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. 2003 Mar;9(3):346-54.
doi: 10.1261/rna.2162203.

Viroid RNA Systemic Spread May Depend on the Interaction of a 71-nucleotide Bulged Hairpin With the Host Protein VirP1

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Free PMC article

Viroid RNA Systemic Spread May Depend on the Interaction of a 71-nucleotide Bulged Hairpin With the Host Protein VirP1

Elsa Maniataki et al. RNA. .
Free PMC article

Erratum in

  • RNA. 2003 Apr;9(4):502.

Abstract

Viroids are noncoding circular single-stranded RNAs that are propagated systemically in plants. VirP1 is a protein from tomato, which is an excellent host for potato spindle tuber viroid (PSTVd), and it has been isolated by virtue of its specific in vitro binding to PSTVd RNA. We report on the specific in vivo interaction of VirP1 with full-length viroid RNA as well as with subfragments in the three-hybrid system. The terminal right domain (TR) of PSTVd was identified as a strong interacting partner for VirP1. A weaker partner is provided by a right-hand subfragment of hop stunt viroid (HSVd), a viroid that infects tomato poorly. We present a sequence and structural motif of the VirP1-interacting subfragments. The motif is disturbed in the replicative but nonspreading R+ mutant of the TR. According to our in vivo and in vitro binding assays, the interaction of this mutant with VirP1 is compromised. We propose that the AGG/CCUUC motif bolsters recognition of the TR by VirP1 to achieve access of the viroid to pathways that propagate endogenous RNA systemic signals in plants. Systemic trafficking has been suggested for miRNA precursors, of which the TR, as a stable bulged hairpin 71 nt long, is quite reminiscent.

Figures

FIGURE 1.
FIGURE 1.
Specific interaction between full-length PSTVd RNA and the tomato protein VirP1. (A) The three full-length PSTVd RNAs that were tested in the three-hybrid system for VirP1 binding. The drawings show where PSTVd(Ha106), PSTVd(Sma), and PSTVd(Sty) are linearized. (B) Results of the β-gal assay. Five RNA hybrids were combined with three protein hybrids, and the respective S. cerevisiae strains were tested for β-gal activity. All five RNA hybrids contain MS2 RNA. Three of them, MS2-PSTVd(Ha106), MS2-PSTVd(Sma), and MS2-PSTVd(Sty), contain additionally PSTVd(Ha106), PSTVd(Sma), and PSTVd(Sty) RNA, respectively. MS2-IRE contains the iron responsive element. All the three protein hybrids contain the Gal4 activation domain. AD-VirP1ct additionally contains a 313-amino-acid carboxy-terminal fragment of VirP1. AD-IRP contains the iron responsive protein.
FIGURE 2.
FIGURE 2.
The TR is responsible for VirP1 binding to PSTVd. (A) The four overlapping PSTVd RNA subfragments that were tested for VirP1 binding in the three-hybrid system. L170 and L280 are left-hand fragments of approximately 170 and 280 bases, respectively. R190 and R80 are right-hand fragments of 190 and 80 bases, respectively. R80 contains the 71-nt TR of PSTVd. (B) Results of the β-gal assay. The RNA hybrids, MS2-L170, MS2-L280, MS2-R190, and MS2-R80 contain L170, L280, R190, and R80, respectively. They were combined with the protein hybrids AD, AD-VirP1ct, and AD-IRP.
FIGURE 3.
FIGURE 3.
VirP1 binds to a right-hand fragment of HSVd. (A) An HSVd RNA monomer, HSV(Sma), and two HSVd RNA subfragments, L190 and R110, were tested. (B) Results of the β-gal assay. The three RNA hybrids, MS2-HSV(Sma), MS2-L190, and MS2-R110, were combined with the protein hybrids AD and AD-VirP1ct.
FIGURE 4.
FIGURE 4.
Comparison of the nucleotide sequences of the viroid subfragments R80(PSTVd) and R110(HSVd). Each sequence is shown folding back on itself, as predicted for the interrupted double-helical viroid conformation. The repeats that are observed within each strand of the subfragments are presented in bold characters. The sequences shared by PSTVd and HSVd are underlined.
FIGURE 5.
FIGURE 5.
(A) Comparison between the terminal right regions of PSTVd and CEVd. Their 18-nt region of sequence and structural identity is boxed (solid line). The repeated ACAGG/CCUUCCU pair that includes the (underlined) AGG/CCUUC motif is presented in bold characters. (B) The designed R34 RNA. The dashed box indicates the 28-nt part that PSTVd and R34 share.
FIGURE 6.
FIGURE 6.
The effect PSTVd R+ and PSTVd var1 mutations on the interaction of VirP1 with PSTVd RNA. (A) Primary and secondary structure of the mutated area of PSTVd. The nucleotides that vary from the wild-type sequence are in lower case characters. (B) Electrophoretic mobility shift assay (EMSA). The picture shown here is representative of four such experiments. Radiolabeled WTR34Taq, R+R34Taq, and var1R34Taq RNA transcripts were incubated with (+) and without (−) purified his-tagged VirP1. The experiment is duplicated with boiled and snap-cooled RNA transcripts. (C) The three-hybrid test. The WTR34Taq, R+R34Taq, and var1R34Taq RNAs are contained in the RNA hybrids MS2-R34WT, MS2-R34R+, and MS2-R34var1, respectively. Each one of those RNA hybrids, and MS2 as well, were tested with the protein hybrid AD-VirP1ct. In each case, two independent double transformants of the S. cerevisiae strain L40-coat were assayed for β-gal activity.

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