Nucleic Acids Research

Plant growth conditions

Homozygous, silenced and hemizygous, expressing T17 plants (transgenic N.tabacum cv SR1) (24) and wild-type N.tabacum SR1 plants were germinated in soil and grown in a greenhouse (25°C, 70 µmol/m²/s light intensity, 14 h light/10 h dark period).

RNA isolation and analysis

RNA was isolated from leaf tissue as described by Jones et al. (25). Poly(A)⁺ and poly(A)^- RNA was isolated with the Oligotex mRNA mini kit (Qiagen Hilden, Germany). Northern blot analysis was performed with total RNA (10 µg for silenced T17 plants and 2.5 µg for expressing T17 plants), poly(A)^- RNA (10 µg) and poly(A)⁺ RNA (0.15 µg). The RNA concentration was determined spectrophotometrically. RNA was separated in a 1.5% agarose-formaldehyde gel according to Sambrook et al. (26). After electrophoresis, the integrity of the RNA was checked by ethidiumbromide staining, and the gels were blotted onto Genescreen membranes (NEN Research Products, Dupont Wilmington, DE) by capillary transfer in 10× SSC and the RNA was subsequently crosslinked in a UV crosslinker (GS gene linker, UV chamber, Biorad Hercules, CA) according to the manufacturer's instructions. Radiolabelled DNA probes were generated by the Rediprime DNA labelling system (Amersham, UK) using DNA fragments corresponding to the gn1 cDNA, the 35S promoter, intron 1 and 2 and the 3[prime]-end of gn1 genomic sequence as template. Membranes were hybridised for 16 h at 65°C in 500 mM phosphate buffer, pH 7.2, 1 mM EDTA, 7% SDS and 1% BSA. Membranes were washed three times in 100 mM phosphate buffer, pH 7.2, 1 mM EDTA and 1% SDS at 65°C. Signals were visualised with a PhosphorImager (Molecular Dynamics Sunnyvale, CA) using ImageQuant software.

Cloning and characterisation of gn1 RNA species

The Marathon cDNA Amplification kit from Clontech (Palo Alto, CA) was used to characterise the 5[prime]-ends of the RNAs from region 3. Briefly, poly (A)⁺ RNA was isolated from silenced T17 plants. First strand cDNA was primed with a modified lock-docking oligo-dT primer. Second strand synthesis was performed according to the method of Gubler and Hoffmann (27) with a cocktail of Escherichia coli DNA polymerase I, RNase H and E.coli DNA ligase. After making the ends blunt with T4 DNA polymerase, the Marathon cDNA adaptor was ligated to the double-stranded cDNA with T4 DNA ligase. After adaptor ligation, the gn1-specific sequences were amplified by PCR using the AP1 primer complementary to the ligated adaptor and the gn1-specific primer GE5 corresponding to nucleotides 3544-3566 (numbering according to sequence M38281 of the EMBL database).

The 3[prime]-ends of the proximal gn1 RNA species were characterised by the RNA ligation-mediated rapid amplification of cDNA ends (RLM-RACE) method according to Liu and Gorovsky (28). In short, total RNA was isolated from expressing and silenced T17 plants and an arbitrary DNA primer (KL29) was ligated to the 3[prime]-OH end of the RNAs with T4 RNA ligase. Subsequently, first strand cDNA synthesis was primed with primer KL28, which is complementary to KL29. The 3[prime]-ends of the proximal product were amplified by PCR using the first strand cDNA as a template with KL28 and a gn1-specific primer GE8, located in exon 2 at position 2593-2617 (numbering according to sequence M38281 of the EMBL database).

PCR fragments were cloned (pGEM-T Easy vector system; Promega Madison, WI) and positive clones were identified by colony hybridisation as described by Sambrook et al. (26) using a DNA probe corresponding to the 3[prime] part (distal fragments) or the 5[prime] part (proximal fragments) of the gn1 cDNA. Nucleotide sequences were determined using automated dideoxy-sequencing systems (A.L.F. DNA Sequencer, Pharmacia Uppsala, Sweden).

Primer extension analysis

Primer extension analysis was performed by using the Primer Extension System-AMV Reverse Transcriptase (Promega). In short, primer GE10 (position 1711-1746, numbering according to sequence M38281 of the EMBL database) was labelled with [[gamma]-³²P]ATP, annealed to the total RNA samples (50 µg) at 65°C and primer extension was carried out with AMV reverse transcriptase at 42°C for 30 min. The DNA marker ([phiv]X174 DNA digested with Hinf I) was generated by end-labelling with [[gamma]-³³P]ATP. A sequence ladder was used to determine the exact length of the bands. The samples were separated on a 17 cm 8% polyacrylamide sequencing gel at 250 V for 3 h. Relative band intensities were quantified using a PhosphorImager (Molecular Dynamics) and ImageQuant software.

Three additional silencing-related gn1 RNA species are present in silenced T17 plants

To gain an insight into the molecular details of silencing-related turnover of gn1 mRNA, a search for RNA degradation products related to silencing was initiated. Jacobs et al. (7) showed that total RNA of homozygous, silenced T17 plants contains, besides the full-length gn1 RNA transcript, additional RNAs. These RNAs migrate on northern blots in three different regions and are hardly detectable in samples of hemizygous, expressing T17 plants (Fig. 1B). RNA species in region 1 migrated slower than the full-length gn1 messenger, whereas RNA species in regions 3 and 4 migrated faster (Fig. 1B). A difference between the relative intensities of the signals in the four regions is regularly observed when RNA preparations from different silenced plants, grown at different times, are used, indicating that the relative abundance of the corresponding RNAs varies. This most probably reflects variations in the intensity of the gene-silencing.

A    B, C, D

Figure 1. Northern blot analysis of gn1-related RNA species. (A) Schematic representation of the full-length gn1 RNA, and the different probes [gn1 cDNA, 5[prime] part (5[prime] probe) and 3[prime] part (3[prime] probe) of the gn1 cDNA] used for hybridisation. (B) Total RNA isolated from hemizygous, expressing T17 plants (he), homozygous, silenced T17 plants (ho) and wild-type SR1 plants (SR1). Northern blots were hybridised with a gn1 cDNA probe. (C) Total RNA, poly(A)⁺ and poly(A)^- RNA isolated from silenced T17 plants. Northern blots were hybridised with a gn1 cDNA probe. (D) Northern blots containing poly(A)⁺ and poly(A)^- RNA from silenced T17 plants hybridised with probes corresponding to the 5[prime] region (5[prime] probe) and the 3[prime] region (3[prime] probe) of the gn1 cDNA sequence, respectively. Arrows indicate the different regions that hybridise to the different probes. total, total RNA; A⁺, poly(A)⁺ RNA; A^-, poly(A)^- RNA.

The novel silencing-related gn1 RNA species result from at least two separate processing events

The gn1 RNA species from region 1 were ~600 nucleotides longer than the full-length gn1 messenger and co-purified with the poly(A)⁺ RNA fraction, indicating that they contain a poly(A) tail (Fig. 1C). We investigated whether additional transgene-derived sequences, besides those present in the mature gn1 mRNA sequence, are present in these large RNA species. Northern blots of leaf tissue from expressing and silenced T17 plants were hybridised to probes corresponding to the 35S promoter, intron 1 and 2 of the gn1 gene and gn1 genomic sequences located downstream of the published 3[prime]-end formation site of the gn1 mRNA (29). The 35S promoter probe did not hybridise with any of the additional gn1 RNA species. Surprisingly, two new RNA species were identified with the 35S promoter probe both in expressing and silenced T17 plants, the abundance of which did not appear to be silencing-related (data not shown). Hybridisation with the intron and 3[prime]-gene probes failed to generate a signal (data not shown), suggesting that the large gn1 RNAs in region 1 do not contain either of the two introns or sequences downstream of the reported polyadenylation site of the gn1 gene (29). Thus, it appears that the additional 600 nucleotides present in the larger-than-mature gn1 transcripts originate either from a silencing-related sequence duplication, or from a long-range splicing event.

The RNA species from region 4 were more abundant than those from region 3 in total RNA preparations of silenced plants. Furthermore, RNA species in region 3 were more readily apparent in the poly(A)⁺ RNA fraction, whereas RNA species in region 4 were only detected in the total RNA and poly(A)^- RNA fractions (Fig. 1C). This indicated that RNA species from region 3 contain a poly(A) tail whereas those of region 4 do not. To investigate to which part of the mature gn1 mRNA the RNA species of regions 3 and 4 correspond, northern blots containing poly(A)⁺ and poly(A)^- RNA from silenced T17 plants were probed with the 5[prime] and the 3[prime] part of the gn1 cDNA. Figure 1D shows that both the 5[prime] and the 3[prime] probe detected the mature, polyadenylated gn1 transcripts. In addition, the 5[prime] probe hybridised to gn1 RNA species in region 4 that are poly(A)^- and the 3[prime] probe hybridised to polyadenylated gn1 RNAs in region 3. This implies that gn1 RNA species from region 4 correspond to the proximal end of gn1 messenger and that gn1 RNA species from region 3 correspond to the polyadenylated, distal end of the gn1 messenger.

Mapping of the 5[prime]-ends of full-length and proximal RNA products

The observation that the RNAs in region 4 appeared as a small smear rather than as a discrete product, suggested that these RNAs were heterogeneous in size. To determine the exact 5[prime]-ends of RNAs containing proximal sequences of the gn1 transgene, primer extensions were performed with a primer in exon 1 of the gn1 transgene (Fig. 2). Two primer extension products, 125 and 103 nucleotides, were generated, both in expressing and in silenced T17 plants. In both expressing and silenced T17 plants the product of 125 nucleotides gave a 1.3-fold stronger signal than the product of 103 nucleotides. No extension products were generated with mRNA of SR1 tobacco plants indicating that the two products obtained are the result of a primer extension reaction on the transgene gn1 mRNA rather than on an endogenous glucanase mRNA. The product of 125 nucleotides extended exactly up to the expected transcription start site of the gn1 transgene, indicating that this is also the predominant transcription start site in the transgenic T17 plants. The presence of the same additional product, both in expressing and in silenced T17 plants indicates that the 35S promoter has two transcription start sites in the transgenic T17 tobacco line. The results demonstrate that no extensive degradation of the gn1 mRNA occurs at the 5[prime]-end, most likely due to the presence of the 5[prime] cap.

Figure 2. Primer extension analysis of full-length and 3[prime] truncated gn1 transcripts. The 5[prime]-ends of the full-length and the proximal RNAs were identified by primer extension. Primer GE10 was end-labelled and annealed to total RNA of hemizygous, expressing (he) and homozygous, silenced (ho) T17 plants and wild-type (SR1) tobacco plants. A sample without RNA was used as a control (C). The primer extension products are indicated by an arrow. The size (nucleotides) of a DNA marker is indicated at the left (M).

Proximal gn1 RNA fragments contain truncated 3[prime]-ends

Since the 5[prime]-end appears relatively fixed, the size heterogeneity of proximal (region 4) RNAs must reside in their 3[prime]-ends. To determine whether this is the case, the 3[prime]-ends of proximal gn1 fragments were identified. For this purpose, the 3[prime] parts of these RNAs were cloned using an RLM-RACE technique (28,30). In total, 26 clones from RNA of silenced T17 plants and 54 clones from RNA of expressing T17 plants were obtained. None of these clones corresponded to endogenous [beta]-1,3-glucanase transcripts.

For the expressing T17 plants, 80% of the clones (44) corresponded to the full-length gn1 messenger (Fig. 3). In contrast, no clones corresponding to the full-length gn1 messenger were obtained from silenced T17 plants. Figure 3 shows that all 26 clones of silenced T17 plants and 10 of 54 expressing T17 plants corresponded to gn1 RNA species lacking parts of the 3[prime]-end of the gn1 messenger. For silenced T17 plants, the 3[prime]-ends of the clones were situated between nucleotide 401 (numbering relative to the transcription start site) and nucleotide 616; in expressing T17 plants, the end points ranged from nucleotide 407 to 802. This result is consistent with a progressive degradation of the gn1 messenger in a 3[prime] to 5[prime] direction. Sequence analysis provided no indication for sequence aberrancies in the proximal gn1 RNAs indicating that their occurrence was not related to unfaithful transcription and that silencing-related RNA degradation is not due to abnormalities in the mRNA sequence (data not shown).

Figure 3. Characterisation of the 3[prime] truncated gn1 transcripts in silenced and expressing T17 plants. The 3[prime]-ends of the 3[prime] truncated gn1 transcripts were mapped through cloning using the RLM-RACE technique. The gn1 transcript is schematically represented and the numbers indicate the position in the cDNA sequence. The position of the introns is indicated (introns are not drawn in scale). The two cDNA clones containing intron 2 are indicated by a plus (+), whereas the seven cDNA clones which terminate at the exon 2/intron 2 boundary are indicated by an asterisks (*). The cDNA clones are represented by lines. The thick arrow indicates the 44 cDNA clones representing full-length gn1 RNA species. The gn1 gene-specific primer (GE8) used in the procedure is indicated by an arrowhead.

The sequence of four clones obtained from silenced T17 plants and of three clones obtained from expressing T17 plants terminated exactly at the last nucleotide of exon 2 of the gn1 transcript. Furthermore, two clones were obtained from silenced T17 plants representing gn1 RNAs that contain intron 2 and at the same time are truncated at the 3[prime]-end. The presence of these two types of clones indicates that splicing of the transgene gn1 transcript may be not efficient in the transgenic T17 plants.

The fact that exclusively 3[prime] truncated cDNAs were obtained from silenced plants, whereas most clones from expressing plants corresponded to the full-length gn1 mRNA indicates that the proximal, truncated RNAs are much more abundant in silenced T17 plants than in expressing T17 plants. This is also consistent with the northern blot analysis (Fig. 1B).

Distal gn1 RNA fragments are truncated at the 5[prime]-end

The truncated gn1 RNAs from silenced T17 plants in region 3 on northern blot corresponded to a distal region of the gn1 mRNA and contained a poly(A) tail. To determine whether truncated, polyadenylated gn1 RNAs contained any sequence aberrancies and where these gn1 RNA species terminate, poly(A)⁺ gn1 RNAs were cloned from silenced plants using the Marathon cDNA Amplification technique (Clontech) and their 5[prime]-termini were determined. Analysis of the 16 clones obtained gave no indication for the occurrence of sequence deviations compared with the published gn1 sequence (data not shown). Figure 4 reveals that the 16 clones from silenced T17 plants represent gn1 RNAs that lack increasing parts of the 5[prime]-end of the gn1 mRNA. The longest clone terminates at nucleotide 701 and the shortest clone at nucleotide 1013 (numbering relative to the predicted transcription start site). Thus, the 5[prime]-termini are scattered over a range of 300 nucleotides. Consistent with this observation, primer extension analyses did not yield any discrete extension products (data not shown). These results are in agreement with the idea that the gn1-derived distal RNAs are the result of on-going degradation by 5[prime] to 3[prime] exonucleases.

Figure 4. Characterisation of the 5[prime] truncated gn1 transcripts in silenced T17 plants. The 5[prime]-ends of the 5[prime] truncated gn1 transcripts were mapped through cloning of polyadenylated gn1 RNA species of silenced T17 plants. The gn1 mRNA transcript is schematically represented and the numbers indicate the position in the mRNA sequence. The position of the introns is indicated (introns are not drawn in scale). The cDNA clones are represented by lines. The gn1 gene-specific primer (GE5) used in the procedure is indicated by an arrow.

DISCUSSION

The present study focuses on the characterisation of gn1 RNAs specifically found in silenced T17 plants. It is shown that silencing of the [beta]-1,3-glucanase genes positively correlates with the accumulation of additional gn1 RNA species in three size classes which result from at least two processing events. The longer-than-full-length gn1 RNAs are polyadenylated and do not contain intronic or surrounding genomic sequences of the gn1 transgene. The RNA sequences that makes these gn1 RNAs longer-than-full-length also seems not to reside within the region spanned by the primers used for cDNA cloning since no cDNA clones that could correspond to these longer-than-full-length RNAs were detected. We conclude that these longer-than-full-length gn1 RNAs must originate from a silencing-related sequence duplication, or from a long-range splicing event.

The less-than-full-length gn1 RNA species correspond to the 5[prime] part (proximal fragments) and to the 3[prime] part (distal fragments) of the gn1 mRNA, and are most probably silencing-related gn1 degradation intermediates which result from an endonucleolytic cleavage followed by exonuclease digestion. Less-than-full-length RNA species have also been described for co-suppression of polygalacturonase and ACC synthase genes and for homology dependent resistance to TEV (13,14,22,23). However, this is the first report describing the cloning and sequence analysis of less-than-full-length RNA species implicated in silencing-related mRNA turnover.

The proximal and distal gn1 RNA species were further analysed to understand whether these could be relatively stable intermediates of an on-going silencing-related RNA degradation mechanism. In the case of T17 plants expressing gn1, >80% of the proximal clones represented full-length, polyadenylated gn1 transcripts. In contrast, all of the proximal cDNA clones from silenced T17 plants were truncated at the 3[prime]-end. The absence of clones representing full-length RNA species in silenced T17 plants is consistent with the substantial (20-40-fold) reduction in the steady-state level of full-length gn1 messengers in silenced compared with expressing T17 plants (30).

The isolation of two cDNA clones from silenced T17 plants that contain the intron 2 sequence, and at the same time are truncated at the 3[prime]-end could indicate that the processing of pre-gn1-RNAs in the nucleus is disturbed in silenced plants and that the presence of intron sequences could `mark' these RNA molecules for degradation in the cytoplasm. However, such aberrant splicing does not seem essential for silencing, since most of the cDNA clones obtained from silenced plants correspond to RNAs that were normally spliced over intron 2.

Importantly, cDNA cloning and sequence analysis did show that truncated proximal gn1 RNAs are present in expressing T17 plants. However, both northern blot and sequence analysis of a large number of cDNA clones indicated that such products are much more abundant in silenced T17 plants. In principle, these products could arise from premature termination of transcription. However, nuclear-runoff transcription has previously shown that transcription proceeds normally over the entire gn1 coding sequence (31). Therefore, we prefer the interpretation that the truncated, proximal RNA species represent intermediates of an on-going RNA degradation mechanism. It is possible that the identified gn1 degradation intermediates are relatively stable due to the protection from RNases by interacting proteins or because they are trapped in the translation complex as described for other silencing-related RNA degradation intermediates (13,14,32).

The observation of two discrete 5[prime]-ends, that correspond to two transcription start sites, in the proximal region of gn1 transcripts indicates that no 5[prime] to 3[prime] exonuclease activity is occurring at the 5[prime]-end of the gn1 transcript. Similarly, the presence of a poly(A) tail at the truncated distal gn1 products suggests that these RNAs are not degraded from their 3[prime]-ends. Together, these results imply that the mature gn1 mRNA is not degraded from the 5[prime] or 3[prime]-termini, but that RNA degradation is initiated via an endonucleolytic cleavage. The range of 3[prime] and 5[prime]-end points could be interpreted in several ways: one possibility is that endonucleolytic cleavage occurs at one specific internal site and is immediately followed by 5[prime] to 3[prime] and 3[prime] to 5[prime] exonuclease activity. Alternatively, endonucleolytic cleavage may occur at many relatively random internal sites of the gn1 mRNA, followed by both 5[prime] to 3[prime] and 3[prime] to 5[prime] exonuclease activity or followed by either one of the exonuclease activities alone. Thus, silencing-related gn1 mRNA degradation in transgenic tobacco appears not to involve decapping and deadenylation, steps that are generally assumed to be important for normal mRNA decay (16-19,33). A similar scenario was suggested previously for silencing-related degradation of chsA mRNA in petunia (5) and for silencing-independent degradation of rbcS SRS4 RNAs (19).

The observation that the proximal and distal gn1 RNA degradation products are much more abundant in silenced T17 plants than in expressing T17 plants, could indicate that these RNAs are more stable in silenced plants. More likely, however, these products are a direct result of the silencing-related increased turn-over of gn1 RNAs (7). The abundance of any given mRNA species is primarily determined by its rate of synthesis and its rate of decay. As the overall gn1 transcription rate in silenced and expressing T17 plants is similar (31), and the truncated gn1 RNA species are most likely not more stable in silenced T17 plants, the only way to explain their higher levels would be to assume that in silenced T17 plants a larger fraction of gn1 RNAs is shuttled into a pathway that specifically generates these truncated RNAs. Therefore, we suggest that various RNA degradation pathways are active in tobacco cells at the same time, and that during silencing, the partitioning of gn1 RNAs over these pathways is shifted in favour of a more active, silencing-related (not necessarily silencing-specific) pathway. This would be in agreement with the results obtained by Tanzer et al. (14), who propose distinctive silencing-related and a silencing-independent decay mechanism for TEV messengers. It remains to be determined to what extent such a silencing-related RNA degradation pathway operating in silenced T17 plants is active in non-silenced plants. Some level of activity in non-silenced plants is expected as this would explain why some of the truncated products are also present in non-silenced T17 plants expressing the [beta]-1,3-glucanase genes at normal levels.

ACKNOWLEDGEMENTS

We thank Raimundo Villarroel for sequence analysis. This work was supported by an EC grant in the context of the EC Network `Control of gene expression and silencing in transgenic plants'.

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*To whom correspondence should be addressed. Tel: +32 9 2645170; Fax: +32 9 2645349; Email: mamon@gengenp.rug.ac.be
⁺Present address: Wellcome/CRC Institute, Tennis Court Road, Cambridge CB2 1QR, UK

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