Cis-acting regulatory elements in the potato virus X 3' non-translated region differentially affect minus-strand and plus-strand RNA accumulation

Abstract

The 72nt 3' non-translated region (NTR) of potato virus X (PVX) RNA is identical in all sequenced PVX strains and contains sequences that are conserved among all potexviruses. Computer folding of the 3' NTR sequence predicted three stem-loop structures (SL1, SL2, and SL3 in the 3' to 5' direction), which generally were supported by solution structure analyses. The importance of these sequence and/or structural elements to PVX RNA accumulation was further analyzed by inoculation of Nicotiana tabacum (NT-1) protoplasts with PVX transcripts containing mutations in the 3' NTR. Analyses of RNA accumulation by S(1) nuclease protection indicated that multiple sequence elements throughout the 3' NTR were important for minus-strand RNA accumulation. Formation of SL3 was required for accumulation of minus-strand RNA, whereas SL1 and SL2 formation were less important. However, sequences within all of these predicted structures were required for minus-strand RNA accumulation, including a conserved hexanucleotide sequence element in the loop of SL3, and the CU nucleotide in a U-rich sequence within SL2. In contrast, 13 nucleotides that were predicted to reside in SL1 could be deleted without any significant reduction in minus or plus-strand RNA levels. Potential polyadenylation signals (near upstream elements; NUEs) in the 3' NTR of PVX RNA were more important for plus-strand RNA accumulation than for minus-strand RNA accumulation. In addition, one of these NUEs overlapped with other sequence required for optimal minus-strand RNA levels. These data indicate that the PVX 3' NTR contains multiple, overlapping elements that influence accumulation of both minus and plus-strand RNA.

Figure 1

A representation of the PVX genome. (A) The five ORFs of the PVX genome are denoted by open boxes. The relative positions of the plus-strand probe (P1) and the minus-strand probe (P3) used for S₁ nuclease protection assays are indicated below and above the genome, respectively, with asterisks (∗) denoting the ³²P-labeled positions. (B) Within the 3′ NTR sequence, symbols are used to depict the hexanucleotide sequence (filled diamonds), the U-rich sequence (open circles), the first potential near-upstream element (NUE1; asterisk), and the second potential NUE (NUE2; plus sign). The relative positions of the primers P5 and P6 used for RNA sequencing and primer extension experiments are depicted below the 3′ NTR sequence.

Figure 2

Summary of structural analyses of the 3′ NTR of PVX RNA. (A) An energy dot plot was derived for secondary structures in the 250 nt 3′region of PVX RNA predicted by the Zuker program (http://www.bioinfo.rpi.edu/applications/mfold/). The axes are labeled to denote the position of each nucleotide in the 3′ region of PVX RNA. The three stem-loop structures predicted to form in the 3′ NTR are boxed and denoted as SL1, SL2 and SL3. (B) A summary of the solution structure probing experiments is superimposed on the optimal predicted secondary structures within the 3′ NTR. The positions of RNase T₁ cleavage are indicated by check marks, with larger check marks indicating more reactivity. RNase V₁ cleavage positions are marked by curved arrows. The DMS modification positions are identified by asterisks with intensities of low (), moderate (), or strong (). The intensities of uridine modification by CMCT are denoted as low (), moderate (), or strong (). Filled circles represent ambiguous CMCT reactivity.

Figure 3

Chemical modification patterns of PVX transcripts. Full length transcripts from pMON8453 were incubated either (A) with (+) or without (−) DMS or (B) CMCT. Samples then were subjected to primer extension with primer P5, and run on denaturing 8% polyacrylamide gels. Lanes marked C, U, A, G correspond to PVX RNA sequenced using the same primer noted above. The positions of the modified nucleotides are noted on the right side of the autoradiogram. A filled circle marks an ambiguous CMCT reactivity where there is a signal in the modified lane, but it corresponds to a C rather than a U residue.

Figure 4

Enzymatic cleavage of PVX transcripts. Transcripts from p10 corresponding to nucleotides 6265–6435 (plus 17 A residues and 4 nt of the SpeI site) from the 3′ end of PVX RNA were incubated either (A) with (+) or without (−) RNase T₁ or (B) RNase V₁, and were subjected to primer extension and gel electrophoresis. Consecutively modified nucleotides are denoted as connected arrows to the right of the autoradiogram. All other labeling is similar to that described in the legend to Figure 3.

Figure 5

Effects of deletions extending from the 5′ region of the PVX 3′ NTR. (A) The sequence of the wild-type, pMON8453 3′ NTR is aligned with corresponding sequences from deletion mutants p71Δ10, p71Δ60 and Δint. The vertical arrow indicates the nucleotide mutated in pMON8453 to generate p71. Deleted sequences in p71Δ10, p71Δ60 and Δint are noted by lines. Different elements are illustrated above the wild-type sequence with symbols as described in the legend to Figure 1. (B) Protoplasts inoculated with replication-defective transcripts derived from p32 (p32), wild-type transcripts derived from pMON8453 (w.t.), and mutant transcripts (p71, p71Δ10, p71Δ60 and Δint) were analyzed at 40 hours post-inoculation by digestion with S₁ nuclease. The protected products were separated on denaturing 8% polyacrylamide gels; arrows indicate the direction of electrophoresis. The relative average values (±standard error) for minus-strand (−RNA) and plus-strand (+RNA) accumulation are noted at the outer edges of the autoradiograms.

Figure 6

Analyses of mutations predicted to affect SL3. (A) The 3′ NTR sequences of the wild-type and mutant transcripts are noted, with altered nucleotides underlined. (B) The predicted optimal secondary structures are depicted for wild-type and mutant RNAs between nucleotides 6358 and 6411. The wild-type sequence (CUGC) on the 5′ side of the SL3 stem is marked by arrowheads, and the wild-type sequence (GACG) on the 3′ side of the SL3 stem is indicated by filled squares. Alterations of these sequences are depicted by shading in the mutant structures. The hexanucleotide and U-rich elements are marked as in Figure 1. (C) Protoplasts inoculated with replication-defective transcripts (p32), wild-type (w.t.), and mutant transcripts (SL3-A, SL3-B and SL3-C) were analyzed for RNA accumulation as described in the legend to Figure 5(B).

Figure 7

Effects of mutations in U-rich sequence and predicted SL2. (A) The 3′ NTR sequences of the wild-type and mutant transcripts are noted, with. altered nucleotides underlined. (B) The predicted secondary structures of wild-type and mutant RNA between nucleotides 6358 and 6411 are illustrated, with designations as described in the legend to Figure 6(B). (C) Analysis of RNA accumulation in protoplasts inoculated with transcripts derived from p32 (p32), pMON8453 (w.t.), and mutant (SL2-A, SL2-B, SL3-C and SL2-D) transcripts was carried out as described in the legend to Figure 5(B).

Figure 8

Deletion analyses of sequences between the U-rich element and the potential NUEs. (A) Sequences between nucleotides 6402 and 6438 for p72, p72Δ13, p72Δ16, p72Δ23 and p72Δ21 are aligned, with deleted regions marked as lines. (B) Predicted secondary structures for wild-type and some of the mutant RNAs between nucleotides 6384 and 6438 are illustrated and marked as described in the legend to Figure 1. Although not shown, SL3 is predicted to be unchanged in all transcripts illustrated in A and B. (C) Minus and plus-strand RNA accumulation at 40 h.p.i. in protoplasts inoculated with transcripts derived from p32, pMON8453 (w.t), and mutants p72Δ13, p72Δ16, p72Δ23 and p72Δ21 were analyzed as described in the legend to Figure 5(B).

Figure 9

RNA accumulation in response to modification of potential NUEs. (A) Sequences between nucleotides 6402 and 6438 of wild-type and mutant RNAs are compared. Transcripts derived from all mutants, except Δ15, have the poly(A) tail. Nucleotides mutated in p73, p74 and Δ15 are denoted by vertical arrows and other designations are as described in the legend to Figure 1. (B) The predicted secondary structures from nucleotides 6384–6438 of wild-type and mutant RNAs are illustrated; the predicted structure for Δ15 contains nucleotides 6384–6423. All of these RNA transcripts are predicted to have a wild-type SL3 structure. All markings are as denoted in the legends to Figure 1, Figure 3. (C) Detection of minus and plus-strand RNA accumulation in protoplasts inoculated with p32, w.t, and mutant (p73, p74, ΔAAU, ΔUAUA, ΔUAAUAUA and Δ15) transcripts as described in the legend to Figure 5(B).

Figure 10

Alignments of the 3′ NTR sequences of different potexviruses. Alignments were done by hand and were centered around hexanucleotide elements (in red) conserved among potexviruses. Additional potential hexanucleotide elements found upstream in CYMV, FXMV, PIAMV, and PMV sequences are also noted in red. The sequences aligned include our PVX strain, bamboo mosaic virus (BaMV), cymbidium mosaic virus (CyMV), clover yellow mosaic virus (CYMV), foxtail mosaic virus (FXMV), narcissus mosaic virus (NMV), potato aucuba mosaic virus (PAMV),Plantago asiatica mosaic virus (PIAMV), papaya mosaic virus (PMV), strawberry mild yellow edge-associated virus (SMYEAV) and white clover mosaic virus (WClMV). Palindromic sequences are underlined on either side of the hexanucleotide elements. Similarities in the U-rich (blue) and putative NUE (yellow) sequences among the potexvirus RNAs are indicated.