and nmnat1-rbp7a transcripts. Size of exons (boxes) and distances corresponds to the position in the genome. [B] Semi-quantitative RT-PCR analyses of rbp7a, nmnat1, and fusion isoform; mRNA generated from indicated embryonic stages and adult tissues. β-actin was used as a control for cDNA input levels. [C-D] Competitive PCRs reveal the ratios of different isoforms expression level during developmental stages. [C] Schematic indicating primers used for competitive amplification of rbp7a (Pr1+Pr3) versus nmnat1-rbp7a (Pn2+Pr3) in the upper panel and nmnat1 (Pn2+Pn5) versus nmnat1-rbp7a in the lower panel. The different isoform exons are colored respectively; purple: nmnat1, green: rbp7a; the unique exon which belongs to nmnat1 or rbp7a is shown in red and brown. [D] Competition RT-PCR in different embryonic stages, note the temporal changes in ratios of long (nmnat1-rbp7a) versus short (rbp7a and nmnat1) PCR products, which were indicated the different dominated expression time windows for isoforms. [E, F] Schematic representation of exon usage of Rbp7, Nmnat1, and the Nmnat1-rbp7 transcriptions in mouse [E] and chicken [F]. [G] RT-PCR analyses of embryonic mRNA form mouse (E14) and chicken (d9) confirming expression of Nmnat1, Rbp7 and Nmnat1-rbp7 transcripts.

Mentions: Upon analyzing the rbp7 EST sequences, we found a unique long rbp7a EST entry, which is lacking the first rbp7a exon and instead contains 4 exons from the 5’ neighboring gene nmnat1 (Fig 3A). To exclude that this EST represents a splice artifact, we performed semi-quantitative RT-PCR analyses with mRNA preparations isolated from different embryonic stages and adult tissues. Using a pair of primers flanking the fusion site, we detected nmnat1-rbp7a fusion specific PCR-products in one-cell stage embryos, segmentation stage embryos (tailbud to 24hpf), and in adult liver and brain (Fig 3B). Further analyses of the same cDNA samples also revealed that the expression of the nmnat1-rbp7a fusion isoform was different from that of nmnat1 and rbp7a (Fig 3B). Consistent with the whole mount expression analyses, rbp7a RT-PCR-products were mainly detected during gastrula stages (30% epiboly to tailbud), while nmnat1 signals were found in all stages analyzed, with the strongest signals peaking during maternally regulated stages (1 cell and 1k-stage). To confirm the different developmental expression profiles and to define the relative expression levels of nmnat1, rbp7a and nmnat1-rbp7a, we performed competitive PCRs for these isoforms with cDNA from different stage embryos. In these studies, the primer pairs flanking the nmnat1-rbp7a fusion site (Pn2 + Pr3) were combined with a third primer that localized either to the last exon of nmnat1 (Pn5) or to the first exon of rbp7a (Pr1) (Fig 3C and 3D). The data revealed nmnat1-rbp7a fusion transcripts as the prevailing rbp7a isoform in zygotes and between 18-somite stage and 24hpf, and they suggested that expression levels of nmnat1-rbp7a at late somite stages were slightly below those of nmnat1.


Nmnat1-Rbp7 Is a Conserved Fusion-Protein That Combines NAD+ Catalysis of Nmnat1 with Subcellular Localization of Rbp7.

Chen H, Babino D, Schoenbichler SA, Arkhipova V, Töchterle S, Martin F, Huck CW, von Lintig J, Meyer D - PLoS ONE (2015)

rbp7a forms a conserved fusion gene with nmnat1.[A] Schematic representation of the exon usage in nmnat1, rbp7a and nmnat1-rbp7a transcripts. Size of exons (boxes) and distances corresponds to the position in the genome. [B] Semi-quantitative RT-PCR analyses of rbp7a, nmnat1, and fusion isoform; mRNA generated from indicated embryonic stages and adult tissues. β-actin was used as a control for cDNA input levels. [C-D] Competitive PCRs reveal the ratios of different isoforms expression level during developmental stages. [C] Schematic indicating primers used for competitive amplification of rbp7a (Pr1+Pr3) versus nmnat1-rbp7a (Pn2+Pr3) in the upper panel and nmnat1 (Pn2+Pn5) versus nmnat1-rbp7a in the lower panel. The different isoform exons are colored respectively; purple: nmnat1, green: rbp7a; the unique exon which belongs to nmnat1 or rbp7a is shown in red and brown. [D] Competition RT-PCR in different embryonic stages, note the temporal changes in ratios of long (nmnat1-rbp7a) versus short (rbp7a and nmnat1) PCR products, which were indicated the different dominated expression time windows for isoforms. [E, F] Schematic representation of exon usage of Rbp7, Nmnat1, and the Nmnat1-rbp7 transcriptions in mouse [E] and chicken [F]. [G] RT-PCR analyses of embryonic mRNA form mouse (E14) and chicken (d9) confirming expression of Nmnat1, Rbp7 and Nmnat1-rbp7 transcripts.
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pone.0143825.g003: rbp7a forms a conserved fusion gene with nmnat1.[A] Schematic representation of the exon usage in nmnat1, rbp7a and nmnat1-rbp7a transcripts. Size of exons (boxes) and distances corresponds to the position in the genome. [B] Semi-quantitative RT-PCR analyses of rbp7a, nmnat1, and fusion isoform; mRNA generated from indicated embryonic stages and adult tissues. β-actin was used as a control for cDNA input levels. [C-D] Competitive PCRs reveal the ratios of different isoforms expression level during developmental stages. [C] Schematic indicating primers used for competitive amplification of rbp7a (Pr1+Pr3) versus nmnat1-rbp7a (Pn2+Pr3) in the upper panel and nmnat1 (Pn2+Pn5) versus nmnat1-rbp7a in the lower panel. The different isoform exons are colored respectively; purple: nmnat1, green: rbp7a; the unique exon which belongs to nmnat1 or rbp7a is shown in red and brown. [D] Competition RT-PCR in different embryonic stages, note the temporal changes in ratios of long (nmnat1-rbp7a) versus short (rbp7a and nmnat1) PCR products, which were indicated the different dominated expression time windows for isoforms. [E, F] Schematic representation of exon usage of Rbp7, Nmnat1, and the Nmnat1-rbp7 transcriptions in mouse [E] and chicken [F]. [G] RT-PCR analyses of embryonic mRNA form mouse (E14) and chicken (d9) confirming expression of Nmnat1, Rbp7 and Nmnat1-rbp7 transcripts.
Mentions: Upon analyzing the rbp7 EST sequences, we found a unique long rbp7a EST entry, which is lacking the first rbp7a exon and instead contains 4 exons from the 5’ neighboring gene nmnat1 (Fig 3A). To exclude that this EST represents a splice artifact, we performed semi-quantitative RT-PCR analyses with mRNA preparations isolated from different embryonic stages and adult tissues. Using a pair of primers flanking the fusion site, we detected nmnat1-rbp7a fusion specific PCR-products in one-cell stage embryos, segmentation stage embryos (tailbud to 24hpf), and in adult liver and brain (Fig 3B). Further analyses of the same cDNA samples also revealed that the expression of the nmnat1-rbp7a fusion isoform was different from that of nmnat1 and rbp7a (Fig 3B). Consistent with the whole mount expression analyses, rbp7a RT-PCR-products were mainly detected during gastrula stages (30% epiboly to tailbud), while nmnat1 signals were found in all stages analyzed, with the strongest signals peaking during maternally regulated stages (1 cell and 1k-stage). To confirm the different developmental expression profiles and to define the relative expression levels of nmnat1, rbp7a and nmnat1-rbp7a, we performed competitive PCRs for these isoforms with cDNA from different stage embryos. In these studies, the primer pairs flanking the nmnat1-rbp7a fusion site (Pn2 + Pr3) were combined with a third primer that localized either to the last exon of nmnat1 (Pn5) or to the first exon of rbp7a (Pr1) (Fig 3C and 3D). The data revealed nmnat1-rbp7a fusion transcripts as the prevailing rbp7a isoform in zygotes and between 18-somite stage and 24hpf, and they suggested that expression levels of nmnat1-rbp7a at late somite stages were slightly below those of nmnat1.

Bottom Line: We find that early embryonic rbp7a expression is negatively regulated by the Nodal/FoxH1-signaling pathway and we show that Nodal/FoxH1 activity has the opposite effect on aldh1a2, which encodes the major enzyme for early embryonic retinoic acid production.Injection experiments in zebrafish further revealed that Nmnat1-Rbp7a and Nmnat1 have similar NAD+ catalyzing activities but a different subcellular localization.HPLC measurements and protein localization analysis highlight Nmnat1-Rbp7a as the only known cytoplasmic and presumably endoplasmic reticulum (ER) specific NAD+ catalyzing enzyme.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Biology/CMBI, University of Innsbruck, Technikerstrasse 25, 6020, Innsbruck, Austria.

ABSTRACT
Retinol binding proteins (Rbps) are known as carriers for transport and targeting of retinoids to their metabolizing enzymes. Rbps are also reported to function in regulating the homeostatic balance of retinoid metabolism, as their level of retinoid occupancy impacts the activities of retinoid metabolizing enzymes. Here we used zebrafish as a model to study rbp7a function and regulation. We find that early embryonic rbp7a expression is negatively regulated by the Nodal/FoxH1-signaling pathway and we show that Nodal/FoxH1 activity has the opposite effect on aldh1a2, which encodes the major enzyme for early embryonic retinoic acid production. The data are consistent with a Nodal-dependent coordination of the allocation of retinoid precursors to processing enzymes with the catalysis of retinoic acid formation. Further, we describe a novel nmnat1-rbp7 transcript encoding a fusion of Rbp7 and the NAD+ (Nicotinamide adenine dinucleotide) synthesizing enzyme Nmnat1. We show that nmnat1-rbp7 is conserved in fish, mouse and chicken, and that in zebrafish regulation of nmnat1-rbp7a is distinct from that of rbp7a and nmnat1. Injection experiments in zebrafish further revealed that Nmnat1-Rbp7a and Nmnat1 have similar NAD+ catalyzing activities but a different subcellular localization. HPLC measurements and protein localization analysis highlight Nmnat1-Rbp7a as the only known cytoplasmic and presumably endoplasmic reticulum (ER) specific NAD+ catalyzing enzyme. These studies, taken together with previously documented NAD+ dependent interaction of RBPs with ER-associated enzymes of retinal catalysis, implicate functions of this newly described NMNAT1-Rbp7 fusion protein in retinol oxidation.

Show MeSH