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Fig. 4.
Isolation of Angiotensinogen 1-14 binding to biotinylated MT-111 RNA. Reactions containing CHO cell S100 extract with or without biotinylated MT-111 were subjected to RNA-affinity chromatography with streptavidin-coated paramagnetic beads. Proteins eluted from the beads were separated by 10% SDS–PAGE. Major protein species are indicated (black arrows); these were excised and subjected to analysis by mass spectrometry.
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In conclusion, we postulate that eEF1α is capable of binding to a specific internal stem-loop region of the 3′UTR of MT-1 mRNA implicated in perinuclear mRNA localization and hypothesise that histidine-tRNA ligase is also a member of this binding complex. Fine mapping of the critical regions involved in complex formation shows that domains I and III of eEF1α protein are sufficient for recognition of a RNA motif in which both the 5-bp stem and CACC repeat sequence are essential. Although these domains of eEF1α can act independently to facilitate this binding, it is likely that other proteins are present in the RNP complex in vivo. Interestingly, eEF1α also binds to the zip-code sequence in actin mRNA and a stem-loop structure in the West Nile virus [3], [18] and [25] and these motifs also contain an ACACC or CAC repeat suggesting that a combination of stem-loop structure and CAC repeat sequence is a conserved motif important in binding of eEF1α to specific transcripts.