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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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Quantification of miR-328, miR-193a, miR-193b, Zip4 and Zip5 mRNA abundance in total RNAs from ZnA and ZnD visceral yolk sac, intestine, pancreas and XEN cells. Embryonic visceral yolk sacs (VYS), intestine and pancreas were harvested on day 14 from pregnant dams fed a ZnA or ZnD diet beginning on day 8 of pregnancy. XEN cells were cultured in ZnA or ZnD medium for 48 h. Total RNA was reverse transcribed and probed via quantitative PCR for miR-328, miR-193a, miR-193b, let-7b miRNA abundance. VYS was also probed for Zip4 and Zip5 mRNA abundance. a miRNA abundance was measured in ZnA (black bars) and ZnD (gray bars) VYS, intestine, pancreas and XEN cells. Raw Ct values are shown. Let-7b (not predicted to target Zip5) is shown as a miRNA known to be associated with polysomes (Maroney et al. 2006). 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. bZip4 and Zip5 mRNA abundance in VYS. The ΔΔCt method was used to quantify qPCR results. Zip5 and Zip4 mRNA abundance was normalized to beta-actin mRNA and are presented as fold change relative to their respective ZnA values. Each bar represents the mean of three independent qPCR runs (where each run was done in triplicate) ± SD. An asterisk indicates a P value <0.05 as determined by Student’s t test
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Fig4: Quantification of miR-328, miR-193a, miR-193b, Zip4 and Zip5 mRNA abundance in total RNAs from ZnA and ZnD visceral yolk sac, intestine, pancreas and XEN cells. Embryonic visceral yolk sacs (VYS), intestine and pancreas were harvested on day 14 from pregnant dams fed a ZnA or ZnD diet beginning on day 8 of pregnancy. XEN cells were cultured in ZnA or ZnD medium for 48 h. Total RNA was reverse transcribed and probed via quantitative PCR for miR-328, miR-193a, miR-193b, let-7b miRNA abundance. VYS was also probed for Zip4 and Zip5 mRNA abundance. a miRNA abundance was measured in ZnA (black bars) and ZnD (gray bars) VYS, intestine, pancreas and XEN cells. Raw Ct values are shown. Let-7b (not predicted to target Zip5) is shown as a miRNA known to be associated with polysomes (Maroney et al. 2006). 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. bZip4 and Zip5 mRNA abundance in VYS. The ΔΔCt method was used to quantify qPCR results. Zip5 and Zip4 mRNA abundance was normalized to beta-actin mRNA and are presented as fold change relative to their respective ZnA values. Each bar represents the mean of three independent qPCR runs (where each run was done in triplicate) ± SD. An asterisk indicates a P value <0.05 as determined by Student’s t test

Mentions: To examine the expression levels of miR-328, miR-193a and miR-193b, we used a sensitive and specific real time quantitative PCR method able to distinguish mature miRNAs differing by a single nucleotide. First, we examined steady state levels of miR-328, miR-193a, miR-193b and let-7b (not predicted to target the Zip5 3′-UTR) in total RNAs from VYS, intestine and pancreas of mice fed a ZnA diet or a ZnD diet and from rat XEN cells cultured in ZnA or ZnD media. Compared to let-7b, a miRNA known to associate with polysomes (Maroney et al. 2006), the three miRNAs of interest had comparable expression levels in total cellular RNA (Fig. 4a). Raw Ct values are plotted for the part a results to show absolute abundance and not relative abundance. No zinc-dependent changes in abundance were observed (Fig. 4a). Because VYS was used for subsequent analyses, the relative abundance of Zip5 and Zip4 mRNAs was also quantified in the ZnA and ZnD VYS RNA samples (Fig. 4b) since the Zip4 mRNA is stabilized and accumulates during zinc deficiency in the mouse VYS while Zip5 mRNA abundance remains constant (Dufner-Beattie et al. 2003, 2004; Weaver et al. 2007). miR-328, miR-193a and miR-193b were each expressed with comparable abundance in the mouse VYS, intestine and pancreas, tissues known to endogenously express and regulate Zip5 which thereby satisfies the third criterion to validate miRNA targets.Fig. 4


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

Weaver BP, Andrews GK - Biometals (2011)

Quantification of miR-328, miR-193a, miR-193b, Zip4 and Zip5 mRNA abundance in total RNAs from ZnA and ZnD visceral yolk sac, intestine, pancreas and XEN cells. Embryonic visceral yolk sacs (VYS), intestine and pancreas were harvested on day 14 from pregnant dams fed a ZnA or ZnD diet beginning on day 8 of pregnancy. XEN cells were cultured in ZnA or ZnD medium for 48 h. Total RNA was reverse transcribed and probed via quantitative PCR for miR-328, miR-193a, miR-193b, let-7b miRNA abundance. VYS was also probed for Zip4 and Zip5 mRNA abundance. a miRNA abundance was measured in ZnA (black bars) and ZnD (gray bars) VYS, intestine, pancreas and XEN cells. Raw Ct values are shown. Let-7b (not predicted to target Zip5) is shown as a miRNA known to be associated with polysomes (Maroney et al. 2006). 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. bZip4 and Zip5 mRNA abundance in VYS. The ΔΔCt method was used to quantify qPCR results. Zip5 and Zip4 mRNA abundance was normalized to beta-actin mRNA and are presented as fold change relative to their respective ZnA values. Each bar represents the mean of three independent qPCR runs (where each run was done in triplicate) ± SD. An asterisk indicates a P value <0.05 as determined by Student’s t test
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Fig4: Quantification of miR-328, miR-193a, miR-193b, Zip4 and Zip5 mRNA abundance in total RNAs from ZnA and ZnD visceral yolk sac, intestine, pancreas and XEN cells. Embryonic visceral yolk sacs (VYS), intestine and pancreas were harvested on day 14 from pregnant dams fed a ZnA or ZnD diet beginning on day 8 of pregnancy. XEN cells were cultured in ZnA or ZnD medium for 48 h. Total RNA was reverse transcribed and probed via quantitative PCR for miR-328, miR-193a, miR-193b, let-7b miRNA abundance. VYS was also probed for Zip4 and Zip5 mRNA abundance. a miRNA abundance was measured in ZnA (black bars) and ZnD (gray bars) VYS, intestine, pancreas and XEN cells. Raw Ct values are shown. Let-7b (not predicted to target Zip5) is shown as a miRNA known to be associated with polysomes (Maroney et al. 2006). 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. bZip4 and Zip5 mRNA abundance in VYS. The ΔΔCt method was used to quantify qPCR results. Zip5 and Zip4 mRNA abundance was normalized to beta-actin mRNA and are presented as fold change relative to their respective ZnA values. Each bar represents the mean of three independent qPCR runs (where each run was done in triplicate) ± SD. An asterisk indicates a P value <0.05 as determined by Student’s t test
Mentions: To examine the expression levels of miR-328, miR-193a and miR-193b, we used a sensitive and specific real time quantitative PCR method able to distinguish mature miRNAs differing by a single nucleotide. First, we examined steady state levels of miR-328, miR-193a, miR-193b and let-7b (not predicted to target the Zip5 3′-UTR) in total RNAs from VYS, intestine and pancreas of mice fed a ZnA diet or a ZnD diet and from rat XEN cells cultured in ZnA or ZnD media. Compared to let-7b, a miRNA known to associate with polysomes (Maroney et al. 2006), the three miRNAs of interest had comparable expression levels in total cellular RNA (Fig. 4a). Raw Ct values are plotted for the part a results to show absolute abundance and not relative abundance. No zinc-dependent changes in abundance were observed (Fig. 4a). Because VYS was used for subsequent analyses, the relative abundance of Zip5 and Zip4 mRNAs was also quantified in the ZnA and ZnD VYS RNA samples (Fig. 4b) since the Zip4 mRNA is stabilized and accumulates during zinc deficiency in the mouse VYS while Zip5 mRNA abundance remains constant (Dufner-Beattie et al. 2003, 2004; Weaver et al. 2007). miR-328, miR-193a and miR-193b were each expressed with comparable abundance in the mouse VYS, intestine and pancreas, tissues known to endogenously express and regulate Zip5 which thereby satisfies the third criterion to validate miRNA targets.Fig. 4

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