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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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Examination of steady state levels for firefly and renilla encoding mRNAs in transiently transfected XEN cells. XEN cells were cultured and transfected as described in the legend to Fig. 7. The following constructs were examined: The wild-type Zip5 3′-UTR (WT); this UTR with scrambled seed sites (SS); this UTR bearing a deletion of the stem-loop (SL); a concatamer of wild-type Zip5 3′-UTR (WT 3×) and a concatamer of this UTR with scrambled seed sites (SS 3×) (see Fig. 6 for details). Total RNA from ZnA (dark gray bars) or ZnD (light gray bars) transfected XEN cells was reverse transcribed and amplified via qPCR to measure relative abundance of firefly and renilla luciferase mRNAs. The ΔΔCt method was used to interpret qPCR results. All firefly 2−ΔΔCt values were first normalized to renilla 2−ΔΔCt values. The WT ZnA normalized value was set to 1 and the 2−ΔΔCt values for all other samples were reported as relative fold changes to the WT 1 × ZnA value. Each bar represents the mean of three independent qPCR runs (where each run was done in triplicate) ± SD. Student’s t test was used to determine significance
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Fig8: Examination of steady state levels for firefly and renilla encoding mRNAs in transiently transfected XEN cells. XEN cells were cultured and transfected as described in the legend to Fig. 7. The following constructs were examined: The wild-type Zip5 3′-UTR (WT); this UTR with scrambled seed sites (SS); this UTR bearing a deletion of the stem-loop (SL); a concatamer of wild-type Zip5 3′-UTR (WT 3×) and a concatamer of this UTR with scrambled seed sites (SS 3×) (see Fig. 6 for details). Total RNA from ZnA (dark gray bars) or ZnD (light gray bars) transfected XEN cells was reverse transcribed and amplified via qPCR to measure relative abundance of firefly and renilla luciferase mRNAs. The ΔΔCt method was used to interpret qPCR results. All firefly 2−ΔΔCt values were first normalized to renilla 2−ΔΔCt values. The WT ZnA normalized value was set to 1 and the 2−ΔΔCt values for all other samples were reported as relative fold changes to the WT 1 × ZnA value. Each bar represents the mean of three independent qPCR runs (where each run was done in triplicate) ± SD. Student’s t test was used to determine significance

Mentions: The full-length wild-type Zip5 3′-UTR (Fig. 6b; WT) and a mutant with scrambled miRNA seed sites (Fig. 6b; SS) were examined (Fig. 7a). Disruption of the miRNA seed sites alone significantly reduced expression of firefly luciferase vector when zinc was adequate in the culture medium. The level of expression of the mutant with scrambled miRNA seed sites in ZnA medium was indistinguishable from that of the wild-type 3′-UTR reporter in ZnD medium. Zinc deficiency caused about a 40% reduction in the activity of the wild-type UTR expression vector, but did not significantly further reduce the level of expression of the mutant with scrambled seed sites (Fig. 7a). Restoring zinc in the culture medium restored expression of the vector with the wild-type Zip5 3′-UTR but had little effect on the expression of the vector with scrambled miRNA seed sites. Quantitative real time PCR analysis demonstrated that changes in firefly and renilla luciferase activity in the transfected cells did not reflect changes in the abundance of these mRNAs (Fig. 8) but rather reflected changes in the accumulation of these proteins, suggestive of translation regulation.Fig. 8


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

Weaver BP, Andrews GK - Biometals (2011)

Examination of steady state levels for firefly and renilla encoding mRNAs in transiently transfected XEN cells. XEN cells were cultured and transfected as described in the legend to Fig. 7. The following constructs were examined: The wild-type Zip5 3′-UTR (WT); this UTR with scrambled seed sites (SS); this UTR bearing a deletion of the stem-loop (SL); a concatamer of wild-type Zip5 3′-UTR (WT 3×) and a concatamer of this UTR with scrambled seed sites (SS 3×) (see Fig. 6 for details). Total RNA from ZnA (dark gray bars) or ZnD (light gray bars) transfected XEN cells was reverse transcribed and amplified via qPCR to measure relative abundance of firefly and renilla luciferase mRNAs. The ΔΔCt method was used to interpret qPCR results. All firefly 2−ΔΔCt values were first normalized to renilla 2−ΔΔCt values. The WT ZnA normalized value was set to 1 and the 2−ΔΔCt values for all other samples were reported as relative fold changes to the WT 1 × ZnA value. Each bar represents the mean of three independent qPCR runs (where each run was done in triplicate) ± SD. Student’s t test was used to determine significance
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3299966&req=5

Fig8: Examination of steady state levels for firefly and renilla encoding mRNAs in transiently transfected XEN cells. XEN cells were cultured and transfected as described in the legend to Fig. 7. The following constructs were examined: The wild-type Zip5 3′-UTR (WT); this UTR with scrambled seed sites (SS); this UTR bearing a deletion of the stem-loop (SL); a concatamer of wild-type Zip5 3′-UTR (WT 3×) and a concatamer of this UTR with scrambled seed sites (SS 3×) (see Fig. 6 for details). Total RNA from ZnA (dark gray bars) or ZnD (light gray bars) transfected XEN cells was reverse transcribed and amplified via qPCR to measure relative abundance of firefly and renilla luciferase mRNAs. The ΔΔCt method was used to interpret qPCR results. All firefly 2−ΔΔCt values were first normalized to renilla 2−ΔΔCt values. The WT ZnA normalized value was set to 1 and the 2−ΔΔCt values for all other samples were reported as relative fold changes to the WT 1 × ZnA value. Each bar represents the mean of three independent qPCR runs (where each run was done in triplicate) ± SD. Student’s t test was used to determine significance
Mentions: The full-length wild-type Zip5 3′-UTR (Fig. 6b; WT) and a mutant with scrambled miRNA seed sites (Fig. 6b; SS) were examined (Fig. 7a). Disruption of the miRNA seed sites alone significantly reduced expression of firefly luciferase vector when zinc was adequate in the culture medium. The level of expression of the mutant with scrambled miRNA seed sites in ZnA medium was indistinguishable from that of the wild-type 3′-UTR reporter in ZnD medium. Zinc deficiency caused about a 40% reduction in the activity of the wild-type UTR expression vector, but did not significantly further reduce the level of expression of the mutant with scrambled seed sites (Fig. 7a). Restoring zinc in the culture medium restored expression of the vector with the wild-type Zip5 3′-UTR but had little effect on the expression of the vector with scrambled miRNA seed sites. Quantitative real time PCR analysis demonstrated that changes in firefly and renilla luciferase activity in the transfected cells did not reflect changes in the abundance of these mRNAs (Fig. 8) but rather reflected changes in the accumulation of these proteins, suggestive of translation regulation.Fig. 8

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