Abstract
Isolation of biologically active cell components from multicellular eukaryotic organisms often poses difficult challenges such as low yields and inability to retain the integrity and functionality of the purified compound. We previously identified a cap-independent translation enhancer (3′CITE) in the 3′UTR of Turnip crinkle virus (TCV) that structurally mimics a tRNA and binds to yeast 80S ribosomes and 60S subunits in the P-site. Yeast ribosomes were used for these studies due to the lack of methods for isolation of plant ribosomes with high yields and integrity. To carry out studies with more natural components, a simple and efficient procedure has been developed for the isolation of large quantities of high quality ribosomes and ribosomal subunits from Arabidopsis thaliana protoplasts prepared from seed-derived callus tissue. Attempts to isolate high quality ribosomes from wheat germ, bean sprouts, and evacuolated protoplasts were unsuccessful. Addition of purified Arabidopsis 80S plant ribosomes to ribosome-depleted wheat germ lysates resulted in a greater than 1200-fold enhancement in in vitro translation of a luciferase reporter construct. The TCV 3′CITE bound to ribosomes with a three to sevenfold higher efficiency when using plant 80S ribosomes compared with yeast ribosomes, indicating that this viral translational enhancer is adapted to interact more efficiently with host plant ribosomes.
Highlights
Studies of translation initiation using plant positive, singlestranded RNA viruses that lack a 5 7-methyl guanosine cap have revealed a wide range of mechanisms centered on highly structured, 3 proximal cap-independent translation enhancers (3 CITEs) that bind to various host translation initiation factors (Simon and Miller, 2013)
We describe a simple, efficient method for isolation of plant ribosomes and ribosomal subunits with high yield and quality from Arabidopsis thaliana protoplasts prepared from seed-derived callus tissue
1st spin plant ribosomes were resuspended in 2.5 ml of buffer B [10% glycerol, 20 mM Tris–HCl pH 7.5, 5 mM Mg(CH3COO)2, 0.5 M KCl, 1 mg/ml heparin, and 1 mM DTT], GTP, and puromycin added to final concentrations of 1 mM, and the ribosome suspension incubated at 30◦C for 30 min
Summary
Studies of translation initiation using plant positive, singlestranded RNA viruses that lack a 5 7-methyl guanosine cap have revealed a wide range of mechanisms centered on highly structured, 3 proximal cap-independent translation enhancers (3 CITEs) that bind to various host translation initiation factors (Simon and Miller, 2013). We describe a simple, efficient method for isolation of plant ribosomes and ribosomal subunits with high yield and quality from Arabidopsis thaliana protoplasts prepared from seed-derived callus tissue.
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