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. 2013 Jul 22:4:271.
doi: 10.3389/fpls.2013.00271. eCollection 2013.

Preparation of biologically active Arabidopsis ribosomes and comparison with yeast ribosomes for binding to a tRNA-mimic that enhances translation of plant plus-strand RNA viruses

Vera A Stupina  1 , Anne E Simon
Affiliations

Preparation of biologically active Arabidopsis ribosomes and comparison with yeast ribosomes for binding to a tRNA-mimic that enhances translation of plant plus-strand RNA viruses

Vera A Stupina et al. Front Plant Sci. .

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.

Keywords: 3′CITE; Arabidopsis thaliana protoplasts; TCV; TSS; plant ribosomes; virus translation.

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Figures

FIGURE 1
FIGURE 1
Arabidopsis protoplasts serve as the optimal source for the preparation of plant ribosomes and their subunits. (A) In situ RNA gel electrophoresis analysis of small-scale ribosomal preparations (1st spin) from evacuolated Arabidopsis protoplast extracts (lane 2), bean sprouts (lane 3), Arabidopsis protoplasts (preparations 1 and 2, lanes 4 and 5). Yeast ribosomal preparation (lane 1) and aliquot of commercial WGL (Promega; lane 6) served as controls. Each preparation (1 ml, between 2.5 and 5 pmol of ribosomes) was resuspended in an equal volume of 100% formamide and the mixture directly loaded onto a 1.2%, 0.5×TBE agarose gel. rRNA was detected by staining gel with ethidium bromide. Note that although 80S ribosomes were loaded, the ribosome subunits disengage in the gel and thus the large and small rRNAs become separated. (B) In situ RNA gel electrophoresis analysis of the large-scale plant ribosomal preparation from Arabidopsis protoplasts: 80S 1st spin (left panel) and sw 80S (right panel). Three different concentrations (0.5, 0.75, 1 pmol) were loaded for each panel. (C) Sucrose-gradient separation of plant 40S and 60S ribosomal subunits. After centrifugation, the first 1.5 ml of the gradient were removed from the top of the tube and discarded. The remaining sucrose gradient containing subunits was subdivided into 0.5 ml fractions. Each fraction (4 ml) was resolved on a 1.2%, 0.5×TBE agarose gel for detection of rRNA from 40S and 60S subunits in situ (left panel). Fractions containing 40S and 60S subunits were combined into two pools. After buffer exchange and sample concentration, 2 μl of each pool were loaded onto the agarose gel to inspect the quality of the rRNA (right panel).
FIGURE 2
FIGURE 2
RNA stability assays. Frozen protoplast pellets were re-suspended in either yeast buffer A or plant buffer A. Suspensions were incubated at either 4°C (left panel) or 10°C (right panel). Aliquots were taken at 1, 2, and 4 h time points to extract rRNA. RNA (1 μg) was inspected for integrity on 1.2%, 0.5×TBE agarose gels.
FIGURE 3
FIGURE 3
Salt-washed plant ribosomes support cap-dependent and cap-independent translation in ribosome-depletedWGL. (A) Map of 5′UTR-Fluc-3661 and Cap-Rluc-A reporter constructs. 5′UTR-Fluc-3661 contains the firefly luciferase ORF flanked by the TCV 5′UTR and positions 3661–4054. Cap-Rluc-A contains a capped, polyadenylated renilla luciferase ORF. (B) In vitro translation of 3′UTR-Fluc-3661 using ribosome-depleted WGL supplemented with either WGL ribosomes or Arabidopsis ribosomes or ribosomal subunits. None, no ribosomes added. (C) In vitro translation of 3′UTR-Fluc-3661 using ribosome-depleted WGL supplemented with different concentrations of sw Arabidopsis ribosomes. (D) Translation of Cap-Rluc-A using different concentrations of sw Arabidopsis ribosomes. (E) Comparison of cap-dependent (Cap-Rluc-A) and cap-independent (5′UTR-Fluc-3661) translation using 0.5× sw Arabidopsis ribosomes.
FIGURE 4
FIGURE 4
Plant ribosomes associate with theTCVTSS at higher efficiency compared with yeast ribosomes. One to 60 pmol of [32P] 5′-end labeled TSS were combined with 15 pmol of sw yeast ribosomes (A) or sw plant ribosomes (B). Y-axis denotes the fraction of TSS bound per ribosome. X-axis indicates the ratio of input (free) TSS to ribosomes. Scatchard plots are included for each saturation curve. To generate a saturation curve and to calculate Kd values, each filter-binding experiment was conducted in triplicate.
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