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Fine mapping of interactions between eEF1alpha protein and 3'UTR of metallothionein-1 mRNA.

Fan K, Chrzanowska-Lightowlers ZM, Hesketh JE - Biochem. Biophys. Res. Commun. (2009)

Bottom Line: Full length recombinant rat eEF1alpha, and independently domains I and III, formed complexes with the mRNA.Proteins binding to biotinylated MT-1 3'UTR sequences were isolated using RNA-affinity techniques, and mass spectrometry identified histidine-tRNA ligase as one of the major MT-1 3'UTR binding proteins.We conclude that a 5-bp internal stem in the MT-1 3'UTR is critical for binding of eEF1alpha and histidine-tRNA ligase, and that binding of eEF1alpha is facilitated through domains I and III.

View Article: PubMed Central - PubMed

Affiliation: Institute for Cell and Molecular Biosciences, Newcastle University, The Medical School, Framlington Place, Newcastle upon Tyne NE2 4HH, UK.

ABSTRACT
The localization of metallothionein-1 (MT-1) mRNA to the perinuclear cytoskeleton is determined by a signal in the 3'untranslated region (3'UTR) and trans-acting binding proteins. The present study carried out detailed mapping of this signal and further characterized the binding to elongation factor 1 alpha (eEF1alpha) and other interacting proteins. Electrophoresis mobility shift assays demonstrated that shortening of a stem region proximal to nucleotides 66-76 abrogated binding. Full length recombinant rat eEF1alpha, and independently domains I and III, formed complexes with the mRNA. Proteins binding to biotinylated MT-1 3'UTR sequences were isolated using RNA-affinity techniques, and mass spectrometry identified histidine-tRNA ligase as one of the major MT-1 3'UTR binding proteins. We conclude that a 5-bp internal stem in the MT-1 3'UTR is critical for binding of eEF1alpha and histidine-tRNA ligase, and that binding of eEF1alpha is facilitated through domains I and III.

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Predicted secondary structure for mutant MT-1 3′UTR sequences. The secondary structure of the various mutant transcripts produced by site-directed mutagenesis was predicted using Mfold (http://mobyle.pasteur.fr/cgi-bin/portal.py?form=mfold). (A–D) Show mutants in which the internal stem is predicted to be shortened; the remaining stem is indicated by the rectangle in each case: pcMT-Δ31,66 has 1 bp deleted (A), pcMT-Δ30,31,66,67 has 2 bp deleted (B), pcMT-Δ29–31,66–68 3 bp deleted (C) and in pcMT-Δ27,28,69,70 the 2 bp close to the loop region are deleted (D). (E and F) Show mutants in which the CACC repeat sequence is altered but base pairing in the stem maintained: pcMT-DS3 had two nucleotides in the stem region CACC sequence substituted (E) and pcMT-TS had both CACC sequences, one in the stem region and one in the loop, interrupted by nucleotide substitution (F).
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fig2: Predicted secondary structure for mutant MT-1 3′UTR sequences. The secondary structure of the various mutant transcripts produced by site-directed mutagenesis was predicted using Mfold (http://mobyle.pasteur.fr/cgi-bin/portal.py?form=mfold). (A–D) Show mutants in which the internal stem is predicted to be shortened; the remaining stem is indicated by the rectangle in each case: pcMT-Δ31,66 has 1 bp deleted (A), pcMT-Δ30,31,66,67 has 2 bp deleted (B), pcMT-Δ29–31,66–68 3 bp deleted (C) and in pcMT-Δ27,28,69,70 the 2 bp close to the loop region are deleted (D). (E and F) Show mutants in which the CACC repeat sequence is altered but base pairing in the stem maintained: pcMT-DS3 had two nucleotides in the stem region CACC sequence substituted (E) and pcMT-TS had both CACC sequences, one in the stem region and one in the loop, interrupted by nucleotide substitution (F).

Mentions: A combination of chemical cleavage analysis and secondary structure prediction of the MT-1 3′UTR has implicated a CACC repeat within a region that contains a internal stem-loop region as being a critical element required for the localization of MT-1 mRNA [6]. The subcellular distribution of the transcript is facilitated by interaction with protein(s) binding to its 3′UTR. To characterise the significance of the CACC repeat and the stem-loop in these protein interactions, a series of mutant MT-1 transcripts were generated by site-directed mutagenesis. The internal stem is predicted to contain 5 bp (nt 27–31 interacting with 66–70) as shown in Fig. 1. Based on this predicted structure four mutants were made in which, as shown in Fig. 2, the deletions were predicted by Mfold to shorten the stem by one, two or three base pairing nucleotides. Thus, the mutant pcMT-Δ31,66 was predicted to produce transcripts with a stem shortened by 1 bp: similarly the stem in MT-Δ30,31,66,67 was 2 bp shorter, and MT-Δ29–31,66–68 by three, whilst MT-Δ27,28,69,70 was predicted to remove 2 bp positioned proximal to the loop region. Two further mutants were made that were predicted to maintain a 5-bp stem but in which the CACC sequence in either the stem or the adjacent loop had been replaced. In MT-DS3 the CACC sequence in the stem region (nt 67–70) was substituted with CGGC and mutant MT-TS had the CACC sequences in both the stem region and loop region replaced with CGGC and CTTC, respectively.


Fine mapping of interactions between eEF1alpha protein and 3'UTR of metallothionein-1 mRNA.

Fan K, Chrzanowska-Lightowlers ZM, Hesketh JE - Biochem. Biophys. Res. Commun. (2009)

Predicted secondary structure for mutant MT-1 3′UTR sequences. The secondary structure of the various mutant transcripts produced by site-directed mutagenesis was predicted using Mfold (http://mobyle.pasteur.fr/cgi-bin/portal.py?form=mfold). (A–D) Show mutants in which the internal stem is predicted to be shortened; the remaining stem is indicated by the rectangle in each case: pcMT-Δ31,66 has 1 bp deleted (A), pcMT-Δ30,31,66,67 has 2 bp deleted (B), pcMT-Δ29–31,66–68 3 bp deleted (C) and in pcMT-Δ27,28,69,70 the 2 bp close to the loop region are deleted (D). (E and F) Show mutants in which the CACC repeat sequence is altered but base pairing in the stem maintained: pcMT-DS3 had two nucleotides in the stem region CACC sequence substituted (E) and pcMT-TS had both CACC sequences, one in the stem region and one in the loop, interrupted by nucleotide substitution (F).
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2719692&req=5

fig2: Predicted secondary structure for mutant MT-1 3′UTR sequences. The secondary structure of the various mutant transcripts produced by site-directed mutagenesis was predicted using Mfold (http://mobyle.pasteur.fr/cgi-bin/portal.py?form=mfold). (A–D) Show mutants in which the internal stem is predicted to be shortened; the remaining stem is indicated by the rectangle in each case: pcMT-Δ31,66 has 1 bp deleted (A), pcMT-Δ30,31,66,67 has 2 bp deleted (B), pcMT-Δ29–31,66–68 3 bp deleted (C) and in pcMT-Δ27,28,69,70 the 2 bp close to the loop region are deleted (D). (E and F) Show mutants in which the CACC repeat sequence is altered but base pairing in the stem maintained: pcMT-DS3 had two nucleotides in the stem region CACC sequence substituted (E) and pcMT-TS had both CACC sequences, one in the stem region and one in the loop, interrupted by nucleotide substitution (F).
Mentions: A combination of chemical cleavage analysis and secondary structure prediction of the MT-1 3′UTR has implicated a CACC repeat within a region that contains a internal stem-loop region as being a critical element required for the localization of MT-1 mRNA [6]. The subcellular distribution of the transcript is facilitated by interaction with protein(s) binding to its 3′UTR. To characterise the significance of the CACC repeat and the stem-loop in these protein interactions, a series of mutant MT-1 transcripts were generated by site-directed mutagenesis. The internal stem is predicted to contain 5 bp (nt 27–31 interacting with 66–70) as shown in Fig. 1. Based on this predicted structure four mutants were made in which, as shown in Fig. 2, the deletions were predicted by Mfold to shorten the stem by one, two or three base pairing nucleotides. Thus, the mutant pcMT-Δ31,66 was predicted to produce transcripts with a stem shortened by 1 bp: similarly the stem in MT-Δ30,31,66,67 was 2 bp shorter, and MT-Δ29–31,66–68 by three, whilst MT-Δ27,28,69,70 was predicted to remove 2 bp positioned proximal to the loop region. Two further mutants were made that were predicted to maintain a 5-bp stem but in which the CACC sequence in either the stem or the adjacent loop had been replaced. In MT-DS3 the CACC sequence in the stem region (nt 67–70) was substituted with CGGC and mutant MT-TS had the CACC sequences in both the stem region and loop region replaced with CGGC and CTTC, respectively.

Bottom Line: Full length recombinant rat eEF1alpha, and independently domains I and III, formed complexes with the mRNA.Proteins binding to biotinylated MT-1 3'UTR sequences were isolated using RNA-affinity techniques, and mass spectrometry identified histidine-tRNA ligase as one of the major MT-1 3'UTR binding proteins.We conclude that a 5-bp internal stem in the MT-1 3'UTR is critical for binding of eEF1alpha and histidine-tRNA ligase, and that binding of eEF1alpha is facilitated through domains I and III.

View Article: PubMed Central - PubMed

Affiliation: Institute for Cell and Molecular Biosciences, Newcastle University, The Medical School, Framlington Place, Newcastle upon Tyne NE2 4HH, UK.

ABSTRACT
The localization of metallothionein-1 (MT-1) mRNA to the perinuclear cytoskeleton is determined by a signal in the 3'untranslated region (3'UTR) and trans-acting binding proteins. The present study carried out detailed mapping of this signal and further characterized the binding to elongation factor 1 alpha (eEF1alpha) and other interacting proteins. Electrophoresis mobility shift assays demonstrated that shortening of a stem region proximal to nucleotides 66-76 abrogated binding. Full length recombinant rat eEF1alpha, and independently domains I and III, formed complexes with the mRNA. Proteins binding to biotinylated MT-1 3'UTR sequences were isolated using RNA-affinity techniques, and mass spectrometry identified histidine-tRNA ligase as one of the major MT-1 3'UTR binding proteins. We conclude that a 5-bp internal stem in the MT-1 3'UTR is critical for binding of eEF1alpha and histidine-tRNA ligase, and that binding of eEF1alpha is facilitated through domains I and III.

Show MeSH