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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 of MT-1 3′UTR. The secondary structure was predicted using Mfold (http://mobyle.pasteur.fr/cgi-bin/portal.py?form=mfold). The region studied in this work (magnified to increase clarity) was the stem formed by nt 26–31 and 66–70 and the CACC repeat between nt 66 and 76.
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fig1: Predicted secondary structure of MT-1 3′UTR. The secondary structure was predicted using Mfold (http://mobyle.pasteur.fr/cgi-bin/portal.py?form=mfold). The region studied in this work (magnified to increase clarity) was the stem formed by nt 26–31 and 66–70 and the CACC repeat between nt 66 and 76.

Mentions: In general mRNA localization is brought about by cis-acting signals within the 3′untranslated region (3′UTR) that interact with RNA-binding proteins [1,2,12]. Localized mRNAs appear to be transported in RNA granules where they are associated with several proteins [13,14]. The nature of such complexes remains to be defined. In the case of perinuclear mRNAs neither the precise nature of the signal within the 3′UTR nor the nature of the binding complex have been characterised. However, for MT-1 mRNA, there is evidence that the region of the 3′UTR between nucleotides (nt) 45 and 76, particularly nt 66–76, is required for localization [15]. This region contains CACC repeats and interestingly such repeats have been implicated in other localization signals [16,17]. Data derived from a combination of bioinformatic folding predictions and chemical cleavages indicate that this region is part of an internal stem-loop (see Fig. 1) and initial mutagenesis studies suggested that the secondary structure is important for localization [6]. This earlier study indicated that elongation factor 1 alpha (eEF1α) binds to this region [6], and furthermore eEF1α has also been reported to bind to the localization signal of β-actin mRNA [18]. To date neither the relative importance of the CACC repeat in combination with the stem-loop structure nor the domains of eEF1α required for this binding have been fully elucidated. Moreover, it is not known which proteins, in addition to eEF1α, are present as part of the localization complex.


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 of MT-1 3′UTR. The secondary structure was predicted using Mfold (http://mobyle.pasteur.fr/cgi-bin/portal.py?form=mfold). The region studied in this work (magnified to increase clarity) was the stem formed by nt 26–31 and 66–70 and the CACC repeat between nt 66 and 76.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: Predicted secondary structure of MT-1 3′UTR. The secondary structure was predicted using Mfold (http://mobyle.pasteur.fr/cgi-bin/portal.py?form=mfold). The region studied in this work (magnified to increase clarity) was the stem formed by nt 26–31 and 66–70 and the CACC repeat between nt 66 and 76.
Mentions: In general mRNA localization is brought about by cis-acting signals within the 3′untranslated region (3′UTR) that interact with RNA-binding proteins [1,2,12]. Localized mRNAs appear to be transported in RNA granules where they are associated with several proteins [13,14]. The nature of such complexes remains to be defined. In the case of perinuclear mRNAs neither the precise nature of the signal within the 3′UTR nor the nature of the binding complex have been characterised. However, for MT-1 mRNA, there is evidence that the region of the 3′UTR between nucleotides (nt) 45 and 76, particularly nt 66–76, is required for localization [15]. This region contains CACC repeats and interestingly such repeats have been implicated in other localization signals [16,17]. Data derived from a combination of bioinformatic folding predictions and chemical cleavages indicate that this region is part of an internal stem-loop (see Fig. 1) and initial mutagenesis studies suggested that the secondary structure is important for localization [6]. This earlier study indicated that elongation factor 1 alpha (eEF1α) binds to this region [6], and furthermore eEF1α has also been reported to bind to the localization signal of β-actin mRNA [18]. To date neither the relative importance of the CACC repeat in combination with the stem-loop structure nor the domains of eEF1α required for this binding have been fully elucidated. Moreover, it is not known which proteins, in addition to eEF1α, are present as part of the localization complex.

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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