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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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Alignment of Slc39a5 (Zip5) 3′-UTRs across five mammalian species showing an mFOLD (Zuker 2003) predicted stable stem-loop structure and Venn-diagram of candidate miRNAs predicted by three algorithms to target this UTR. The full-length sequences of Zip5 3′ UTRs from human, chimp, dog, mouse, and rat were aligned and annotated with empirical information from available Zip5 cDNAs (Dufner-Beattie et al. 2004). The polyadenylation signal (polyA signal) and the polyadenylation site (polyA site) are indicated. Yellow indicates nucleotides conserved in all species shown; green and cyan indicate nucleotides conserved in two or more species shown. The most stable stem-loop predicted by mFOLD (Zuker 2003) is shown. Stem-loop residue A17 is A44 in the full length UTR sequence. Two conserved single nucleotide bulges are indicated by heavy arrowheads. miR-328 and miR-193 seed sites are indicated with black bars. Venn-diagram: light gray shaded  region, miRNAs predicted by the miRBase algorithm (Sanger); dark gray shaded region, miRNAs predicted by TargetScan; black shaded region, miRNAs predicted by PicTar. Only miR-328, miR-193, and miR-137 are predicted by all three algorithms to target the 3′-UTR of Zip5 mRNA
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Fig1: Alignment of Slc39a5 (Zip5) 3′-UTRs across five mammalian species showing an mFOLD (Zuker 2003) predicted stable stem-loop structure and Venn-diagram of candidate miRNAs predicted by three algorithms to target this UTR. The full-length sequences of Zip5 3′ UTRs from human, chimp, dog, mouse, and rat were aligned and annotated with empirical information from available Zip5 cDNAs (Dufner-Beattie et al. 2004). The polyadenylation signal (polyA signal) and the polyadenylation site (polyA site) are indicated. Yellow indicates nucleotides conserved in all species shown; green and cyan indicate nucleotides conserved in two or more species shown. The most stable stem-loop predicted by mFOLD (Zuker 2003) is shown. Stem-loop residue A17 is A44 in the full length UTR sequence. Two conserved single nucleotide bulges are indicated by heavy arrowheads. miR-328 and miR-193 seed sites are indicated with black bars. Venn-diagram: light gray shaded region, miRNAs predicted by the miRBase algorithm (Sanger); dark gray shaded region, miRNAs predicted by TargetScan; black shaded region, miRNAs predicted by PicTar. Only miR-328, miR-193, and miR-137 are predicted by all three algorithms to target the 3′-UTR of Zip5 mRNA

Mentions: The full length 113 nucleotide mouse Zip5 3′-UTR was generated by annealing and ligating synthetic overlapping oligonucleotides with free 3′A residues into the pGEMT vector (mouse; Fig. 1). A sequence verified clone was linearized with Nco I and SP6 RNA polymerase was used to synthesize a 32P body-labeled sense-strand RNA or a non-radioactive sense-strand RNA competitor. The RNA synthesis reactions (30 μl) were as follows: Linear DNA template (1 μg) in transcription buffer (Agilent) containing 10 mM DTT, 0.5 mM each of ATP, GTP, UTP and 0.5 mM CTP for non-radioactive RNA competitor or 110 μCi of α32P-CTP (800 Ci/mmol) for radioactive RNA. SP6 RNA polymerase (10 U) was added and the reaction mixture was incubated at 37°C for 1 h. The DNA template was removed by the addition of 4 units of RNAse-free DNAseI (NEB) for 15 min at 37°C. The synthesized RNAs were precipitated by the addition of 1.5 μg of heat-denatured, fragmented salmon sperm DNA (ssDNA), 0.3 M ammonium acetate, and 2.5 volumes of cold ethanol. RNA pellets were washed twice with 70% ethanol, briefly dried, and diluted into 50% glycerol. ssDNA was used to prevent non-specific binding and served as a carrier that did not interfere with the T1 RNAse digestion. Reactions with ssDNA omitted generated similar T1 digests but had variability in loading due to non-quantitative transfer.Fig. 1


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

Weaver BP, Andrews GK - Biometals (2011)

Alignment of Slc39a5 (Zip5) 3′-UTRs across five mammalian species showing an mFOLD (Zuker 2003) predicted stable stem-loop structure and Venn-diagram of candidate miRNAs predicted by three algorithms to target this UTR. The full-length sequences of Zip5 3′ UTRs from human, chimp, dog, mouse, and rat were aligned and annotated with empirical information from available Zip5 cDNAs (Dufner-Beattie et al. 2004). The polyadenylation signal (polyA signal) and the polyadenylation site (polyA site) are indicated. Yellow indicates nucleotides conserved in all species shown; green and cyan indicate nucleotides conserved in two or more species shown. The most stable stem-loop predicted by mFOLD (Zuker 2003) is shown. Stem-loop residue A17 is A44 in the full length UTR sequence. Two conserved single nucleotide bulges are indicated by heavy arrowheads. miR-328 and miR-193 seed sites are indicated with black bars. Venn-diagram: light gray shaded  region, miRNAs predicted by the miRBase algorithm (Sanger); dark gray shaded region, miRNAs predicted by TargetScan; black shaded region, miRNAs predicted by PicTar. Only miR-328, miR-193, and miR-137 are predicted by all three algorithms to target the 3′-UTR of Zip5 mRNA
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3299966&req=5

Fig1: Alignment of Slc39a5 (Zip5) 3′-UTRs across five mammalian species showing an mFOLD (Zuker 2003) predicted stable stem-loop structure and Venn-diagram of candidate miRNAs predicted by three algorithms to target this UTR. The full-length sequences of Zip5 3′ UTRs from human, chimp, dog, mouse, and rat were aligned and annotated with empirical information from available Zip5 cDNAs (Dufner-Beattie et al. 2004). The polyadenylation signal (polyA signal) and the polyadenylation site (polyA site) are indicated. Yellow indicates nucleotides conserved in all species shown; green and cyan indicate nucleotides conserved in two or more species shown. The most stable stem-loop predicted by mFOLD (Zuker 2003) is shown. Stem-loop residue A17 is A44 in the full length UTR sequence. Two conserved single nucleotide bulges are indicated by heavy arrowheads. miR-328 and miR-193 seed sites are indicated with black bars. Venn-diagram: light gray shaded region, miRNAs predicted by the miRBase algorithm (Sanger); dark gray shaded region, miRNAs predicted by TargetScan; black shaded region, miRNAs predicted by PicTar. Only miR-328, miR-193, and miR-137 are predicted by all three algorithms to target the 3′-UTR of Zip5 mRNA
Mentions: The full length 113 nucleotide mouse Zip5 3′-UTR was generated by annealing and ligating synthetic overlapping oligonucleotides with free 3′A residues into the pGEMT vector (mouse; Fig. 1). A sequence verified clone was linearized with Nco I and SP6 RNA polymerase was used to synthesize a 32P body-labeled sense-strand RNA or a non-radioactive sense-strand RNA competitor. The RNA synthesis reactions (30 μl) were as follows: Linear DNA template (1 μg) in transcription buffer (Agilent) containing 10 mM DTT, 0.5 mM each of ATP, GTP, UTP and 0.5 mM CTP for non-radioactive RNA competitor or 110 μCi of α32P-CTP (800 Ci/mmol) for radioactive RNA. SP6 RNA polymerase (10 U) was added and the reaction mixture was incubated at 37°C for 1 h. The DNA template was removed by the addition of 4 units of RNAse-free DNAseI (NEB) for 15 min at 37°C. The synthesized RNAs were precipitated by the addition of 1.5 μg of heat-denatured, fragmented salmon sperm DNA (ssDNA), 0.3 M ammonium acetate, and 2.5 volumes of cold ethanol. RNA pellets were washed twice with 70% ethanol, briefly dried, and diluted into 50% glycerol. ssDNA was used to prevent non-specific binding and served as a carrier that did not interfere with the T1 RNAse digestion. Reactions with ssDNA omitted generated similar T1 digests but had variability in loading due to non-quantitative transfer.Fig. 1

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