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Abstract
The control of subcellular mRNA localization and translation is often mediated by protein factors that are directly or indirectly associated with the cytoskeleton. We report the identification and characterization of a rice seed protein that possesses both RNA and microtubule binding activities. In vitro UV cross-linking assays indicated that this protein binds to all mRNA sequences tested, although there was evidence for preferential binding to RNAs that contained A-C nucleotide sequence motifs. The protein was purified to homogeneity using a two-step procedure, and amino acid sequencing identified it as the multifunctional protein (MFP), a peroxisomal enzyme known to possess a number of activities involved in the beta-oxidation of fatty acids. The recombinant version of this rice MFP binds to RNA in UV cross-linking and gel mobility shift experiments, co-sediments specifically with microtubules, and possesses at least two enzymatic activities involved in peroxisomal fatty acid beta-oxidation. Taken together these data suggest that MFP has an important role in mRNA physiology in the cytoplasm, perhaps in regulating the localization or translation of mRNAs through an interaction with microtubules, in addition to its peroxisomal function.
Highlights
The cytoskeleton plays a central role in the organization and function of eukaryotic cells
The protein was purified to homogeneity using a two-step procedure, and amino acid sequencing identified it as the multifunctional protein (MFP), a peroxisomal enzyme known to possess a number of activities involved in the -oxidation of fatty acids
We propose that rice MFP carries out a cytoplasmic role in mRNA localization or translational control, in addition to its enzymatic roles in the -oxidation of fatty acids within the peroxisomal matrix
Summary
Preparation of Soluble Rice Seed Protein—Thirty grams of developing rice seeds (10 –15 days after pollination) were ground to fine powder in liquid nitrogen and ground in 40 ml of extraction buffer (100 mM HEPES, pH 7.6, 5 mM MgSO4, 5 mM EGTA, 10 mM DTT, and 2.5 g/ml each of leupeptin, pepstatin, and aprotinin). For UV cross-linking experiments, varying amounts of the MAP fraction or recombinant protein were preincubated in 20 mM HEPES, pH 7.6, 100 mM KCl, 10% glycerol, 1 mM DTT, 4 g of yeast tRNA, 5 g of heparin, and 1 unit of prime RNase inhibitor (5 Prime 3 3 Prime, Inc., Boulder, CO) for 10 min on ice followed by incubation with 1–3 ng (200,000 cpm) of synthetic radioactively labeled RNA probe for 10 min in a final reaction volume of 20 l. The microtubule cosedimentation assays were performed in 20 mM HEPES, pH 7.6, 50 mM KCl, 1 mM DTT, 0.1 mM GTP, 1 mM EGTA, and 2.5 g/ml each of leupeptin, pepstatin, and aprotinin in a final volume of 20 l and contained varying concentrations of microtubules, BSA, MFP, or labeled RNA.
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