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Regulation of zinc-responsive Slc39a5 (Zip5) translation is mediated by conserved elements in the 3'-untranslated region.

Weaver BP, Andrews GK - Biometals (2011)

Bottom Line: Herein, we examined the mechanisms regulating translation of Zip5.The 3'-untranslated region (UTR) of Zip5 mRNA is well conserved among mammals and is predicted by mFOLD to form a very stable stem-loop structure.Three algorithms predict this structure to be flanked by repeated seed sites for miR-328 and miR-193a.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, KS 66160-7421, USA. Benjamin.Weaver@Colorado.edu

ABSTRACT
Translation of the basolateral zinc transporter ZIP5 is repressed during zinc deficiency but Zip5 mRNA remains associated with polysomes and can be rapidly translated when zinc is repleted. Herein, we examined the mechanisms regulating translation of Zip5. The 3'-untranslated region (UTR) of Zip5 mRNA is well conserved among mammals and is predicted by mFOLD to form a very stable stem-loop structure. Three algorithms predict this structure to be flanked by repeated seed sites for miR-328 and miR-193a. RNAse footprinting supports the notion that a stable stem-loop structure exists in this 3'-UTR and electrophoretic mobility shift assays detect polysomal protein(s) binding specifically to the stem-loop structure in the Zip5 3'-UTR. miR-328 and miR-193a are expressed in tissues known to regulate Zip5 mRNA translation in response to zinc availability and both are polysome-associated consistent with Zip5 mRNA localization. Transient transfection assays using native and mutant Zip5 3'-UTRs cloned 3' to luciferase cDNA revealed that the miRNA seed sites and the stem-loop function together to augment translation of Zip5 mRNA when zinc is replete.

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Functional analyses of the Zip5 3′-UTR in transfected XEN cells. XEN cells were cultured in ZnA or ZnD media for 30–48 h and then transfected with the luciferase Zip5 3′-UTR reporter constructs. The UTR structure is diagramed above each panel (see Fig. 6 for sequence details). Some ZnD transfected cells had 40 μM zinc added during the last 6–8 h of culture (ZnR). All firefly luciferase values were normalized to co-transfected renilla luciferase values. Each bar represents the mean of 3–4 replicas ± SD. An asterisk indicates a P value <0.05. All sample means were compared to the respective wild-type ZnA mean for the given experiment. a The wild-type (WT) and seed site (SS) mutant UTR vectors were also co-transfected with either an antagomiR vector expressing GFP and siRNA targeting LacZ (αLacZ) or antagomiR vectors expressing GFP and siRNAs targeting miR-193a and miR-328 (α193/328), as described in detail in the “Materials and methods” section. b Concatamerized wild-type Zip5 3′-UTR (WT 3×) and concatamerized Zip5 3′-UTR with scrambled seed sites (SS 3×) were transfected with a GFP expression vector as carrier. c The full length wild-type Zip5 3′-UTR (WT) or the Zip5 3′-UTR with deletion of the stem-loop (SL) were transfected with a GFP expression vector as carrier. Two independent experiments are shown. d The full length wild-type Zip5 3′-UTR (WT) or the Zip5 3′-UTR with both scrambled seed sites and deletion of the putative stem-loop (DM) were transfected with a GFP expression vector as carrier
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Fig7: Functional analyses of the Zip5 3′-UTR in transfected XEN cells. XEN cells were cultured in ZnA or ZnD media for 30–48 h and then transfected with the luciferase Zip5 3′-UTR reporter constructs. The UTR structure is diagramed above each panel (see Fig. 6 for sequence details). Some ZnD transfected cells had 40 μM zinc added during the last 6–8 h of culture (ZnR). All firefly luciferase values were normalized to co-transfected renilla luciferase values. Each bar represents the mean of 3–4 replicas ± SD. An asterisk indicates a P value <0.05. All sample means were compared to the respective wild-type ZnA mean for the given experiment. a The wild-type (WT) and seed site (SS) mutant UTR vectors were also co-transfected with either an antagomiR vector expressing GFP and siRNA targeting LacZ (αLacZ) or antagomiR vectors expressing GFP and siRNAs targeting miR-193a and miR-328 (α193/328), as described in detail in the “Materials and methods” section. b Concatamerized wild-type Zip5 3′-UTR (WT 3×) and concatamerized Zip5 3′-UTR with scrambled seed sites (SS 3×) were transfected with a GFP expression vector as carrier. c The full length wild-type Zip5 3′-UTR (WT) or the Zip5 3′-UTR with deletion of the stem-loop (SL) were transfected with a GFP expression vector as carrier. Two independent experiments are shown. d The full length wild-type Zip5 3′-UTR (WT) or the Zip5 3′-UTR with both scrambled seed sites and deletion of the putative stem-loop (DM) were transfected with a GFP expression vector as carrier

Mentions: To study the functional significance of the Zip5 3′-UTR stem-loop and miRNA seeds-sites, we employed a rat extraembryonic endoderm XEN cell line previously shown to express extraembryonic endoderm markers (Soares et al. 1987). Extraembryonic endoderm is known to express and regulate ZIP5 in response to zinc (Dufner-Beattie et al. 2004; Weaver et al. 2007), but the rat XEN cells did not contain significant levels of Zip5 mRNA (data not shown). However, the XEN cells do express miR-328, miR-193a and miR-193b (Fig. 4) and do exhibit specific Zip5 3′-UTR-protein interactions indistinguishable from VYS (Fig. 3). To determine the structure–function relationships in the Zip5 3′-UTR, we engineered expression vectors in which the firefly luciferase coding sequence has the full-length mouse Zip5 3′-UTR cloned downstream of its stop codon (Figs. 6a, 7). In addition to the full-length wild-type mouse Zip5 3′-UTR, this UTR with the miRNA seed-sites scrambled and/or the stem-loop deleted were examined. The nucleotide sequences for the various UTR structures are shown with detail in Fig. 6b and diagrammatically in Fig. 7 for each given experiment. The firefly luciferase-UTR vectors were transfected into XEN cells along with a renilla luciferase expression vector internal control and the effects of zinc were assessed (Fig. 7). Raw renilla luciferase values are shown since zinc deficiency impacts global translation and firefly luciferase values are normalized to renilla luciferase in these experiments.Fig. 6


Regulation of zinc-responsive Slc39a5 (Zip5) translation is mediated by conserved elements in the 3'-untranslated region.

Weaver BP, Andrews GK - Biometals (2011)

Functional analyses of the Zip5 3′-UTR in transfected XEN cells. XEN cells were cultured in ZnA or ZnD media for 30–48 h and then transfected with the luciferase Zip5 3′-UTR reporter constructs. The UTR structure is diagramed above each panel (see Fig. 6 for sequence details). Some ZnD transfected cells had 40 μM zinc added during the last 6–8 h of culture (ZnR). All firefly luciferase values were normalized to co-transfected renilla luciferase values. Each bar represents the mean of 3–4 replicas ± SD. An asterisk indicates a P value <0.05. All sample means were compared to the respective wild-type ZnA mean for the given experiment. a The wild-type (WT) and seed site (SS) mutant UTR vectors were also co-transfected with either an antagomiR vector expressing GFP and siRNA targeting LacZ (αLacZ) or antagomiR vectors expressing GFP and siRNAs targeting miR-193a and miR-328 (α193/328), as described in detail in the “Materials and methods” section. b Concatamerized wild-type Zip5 3′-UTR (WT 3×) and concatamerized Zip5 3′-UTR with scrambled seed sites (SS 3×) were transfected with a GFP expression vector as carrier. c The full length wild-type Zip5 3′-UTR (WT) or the Zip5 3′-UTR with deletion of the stem-loop (SL) were transfected with a GFP expression vector as carrier. Two independent experiments are shown. d The full length wild-type Zip5 3′-UTR (WT) or the Zip5 3′-UTR with both scrambled seed sites and deletion of the putative stem-loop (DM) were transfected with a GFP expression vector as carrier
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Fig7: Functional analyses of the Zip5 3′-UTR in transfected XEN cells. XEN cells were cultured in ZnA or ZnD media for 30–48 h and then transfected with the luciferase Zip5 3′-UTR reporter constructs. The UTR structure is diagramed above each panel (see Fig. 6 for sequence details). Some ZnD transfected cells had 40 μM zinc added during the last 6–8 h of culture (ZnR). All firefly luciferase values were normalized to co-transfected renilla luciferase values. Each bar represents the mean of 3–4 replicas ± SD. An asterisk indicates a P value <0.05. All sample means were compared to the respective wild-type ZnA mean for the given experiment. a The wild-type (WT) and seed site (SS) mutant UTR vectors were also co-transfected with either an antagomiR vector expressing GFP and siRNA targeting LacZ (αLacZ) or antagomiR vectors expressing GFP and siRNAs targeting miR-193a and miR-328 (α193/328), as described in detail in the “Materials and methods” section. b Concatamerized wild-type Zip5 3′-UTR (WT 3×) and concatamerized Zip5 3′-UTR with scrambled seed sites (SS 3×) were transfected with a GFP expression vector as carrier. c The full length wild-type Zip5 3′-UTR (WT) or the Zip5 3′-UTR with deletion of the stem-loop (SL) were transfected with a GFP expression vector as carrier. Two independent experiments are shown. d The full length wild-type Zip5 3′-UTR (WT) or the Zip5 3′-UTR with both scrambled seed sites and deletion of the putative stem-loop (DM) were transfected with a GFP expression vector as carrier
Mentions: To study the functional significance of the Zip5 3′-UTR stem-loop and miRNA seeds-sites, we employed a rat extraembryonic endoderm XEN cell line previously shown to express extraembryonic endoderm markers (Soares et al. 1987). Extraembryonic endoderm is known to express and regulate ZIP5 in response to zinc (Dufner-Beattie et al. 2004; Weaver et al. 2007), but the rat XEN cells did not contain significant levels of Zip5 mRNA (data not shown). However, the XEN cells do express miR-328, miR-193a and miR-193b (Fig. 4) and do exhibit specific Zip5 3′-UTR-protein interactions indistinguishable from VYS (Fig. 3). To determine the structure–function relationships in the Zip5 3′-UTR, we engineered expression vectors in which the firefly luciferase coding sequence has the full-length mouse Zip5 3′-UTR cloned downstream of its stop codon (Figs. 6a, 7). In addition to the full-length wild-type mouse Zip5 3′-UTR, this UTR with the miRNA seed-sites scrambled and/or the stem-loop deleted were examined. The nucleotide sequences for the various UTR structures are shown with detail in Fig. 6b and diagrammatically in Fig. 7 for each given experiment. The firefly luciferase-UTR vectors were transfected into XEN cells along with a renilla luciferase expression vector internal control and the effects of zinc were assessed (Fig. 7). Raw renilla luciferase values are shown since zinc deficiency impacts global translation and firefly luciferase values are normalized to renilla luciferase in these experiments.Fig. 6

Bottom Line: Herein, we examined the mechanisms regulating translation of Zip5.The 3'-untranslated region (UTR) of Zip5 mRNA is well conserved among mammals and is predicted by mFOLD to form a very stable stem-loop structure.Three algorithms predict this structure to be flanked by repeated seed sites for miR-328 and miR-193a.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, KS 66160-7421, USA. Benjamin.Weaver@Colorado.edu

ABSTRACT
Translation of the basolateral zinc transporter ZIP5 is repressed during zinc deficiency but Zip5 mRNA remains associated with polysomes and can be rapidly translated when zinc is repleted. Herein, we examined the mechanisms regulating translation of Zip5. The 3'-untranslated region (UTR) of Zip5 mRNA is well conserved among mammals and is predicted by mFOLD to form a very stable stem-loop structure. Three algorithms predict this structure to be flanked by repeated seed sites for miR-328 and miR-193a. RNAse footprinting supports the notion that a stable stem-loop structure exists in this 3'-UTR and electrophoretic mobility shift assays detect polysomal protein(s) binding specifically to the stem-loop structure in the Zip5 3'-UTR. miR-328 and miR-193a are expressed in tissues known to regulate Zip5 mRNA translation in response to zinc availability and both are polysome-associated consistent with Zip5 mRNA localization. Transient transfection assays using native and mutant Zip5 3'-UTRs cloned 3' to luciferase cDNA revealed that the miRNA seed sites and the stem-loop function together to augment translation of Zip5 mRNA when zinc is replete.

Show MeSH
Related in: MedlinePlus