Evidence for the existence of the loop E motif of Potato spindle tuber viroid in vivo

Abstract

RNA motifs comprising nucleotides that interact through non-Watson-Crick base pairing play critical roles in RNA functions, often by serving as the sites for RNA-RNA, RNA-protein, or RNA small ligand interactions. The structures of viral and viroid RNA motifs are studied commonly by in vitro, computational, and mutagenesis approaches. Demonstration of the in vivo existence of a motif will help establish its biological significance and promote mechanistic studies on its functions. By using UV cross-linking and primer extension, we have obtained direct evidence for the in vivo existence of the loop E motif of Potato spindle tuber viroid. We present our findings and discuss their biological implications.

FIG. 1.

Secondary structure of PSTVd (upper panel) (14) and non-Watson-Crick base pairs in loop E (lower panel) (37). The symbols that denote each of the specific base edge-edge interactions are defined below the structure (for details, see references and 37). The dashed line indicates G98 and U260, which can be cross-linked by UV irradiation. R1, R2, and R3 indicate the positions of the primers used in the primer extension experiments.

FIG. 2.

RNA gel blot showing the appearance of a UV-cross-linked product (arrow) of PSTVd RNAs from infected tomato leaves. Treatment with proteinase K (0.5 units for 30 min; Invitrogen) indicates that this cross-linked product is not attributed to protein binding. c-PSTVd and l-PSTVd denote circular and linear PSTVd RNAs, respectively. For gel blotting, total RNAs were extracted from infected tomato leaves that were untreated or irradiated with UV for 80 min (10 J/cm²) by using the RNeasy plant mini kit (QIAGEN, Valencia, CA). RNA samples were run on 5% polyacrylamide-8 M urea gels, transferred to Hybond-XL nylon membranes (Amersham Biosciences, Piscataway, NJ) using a vacuum blotting system (Amersham), and immobilized by UV cross-linking. After overnight hybridization with [α-³²P]UTP-labeled riboprobes at 65°C in ULTRAhyb reagent (Ambion, Austin, TX), the membranes were washed twice at 65°C in 2× SSC-0.1% sodium dodecyl sulfate (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate) and 0.2× SSC-0.1% sodium dodecyl sulfate and exposed to a Storage phosphor screen (Kodak, Rochester, NY).

FIG. 3.

RNA gel blot showing the origin of the in vivo UV-cross-linked product predominantly from the circular PSTVd RNAs. Total RNAs were extracted and run on 5% polyacrylamide-8 M urea gels as described in the legend for Fig. 2. The in vivo circular PSTVd (c-PSTVd) and linear PSTVd (l-PSTVd) RNAs were gel purified and irradiated with UV for 5 min in EXL buffer (600 mM NaCl, 4 M urea, 1 mM cacodylate, 0.1 mM EDTA) as described by Zhong et al. (37). In vitro transcripts of PSTVd were obtained by transcribing HindIII-linearized pRZ6-2 template (17) by utilizing a T7 MAXIscript kit (Ambion). Unit-length l-PSTVd RNAs were gel purified from the 5% polyacrylamide-8 M urea gel. Incubation of the l-PSTVd transcripts in wheat germ extract gave rise to the circular PSTVd RNAs, which were run on a 5% polyacrylamide-8 M urea gel followed by gel purification. Both c-PSTVd and l-PSTVd in vitro transcripts were subjected to UV irradiation for 5 min in the EXL buffer. Approximately 100 to 200 ng of RNA was loaded for each lane.

FIG. 4.

Primer extension mapping of an in vivo cross-linking site to G98 of loop E, using three different primers as indicated (R1, R2, and R3). Lane c, cDNAs from reverse transcription of the circular PSTVd RNA template purified from infected tomato leaves; lane cI, cDNAs from reverse transcription of the UV cross-linked circular PSTVd RNA template purified from infected leaves. The reverse transcription was performed by following the protocols of Baumstark et al. (3) with modifications, using Invitrogen Superscript III RNase H- reverse transcriptase and ³²P-labeled primers for 40 min at 52°C. Lanes U to A, sequencing ladders generated by PCR with pRZ6-2 template and ³²P-labeled primers in the presence of ddATP, ddCTP, ddGTP, and ddUTP by utilizing the Thermo Sequenase cycle sequencing kit (USB, Cleveland, OH). All samples were run on an 8% polyacrylamide-8 M urea sequencing gel. The nucleotide sequence of the (+)-PSTVd is given on the left, and the arrows point to the bands corresponding to reverse transcription termination at A99.