Improperly terminated, unpolyadenylated mRNA of sense transgenes is targeted by RDR6-mediated RNA silencing in Arabidopsis

Abstract

RNA silencing can be induced by highly transcribed transgenes through a pathway dependent on RNA-DEPENDENT RNA POLYMERASE6 (RDR6) and may function as a genome protection mechanism against excessively expressed genes. Whether all transcripts or just aberrant transcripts activate this protection mechanism is unclear. Consistent RNA silencing induced by a transgene with three direct repeats of the beta-glucuronidase (GUS) open reading frame (ORF) is associated with high levels of truncated, unpolyadenylated transcripts, probably from abortive transcription elongation. Truncated, unpolyadenylated transcripts from triple GUS ORF repeats were degraded in the wild type but accumulated in an rdr6 mutant, suggesting targeting for degradation by RDR6-mediated RNA silencing. A GUS transgene without a 3' transcription terminator produced unpolyadenylated readthrough mRNA and consistent RDR6-dependent RNA silencing. Both GUS triple repeats and terminator-less GUS transgenes silenced an expressed GUS transgene in trans in the wild type but not in the rdr6 mutant. Placing two 3' terminators in the GUS transgene 3' reduced mRNA 3' readthrough, decreased GUS-specific small interfering RNA accumulation, and enhanced GUS gene expression. Moreover, RDR6 was localized in the nucleus. We propose that improperly terminated, unpolyadenylated mRNA from transgene transcription is subject to RDR6-mediated RNA silencing, probably by acting as templates for the RNA polymerase, in Arabidopsis thaliana.

Figure 1.

Scheme of the GUS Constructs in the Binary Vector pOCA30. (A) The T-DNA region of the binary vector pOCA30 contains the CaMV 35S promoter with duplicated enhancers. GUS constructs were inserted behind the 35S promoter. LB, left border; RB, right border; E35S promoter, CaMV 35S promoter with duplicated enhancers; GUS, β-glucuronidase; 35S terminator, the CaMV 35S transcription terminator; nos terminator, the transcription terminator from the nos gene of A. tumefaciens; EcoRI/ClaI fragment, the ∼0.8-kb EcoRI-ClaI DNA fragment from pOCA30 located 3′ to the insertion site of the GUS constructs. (B) DNA sequences of the CaMV 35S terminator and the Agrobacterium nos gene terminator used in the GUS gene constructs.

Figure 2.

Effects of Transgene Copy Number on Transgene Expression. (A) GUS activities in the wild-type and sde1-1 transformants with one to five copies of a GUS transgene driven by the CaMV 35S promoter. The means and se of GUS activities were calculated from 8 to 30 T1 transformants for each copy number. GUS activities are expressed in units (nanomoles of 4-methylumbelliferone per minute per milligram of total soluble protein). (B) Ratios of GUS activities in the sde1-1 transformants over those in the wild-type transformants harboring the same copy numbers of the pGt_s transgene construct.

Figure 3.

GUS Activities and GUS mRNA Accumulation in Arabidopsis Wild-Type and sde1-1 Transformants. (A) GUS activities. Average GUS activity and sd for each construct calculated from 30 T1 wild-type or sde1-1 transformants containing a single-copy T-DNA insertion. GUS activities are expressed in units (nanomoles of 4-methylumbelliferone per minute per milligram of total soluble protein). According to Duncan's multiple range test (P = 0.05), means of GUS activities do not differ significantly if they are indicated with the same letters. (B) Accumulation of GUS transcripts. Total RNA was pooled from 9 to 10 randomly selected T1 transformants with a single-copy T-DNA insertion for each construct in the wild-type or sde1-1 background and probed with the full-length GUS gene fragment. The ethidium bromide stain of rRNA is shown for each lane to allow assessment of equal loading.

Figure 4.

Analysis of Total and Full-Length GUS Transcripts in T1 pGt_s and pGGGt_s Transformants in the sde1-1 Mutant Background. Total RNA was isolated from four randomly selected T1 transformants with a single-copy T-DNA insertion for each construct in the sde1-1 background and probed first with the GUS gene fragment (A). After stripping the GUS probe, the blot was rehybridized with the CaMV 35S terminator sequence (B). GUS activities for individual transformants analyzed are shown at the bottom of the blot. The ethidium bromide stain of rRNA is shown for each lane to allow assessment of equal loading.

Figure 5.

Analysis of Total and Polyadenylated GUS mRNA in T1 pGt_s, pGGGt_s, and pG Transformants. (A) Total GUS transcripts. Total RNA was pooled from 10 T1 transformants with a single-copy T-DNA insertion for each construct in the sde1-1 background and probed with the GUS gene fragment to determine the total GUS transcripts in the transformants. The ethidium bromide stain of rRNA is shown for each lane to allow assessment of equal loading. (B) Polyadenylated GUS transcripts. The total RNA used for analysis of total GUS transcripts shown in (A) was mixed with oligo(dT) cellulose. After washing, polyadenylated mRNA was eluted and probed with the GUS gene fragment. The polyadenylated mRNA was reprobed with a β-tubulin gene fragment (TUB8; At5g23860) after stripping of the first probe.

Figure 6.

GUS Activities and GUS mRNA Accumulation in Arabidopsis pGt_s Transformants with or without the pGGGt_s, pG, or pN Construct. F1 progeny in the silencing-competent SDE1/sde1 or silencing-deficient sde1/sde1 background were generated from crosses between a single-copy paternal wild-type or sde1 pGt_s transformant and a single-copy maternal sde1 pGGGt_s, pG, or pN transformant. F1 progeny were genotyped by PCR, and plants containing one or both constructs from their parental lines were identified. Means and se of GUS activities (A) and levels of GUS transcripts (B) were determined from 10 F1 progeny for each genotype containing only the paternal pGt_s construct (column 1 and lane 1), only the maternal pGGGt_s, pG, or pN construct (column 3 and lane 3), or both the paternal pGt_s construct and a corresponding maternal construct (column 2 and lane 2). GUS activities are expressed in units (nanomoles of 4-methylumbelliferone per minute per milligram of total soluble protein).

Figure 7.

Analysis of mRNA 3′ Readthrough in T1 Transformants Transformed with pGt_s, pGt_n, and pGt_st_n. (A) Detection of readthrough transcripts. Total RNA was isolated from T1 transformants for each construct in the wild-type (W) or sde1-1 (s) background and probed with the EcoRI-ClaI fragment corresponding to the region downstream of the GUS construct in the T-DNA part of the binary vector pOCA30 (see Figure 1A). (B) Polyadenylation status of the readthrough transcripts. Total RNA (10 μg for pGt_s and pGt_n and 1 μg for pGECt_s) from T1 sde1-1 transformants was separated and probed with the pOCA30 EcoRI-ClaI DNA fragment (top panel). Poly(A)⁺ mRNA was isolated from 20 μg of total RNA from the transformants. All isolated polyadenylated mRNA for pGt_s and pGt_n and one-tenth for pGECt_s was fractionated and probed with the pOCA30 EcoRI-ClaI fragment and reprobed with the β-tubulin gene fragment after stripping of the first probe (bottom panel).

Figure 8.

Accumulation of siRNA in T1 Transformants Transformed with the pGt_s, pGt_n, pGt_st_n, and pG Constructs. Total RNA was isolated from 10 randomly selected T1 transformants for each construct in the wild-type or the sde1-1 background and enriched for low molecular weight RNA. After fractionation on a polyacrylamide gel, the RNA was probed with the full-length GUS gene fragment. Equivalent loading of the samples was shown by detection of 5S rRNA in the lanes prior to blot transfer.

Figure 9.

Nuclear Localization of RDR6 in Arabidopsis Seedlings. (A) Detection of the RDR6-GFP in the nucleus of Arabidopsis trichome cells. The Arabidopsis rdr6-11 mutant plants were transformed with a 35S:RDR6-GFP or a 35S:GFP construct, and the seedlings of positive transformants were visualized by a confocal microscope. The RDR6-GFP fusion protein is predominantly localized in the nuclei of trichome cells. The low green fluorescence signals around the edges of cells containing the RDR6-GFP fusion protein may result from autofluorescence, since similar levels of signals were also observed in nontransgenic wild-type seedlings. The native GFP is localized in both the cytoplasm and the nuclei due to its small size. More than 10 T1 plants were investigated for each construct, and all showed the same fluorescence pattern. Bars = 100 μm. (B) Leaf morphology of wild-type, sde1-1, and sde1-1 transformants harboring the 35S:RDR6-GFP construct. The photograph was taken 5 weeks after germination. (C) Transcript levels of ARF4 in wild-type, sde1-1, and sde1-1 transformants harboring the 35S:RDR6-GFP construct.