Научная статья на тему 'Tas3 genes for small ta-siARF RNAs in plants belonging to subtribe Senecioninae: occurrence of prematurely terminated RNA precursors'

Tas3 genes for small ta-siARF RNAs in plants belonging to subtribe Senecioninae: occurrence of prematurely terminated RNA precursors Текст научной статьи по специальности «Биологические науки»

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Ключевые слова
MICRO RNA / TRANS-ACTING SIRNA / CDNA / PLANT / SUCCULENT / PHYLOGENY

Аннотация научной статьи по биологическим наукам, автор научной работы — Ozerova L. V., Krasnikova M. S., Troitsky A. V., Solovyev A. G., Morozov S. Y.

The various classes of plant 21to 24-nt siRNAs derive from long dsRNA precursors that are processed by the ribonuclease Dicer-like (DCL). The species of ta-siRNA were originally discovered in Arabidopsis thaliana. Four gene families have been identified in Arabidopsis that each produces a number of ta-siRNAs: TAS1, TAS2, TAS3 and TAS4. The TAS3 genes encode tasiR-ARF species which target the mRNA of three Auxin Response Factor (ARF) genes (ARF2, ARF3/ETT and ARF4) for subsequent degradation. The function of TAS3 precursor RNA is controlled by two miR390 target sites flanking tandem of ta-siARF sequences. In this paper, we have studied the presence of ta-siARF RNA genes in the representatives of subtribe Senecioninae. Senecioneae is the largest tribe of Asteraceae, comprised of ca. 150 genera and 3,000 species which include many common succulents of greenhouses. Approximately one-third of species are placed in genus Senecio, making it one of the largest genera of flowering plants. However, there was no information on the structure of TAS genes in these plants. We revealed that the TAS3 species (TAS3-Sen1) in Senecio representatives was actively transcribed, and its homologues are distributed among many Asteracea plants and found to be similar to Arabidopsis AtTAS3a gene. We revealed several prematurely terminated transcripts of TAS3-Sen1. Finding the alternative shortened transcripts of TAS3-Sen1 lacking the 3 ’ -terminal site cleaved by miR390 and retaining the 5 ’-terminal miR3 90 non-cleaved site suggested their using as decoys for the modulation of miR390 activity to regulate synthesis of ta-siARF RNAs in different Senecioninae species.

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Текст научной работы на тему «Tas3 genes for small ta-siARF RNAs in plants belonging to subtribe Senecioninae: occurrence of prematurely terminated RNA precursors»

ЭКСПЕРИМЕНТАЛЬНЫЕ СТАТЬИ

In a prospective cohort study, 15 HCC cases selected from a prospective cohort of 1,638 high-risk individuals on the basis of available plasma samples spanning the years before and after diagnosis. In this study, T1762/A1764 double mutation was detected in 8 of the 15 cases (53.3%) before cancer. It is suggested T1762/A1764 double mutation in plasma could be a valuable predictive biomarker for HCC development [24]. Shinkai et al., had found T1762/A1764 double mutation in 58 of 80 (73%) and 73 of 80 (91%) in patients without and with HCC, respectively [6]. This workers had also found A1896 mutation in 41 of 80 (51%) and 43 of 80 (54%) in patients without and with HCC, respectively [6]. Ito et al., had found T1762/A1764 double mutation in 31 of 40 (77.5%) and 36 of 40 (90%) in patients without and with HCC, respectively [20]. This workers had also found A1896 mutation in 26 of 40 (65%) and 25 of 40 (62.5%) in patients without and with HCC, respectively.

T1762/A1764 double mutation rate has been detected in 4 of 61 (6.55%) samples without HCC in our study. All of this data are also in agreement with our results, nevertheless our T1762/A1764 double mutation and A1896 mutation rates are also low (6.55%, 16.39% respectively).

Our low mutation rates may be due to our sequencing protocol. Automated sequencing protocol use probably will increase revealing of mutation rates.

In the literature the assosication between HBV mutations and HCC remains controversial because of conflicting data based on different populations. In a meta analysis, 43 studies evaluated a total of 11552 HBV-infected participants of whom 2801 had HCC. According to the study, A1896 mutation was not statistically associated with HCC risk. However, T1653 and T1762/A1764 double mutation are associated with the development of HCC with an increased risk of 2.76 and 3.79 fold, respectively. The authors suggest that these mutations alone and in combination may be predictive for HCC [3].

In conclusion, due to the statistically signicifant association between HBV mutations and HCC, underlying mechanisms are still unclear. However, the associated mutations might be reveal the HCC risks for people in a population and could be used in early detection of HcC. Our findings demonstrated that mutations might be related to HCC such as T1653 and T1762/A1764 double mutation are present in our region. Prospective clinical chord studies should be planned in the

future for better patient management to follow HCC thorough mutational examination of HBV.

acknowledgments

This study was supported by the University of Mersin research grants BAP-SBE FMA (EK) 2010-1 YL.

R E F E R E N C E S

1. Liang T.J., Hepatology. 2009; 49 (5): 13-21.

2. Fen Ji., Li Zhou., Sufang Ma. et al. J. Med. Virol. 2009; 81 (9): 1551-9.

3. Liu S., Zhang H, Gu C. et al. J. Natl. Cancer Inst. 2009; 101 (15): 1066-82.

4. Bosch F.X., Kibes J., Diaz M. et al. Gastroenterology. 2004; 127 (5, suppl. 1): S5-16.

5. Mitri M.S., Cassini K., Bernardi M., Eur. J. Cancer. 2010; 46 (12): 2178-86.

6. Shinkai N., Tanaka Y, Ito K. J. Clin. Microbiol. 2007: 45 (10): 3191-7.

7. Serin M.S., Bekiroglu E., Polat S. Mol. Genet. Microbiol. Virol. 2010; 25 (4): 178-82.

8. Hunt C.M., McGill J.M., AllenM.I., Hepatology. 2000; 31 (5): 103744.

9. Yuen M.F., Tanaka Y., Shinkai N. et al. Gut. 2008; 57 (1): 98-102.

10. Kao J.H., ChenP.J., LaiM.Y. Gastroenterology. 2003; 124 (2): 327-34.

11. Blackberg J., Kidd-LjunggrenK. J. Med. Virol. 1997; 71 (1): 18-23.

12. Sung J.J., Tsui S.K., Tse C.H. et al. J. Virol. 2008; 82 (7): 3604-11.

13. Lin C.L., Lin C.H., Chen W. et al. Liver Int. 2007; 27 (7): 983-8.

14. Chen C.H., Hung C.H., Lee C.M. et al. Gastroenterology. 2007; 133 (5): 1466-74.

15. Huang H.P., Hsu H.Y., Chen C.L. et al. Pediatr. Res. 2010; 67 (1): 90-4.

16. Bozdayi G., TurkyilmazA.K., Idilman K. et al. J. Med. Virol. 2005; 76 (4): 476-81.

17. Bartholomeusz A., Locarnini S. J. Med. Virol. 2006; 78 (suppl. 1): S52-5.

18. Bosch F.X., Kibes J., Díaz M. et al. Gastroenterology. 2004; 127 (5, suppl. 1): S5-16.

19. Yuh C.H., Chang Y.L., TingL.P., J. Virol. 1992; 66 (7): 4073-84.

20. Ito K., Tanaka Y., Orito E. et al. Clin. Infect. Dis. 2006; 42 (1): 1-7.

21. François G., Kew M., Van Damme P. et al. Vaccine. 2001; 19 (2829): 3799-815.

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Поступила 06.09.12

© КОЛЛЕКТИВ АВТОРОВ, 2013

L. V. Ozerova", M. S. Krasnikovab, A. V. Troitskyb, A. G. Solovyevb, and S. Y. MorozoV

tas3 genes for small ta-siarf rnas in plants belonging to

sUBTRIBE SENECIONINAE: occurrence of prematurely terminated RNA

precursors

a Tsytsin Main Botanical Garden, Russian Academy of Sciences, Botanicheskaya 4, 127276 Moscow, Russia;b A.N.Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow, 119992, Russia e-mail: [email protected]

Abstract

The various classes of plant 21- to 24-nt siRNAs derive from long dsRNA precursors that are processed by the ribonuclease Dicer-like (DCL). The species of ta-siRNA were originally discovered in Arabidopsis thaliana. Four gene families have been identified in Arabidopsis that each produces a number of ta-siRNAs: TAS1, TAS2, TAS3 and TAS4. The TAS3 genes encode tasiR-ARF species which target the mRNA of three Auxin Response Factor (ARF) genes (ARF2, ARF3/ETT and ARF4) for subsequent degradation. The function of TAS3 precursor RNA is controlled by two miR390 target sites flanking tandem of ta-siARF sequences. In this paper, we have studied the presence of ta-siARF RNA genes in the representatives of subtribe Senecioninae. Senecioneae is the largest tribe of Asteraceae, comprised of ca. 150 genera and 3,000 species which include many common succulents of greenhouses.

Approximately one-third of species are placed in genus Senecio, making it one of the largest genera of flowering plants. However, there was no information on the structure of TAS genes in these plants. We revealed that the TAS3 species (TAS3-Sen1) in Senecio representatives was actively transcribed, and its homologues are distributed among many Asteracea plants and found to be similar to Arabidopsis AtTAS3a gene. We revealed several prematurely terminated transcripts of TAS3-Sen1. Finding the alternative shortened transcripts of TAS3-Sen1 lacking the 3' -terminal site cleaved by miR390 and retaining the 5 '-terminal miR3 90 non-cleaved site suggested their using as decoys for the modulation of miR390 activity to regulate synthesis of ta-siARF RNAs in different Senecioninae species.

Keywords: micro RNA, trans-acting siRNA, cDNA, plant, succulent, phylogeny.

МОЛЕКУЛЯРНАЯ ГЕНЕТИКА, МИКРОБИОЛОГИЯ И ВИРУСОЛОГИЯ №2, 2013

Introduction

Each higher plant TAS gene so far identified produces a non-protein-coding transcript that gives rise to multiple ta-siRNAs. The species of ta-siRNA were originally discovered in Arabidopsis [24]. Four gene families have been identified in Arabidopsis that each produce a number of ta-siRNAs: TAS1, TAS2, TAS3 and TAS4. Within the TAS1 family, TA-S1a, TASlb and TASlc are very similar in sequence and all produce the siR255 ta-siRNA among others [2, 24]. One of the most important ta-siRNAs is the trans-acting short-interfering RNA targeting auxin response factor mRNA. Auxins are a class of signaling molecules that play a central role in plant development. The tasiR-ARF RNA directs the mRNA of three Auxin Response Factor (ARF) genes (ARF2, ARF3/ ETT and ARF4) for degradation [10, 12, 19, 24]. TasiR-ARF is derived from the TAS3 gene. While Arabidopsis contains several ta-siRNAs not found in other plants, tasiR-ARF is highly conserved in many flowering and lower land plants including mosses [2, 16]. This indicates that, like miRNAs, ta-siRNAs (and tasiR-ARF in particular) have been used to regulate gene expression in plants since before the separation between the seed plant and moss lineages [2, 24].

These ta-siRNAs are lined up one after the other in both sense and anti-sense orientations of RNA precursor. After transcription of the TAS gene, specific miRNAs pair with certain members of the Argonaute (AGO) protein family and bind to the single stranded RNA (ssRNA) at miRNA recognition sites [2, 6, 7, 23]. This specifies site specific cleavage of the primary TAS gene transcript at the beginning of the first ta-siRNA in the series and sets the phase for future processing by DCL с участием DCLs [2, 3, 5, 6, 7, 24]. TAS3 gene transcript contain two miR390 recognition sites. Nevertheless only second (3'-terminal) site is cleaved by AGO7 and a single miRNA-guided cleavage of the transcript is usually required for phased ta-siRNA biogenesis [2, 3, 23, 26]. At this point, Suppressor of Gene Silencing 3 (SGS3) protects the ssRNA from degradation. Next, RNA-dependent Poly-merase 6 (RDR6) produces a complementary strand, turning the transcript into double stranded RNA (dsRNA) until non-cleaved site of TAS3 transcript bound to AGO-miRNA complex [2, 23]. Dicer-like Protein 4 (DCL4) then cleaves the dsRNA in 21-nt increments to generate mature ta-siRNAs [2, 18, 23].

The TAS3 gene-mediated ta-siRNA pathway is a plant-specific miR390-dependent phenomenon conserved in all higher plants and in primitive land plants such as Physcomi-trella and other mosses [2, 16]. In Arabidopsis, miR390-guided cleavage occurs only at the 3' target site of the TAS3 precursor where canonical targeting rules include perfect complementarity between miRNA nucleotides 2-13 (from the 5' end) and the noncoding RNA target [2] (Fig. 1). Importantly, the 5' target site is resistant to cleavage but is important for processing of the tasiRNA precursor transcript. This characteristic feature is attributed to mismatches at nucleotides 9-11 from the 5' end of the miRNA (Fig. 1). These mismatches are highly conserved in the higher plant TAS3-primary transcripts [2, 24].

In this paper, we have studied the presence of tasiARF RNA genes in the representatives of subtribe Senecioninae. It was found that they encode such RNA precursors principally similar to those found in Arabidopsis. We used primers mimicking miR390 [15] and oligo (dT)-based oligonucle-otides to show that one of the TAS3 species (TAS3-Sen1) in Senecio representatives was actively transcribed and found to be similar to AtTAS3a gene of Arabidopsis thaliana. The data obtained may contribute to understanding of the structure and expression regulation of Senecio TAS3-like genes.

Materials and Methods

Plant material. Specimens for Senecio talinoides, Curio repens and Curio articulatus were taken from collections of the N.V. Tsytsin Main Botanical Garden of the Russian Academy of Sciences.

Analysis of Nucleic Acids. Genomic plant DNA was isolated from 200 mg of plant material by DNA extraction kit (Macherey-Nagel) according to the protocol of the manufacturer. TAS3 genes were amplified and sequences as described in [15]. Total RNA was isolated from Nicotiana benthamiana leaves with the Trizol reagent according to the manufacturer's instructions (Invitrogen). Digestion of any contaminating DNA was achieved by treatment of samples with RQI RNase-free DNase (Promega). Reverse transcription was performed with 1 [g of total RNA and oligo (dT)-primer t20-xho (attctcgaggccgaggcggc-cgacatgtttttttttttttttttttttttttv) using the RT system (Invitrogen) according to the protocol of the manufacturer. Primers for dicotyledonous plants were: forward primer: TAS-P (5'-GGTGCTATCCTATCT-GAGCTT-3') and mixture of reverse primers TAS-Mcaa (5'-AGCT-CAGGAGGGATAGCAA-3') and TAS-Maca (5'-AGCTCAGGAGG-GATAGACA-3'). For PCR, 25-35 cycles were used for amplification with a melting temperature of 95°C, an annealing temperature of 58°C and an extending temperature of 72°C, each for 30 seconds, followed by a final extension at 72°C for 3 min. PCR products were separated by electrophoresis of samples in 1.5% agarose gel and purified using the GFXTM PCR DNA and Gel Band Purification Kit (Amersham Biosciences). For cloning, the PCR-amplified DNA bands isolated from gel were ligated into pGEM-T (Promega). Cloned products were used as templates in sequencing reactions with the ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems). DNA sequences were deposited at the NCBI data bank, the accession numbers are JN692262, JN692261, JN692260 and JN692259. The TAS3 cDNA sequences were cut out of the vectors pGEM-T by digestion with HindHI and ligated into HindIII-digested binary vector pLH7000 containing the flanking 35S promoter and transcriptional terminator and provided by Dr L. Hausmann (Federal Centre for Breeding Research on Cultivated Plants, Germany). Agroinfiltration of Nicotiana benthamiana plants was carried out as described [29]. RNA was isolated 4 days after agroinfiltration of plant leaves (see above).

Computational sequence analysis. TAS3-like sequences identified in this paper and NCBI database were compared using multiple alignment tool at MAFFT (version 5.8). The phylogenetic tree was generated according MAFFT6 program (http://mafft.cbrc.jp/align-ment/server). Sequences were additionally analysed at http://blast. ncbi.nlm.nih.gov/BlastAlign.cgi by NCBI Blast.

Results and Discussion

Identification and molecular phylogeny of the most abundant TAS3-like species in representatives of family Asteraceae.

Previously, we described the new method for identification of plant ta-siRNA precursor genes based on PCR with oligodeoxyribonucleotide primers mimicking miR390. The method was found to be efficient for dicotyledonous plants, cycads, conifers and mosses [15, 16]. In this study, PCR-based approach was used as a tool to probe genomic DNA samples derived from three species of tribe Senecioneae: Senecio talinoides, Curio repens and Curio articulatus [4, 20]. PCR amplification of chromosomal DNA from these plants resulted in synthesis of one major band of270 bp (data not shown). Cloning and sequencing of the obtained DNA bands revealed that the amplified sequences are closely related (more than 90% identity) (data not shown). They contained putative ta-siARF site composed of two tandem copies of ARF-specific ta-siR-NAs and located between miR390 target sites corresponding to PCR primers (Fig. 1). This TAS3-like gene was sent by our group to GenBank and named as TAS3-Sen1 (GenBank accession number -JN692259).

Bioinformatic analysis of the putative TAS3-like sequences from Senecio talinoides, Curio repens and Curio articulatus by

ЭКСПЕРИМЕНТАЛЬНЫЕ СТАТЬИ

NCBI Blast revealed closely related sequences in other representatives of the family Asteraceae (data not shown and Fig. 2). Using available sequence databases, more distant TAS3-like sequences were identified also in flowering plants belonging to subclass Rosids, for which complete sequences of TAS3 cDNAs were revealed (see Materials and Methods; data not shown and Fig. 2). The phylogenetic tree based on comparisons of TAS3-like sequences demonstrated that all TAS3-Sen1 sequences ofAsteraceae form a monophyletic group (Fig. 2). On the other hand, the closest branching lineage in eudicots was represented by plants from subclass Asterids, namely, orders Lamiales (Antirrhinum majus) and Solanales (Nico-tiana tabacum and Solanum tuberosum) (Fig. 2). These observations are in agreement with the branching order of flowering plant evolution trees based on protein-coding genes and published previously [8].

The TAS3-Sen1 family like other TAS3 loci is characterized by the dual miR390 binding sites, which are functionally required by TAS3 mRNA to define the phasing register for tasiRNA production [2]. We conducted an exhaustive Blast search using TAS3-Sen1 genes as queries to find additional putative Senecio TAS3 genes in the NCBI databases. The Senecio madagascariensis cDNA contig (GenBank accession number SRR006592) includes the tasiARF region and flanking 5'-terminal miR390 complementary site. However, this cDNA contig was shorter than the full-length TAS3 mRNA precursor and 3'-terminal miR390 site could not be found. Because of this uncertainty, we used sequence of contig SRR006592 only to reconstruct the authentic sequence (not derived from PCR primer mimicking miR390) of 5'-terminal miR390 complementary site in TAS3-Sen1 (Fig. 3A). The tasiARF sequences in TAS3-Sen1 were coaligned with the phases D7(+) and D8(+) defined by the 3' miR390 processing site in their putative precursors as observed in angiosperms (Figs. 1 and 3A) [, 14]. TAS3-Sen1 loci in S.articulatus and S.madagascariensis both had identical region including two closely related 21-nt sequences adjacent to one another, of which one was identical to Arabidopsis tasiARF at phase 5'D8(+) and the other contained three mismatches with respect to the Arabidopsis tasiARF at position 5'D7(+) (Fig. 3B). To verify whether the interaction between Senecio miR390 and the TAS3-Sen1 precursor is similar to what was observed in Arabidopsis, sequence comparison was performed to predict cleavage events at the 5' and 3' target sites on the TAS3 precursor. Canonical targeting rule including nearly perfect complementarity between miR390 nucleotides 2-13 (from the 5' end) and the TAS3 RNA 3'-terminal miR390 target was confirmed (Fig. 3). These results are also consistent with the previous suggestion that miR390 is unable to guide for a cleavage because of mismatches between 9 nt and 11 nt from the 5' end of the miR390 [2, 14] (Fig. 3). Thus our original data on sequencing of TAS3 genes

Fig. 1. Organization of TAS3 tasiRNA precursors according to compilations of the review article [2]. The miR390-guided cleavage site is indicated by the arrow. The tasiRNA region is indicated by brackets. The region of ta-siARF RNAs is shaded. Targeting specificity of

other ta-siRNAs is unknown.

Senecio articulatis

- Artemisia annua

Helianthus annuus

Lactuca sativa

-Barnadesia spinosa

Antirrhinum majus -Nicotiana tabacum

Solanum tuberosum

Arabidopsis thaliana

Cajanus cajan — Glycine max

-Vigna unguiculata

- Cicer arietinum

Liriodendron tulipifera

- Arachis hypogaea

-Prunus pérsica

Malus x domestica

Theobroma cacao

- Citrullus lanatus

Citrus sinensis

Ricinus communis

Manihot esculenta

Populus trochocarps -Vitis vinifera

-Carica papaya

Fig. 2. The minimal evolution phylogenetic tree based on analysis of the aligned TAS3 genes from some flowering plants. This tree was generated according MAFFT6 program (http://mafft.cbrc.jp/alignment/server).

in subtribe Senecioninae indicate their close relationship to the analogous genes of other Asteraceae plants.

Cloning and sequencing of the TAS3-Sen1 cDNA species suggests a new strategy for the involvement of non-cleavable miR390 target site in control of ta-siARF formation.

Processing and polyadenylation of plant TAS3 gene precursors has not been studied in detail excepting splicing of Arabidopsis thaliana gene TAS3a (AT3G17185) [1]. We have studied the polyadenylation sites of TAS3-Sen1 genes in three distinct Senecio species by sequence analysis of multiple cDNA clones. Figure 4 shows the nucleotide sequence of the 3' tail regions following ta-siARF tandem region of TAS3-Sen1 gene in three plant species and the polyadenylation sites of the analyzed cDNA clones. Since the poly A tails of all the cDNA

МОЛЕКУЛЯРНАЯ ГЕНЕТИКА, МИКРОБИОЛОГИЯ И ВИРУСОЛОГИЯ №2, 2013

clones analyzed do not occur in the regions corresponding to a long AT rich area in the genomic sequence we are confident that correct priming occurred with the oligo (dT) during the cDNA preparation, and that the cDNA clones represent true differences in the poly A addition site on the mRNA's.

The two putative full-length St. 41 and Ca. 90 cDNA clones of different plant genera had different poly A sites. Eight out of 12 of the cDNA clones for C. repens and 7 out of 13 clones for C. articulatus had the poly A tail added in the alternative middle position. The sites where polyadenylation occurs in the mRNA are spread over quite large distances (Fig. 4 and data not shown). The two polyadenylation sites detected for C. articulatus are 216 nucleotides apart, those for S. talinoides are separated by 269 nucleotides. However, the three sites were detected for C. repens (Fig. 4). A high degree of variability in the positioning of the poly A tails therefore occurs between these highly related genes in three plant species. Importantly, these data indicate that transcription of TAS3 gene can be prematurely terminated upstream of 3' miR390 targeting site. This phenomenon suggests that shortened TAS3 transcripts are not processed by DCL but still able bind miR390 at the 5' non-cleavable targeting site.

Also illustrated in Figure 4 is sequence in St.41 which represents the G/A cluster positioned immediately upstream of the poly A addition site. The potential importance of ho-mopurine-homopyrimidine sequences in biological processes is suggested by the ubiquity and non-random distribution of sequences such as (CT)n'(GA)n within the genomes of many organisms [22]. GA repeats have been found in promoter regions of many genes, at recombination hotspots and at replication origins [22]. Homopurine-homopyrimidine sequences could influence transcription through several possible mechanisms. It has been suggested that the tendency of these sequences to adopt alternative DNA conformations such as H-DNA could be used to absorb negative supercoils generated in the wake of RNA polymerase, facilitating DNA unwinding within the transcription bubble [17, 22]. Ho-mopurin-homopyrimidine sequences could also influence chromatin structure by serving as binding sites for regulatory proteins or important chromatin assembly factors. This possibility has been particularly well studied in the case of the Drosophila hsp26 gene [17]. GAGA binding factor is believed to affect severalsteps in the process of transcription particularly causing RNA polymerase II pausing in many genes during transcription [17]. This phenomenon may be related to premature termination of gene transcription.

To experimentally test the possibility that homopurine-homopyrimidine sequences may influence premature termination of TAS3 gene transcription, we inserted the clone St.41 into agrobacterial vector for transient expression. Cells of N. benthamiana leaves were agroinfiltrated with this construct where transient transcription was driven by 35S promoter. At 4 days post infiltration total RNA was isolated. Three out of 9 clones for transiently expressed C. articulatus TAS3-Sen1 had the poly (A) tail added in the middle position thus showing premature termination of transcription (similar to clone St.33 - Fig. 4). The rest of the clones were terminated at the position typical for cauliflower mosaic virus 35S terminator (data not shown). These experiments allow us to conclude that G/A cluster may indeed induce premature termination of TAS3 transcription. However most of the transcripts are continued through G/A block that could indicate differences between Curio and Nicotiana systems.

It was shown recently that alternative polyadenylation would generate two transcripts from the Arabidopsis TAS3a locus (At3g17185), only one of which contains both miR390 binding sites flanking ta-siARF region and would be subject to miR390-mediated processing [11, 25]. The shortened tran-

script contains only miR390 binding site which is imperfect and cannot be cleaved by miR390. Such phenomenon allows for more layers of regulation linked to miRNA and ta-siRNA. For example, the non-protein coding gene IPS1 (INDUCED BY PHOSPHATE STARVATION 1) from Arabidopsis thaliana contains a motif with sequence complementarity to the phosphate (Pi) starvation-induced miRNA miR399, but the pairing is interrupted by a mismatched loop at the expected miRNA cleavage site [9]. As a result IPS1 RNA is not cleaved but instead sequesters miR399. Thus, IPS1 overexpression results in increased accumulation of the miR399 target PHO2 mRNA and, concomitantly, in reduced shoot Pi content. Moreover it was found that over-expression of several additional miRNA decoys with non-cleavable miRNA sites result in a certain degree of specific inactivation of individual members of different miRNA families [13]. In principle, the application of alternative transcripts of Senecio TAS3 genes as decoys for the modulation of miR390 activity could be extended to regulate synthesis of ta-siARF RNAs in different Senecio species by permitting varying degrees of miR390 inactivation by their own functional TAS3 target genes.

ACKNOWLEDGEMENTS

This work was supported by Grant of Ministry and Education of Russian Federation № 16.518.11.7076.

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Поступила 1.07.12

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