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RESEARCH COMMUNICATION
1 Department of Plant Sciences, Weizmann Institute of Science, Rehovot 76100, Israel; 2 Department of Life Sciences, Ben Gurion University, Beer Sheva 84105, Israel; 3 Department of Biochemistry, New York University School of Medicine, New York, New York 10016, USA
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Abstract |
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[Keywords: Thiamine pyrophosphate; riboswitch; alternative splicing; higher eukaryotes; metabolite sensing]]
Received June 5, 2007; revised version accepted September 24, 2007.
; Nudler and Mironov 2004; Coppins et al. 2007). Notably, riboswitches exert their functions without the need for protein cofactors (Mironov et al. 2002; Winkler et al. 2002). In most cases, they act in feedback regulation mechanisms; once the level of an end product in a metabolic pathway rises, riboswitch binding occurs, triggering a repression of gene expression in the same pathway. The substrate specificity of riboswitches is extremely high, allowing them to perform their activity amid the presence of numerous related compounds (Sudarsan et al. 2005). In prokaryotes, genetic control mediated by riboswitches is a prevalent phenomenon, and the dozen riboswitches identified to date regulate >3% of all bacterial genes (Nudler 2006).
Thiamin pyrophosphate (TPP) is a coenzyme derived from vitamin B1 (thiamin) that is synthesized by bacteria, fungi, and plants, and serves as a dietary requirement for humans and other animals. It is an obligatory cofactor that plays an important role in amino acid and carbohydrate metabolism; thus, fine control of its levels is essential for the viability of all living cells. TPP-binding riboswitches, first identified in Bacillus subtilis and Escherichia coli (Mironov et al. 2002; Winkler et al. 2002), exist in the genomes of species belonging to most bacterial phyla (Rodionov et al. 2002). Binding of TPP by the riboswitch down-regulates expression of thiamin biosynthesis genes by inducing either the formation of a transcription terminator hairpin or the formation of a Shine-Dalgarno sequester hairpin (Mironov et al. 2002; Rodionov et al. 2002; Winkler et al. 2002). Much less is known about riboswitch-mediated gene control in eukaryotic organisms, and TPP-binding riboswitches are the only ones identified to date (Kubodera et al. 2003; Sudarsan et al. 2003). Very recently, Cheah et al. (2007) demonstrated that a TPP riboswitch controls expression of the THI4 and NMT1 genes in Neurospora crassa by directing the splicing of an intron located in the 5' untranslated region (UTR). Intron retention results in the appearance of upstream and out-of-frame initiation codons, whereas intron splicing generates a complete and correct open reading frame. While the prokaryotic and fungi riboswitches are located in the 5' UTR, a plant TPP riboswitch located in the 3' UTR of the Arabidopsis THIAMINE C SYNTHASE gene (AtTHIC) was recently identified (Sudarsan et al. 2003). This difference in location suggests a unique mode of action for the plant riboswitch. Yet, the mechanisms mediated by riboswitch activity in higher eukaryotes, particularly in the plant kingdom, remain to be elucidated. Here we propose a unique prevalent model for TPP riboswitch-controlled gene expression in all flowering plants, according to which TPP binding to THIC pre-mRNA engenders alternative splicing that leads to the generation of an unstable transcript, which in turn lowers TPP biosynthesis.
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Results and Discussion |
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TopAbstractResults and DiscussionMaterials and methodsAcknowledgmentsReferences |
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The conservation of the TPP-binding riboswitch in the plant kingdom was examined by computational screening of plant EST libraries (http://compbio.dfci.harvard.edu/tgi). We discovered that a TPP riboswitch is present in the 3' UTR of THIAMINE C SYNTHASE (THIC) genes of 13 different plant species (Supplementary Table S2). Moreover, in eight of them, two alternatively spliced variants were detected in which the second intron of the 3' UTR (int2) was either retained or spliced out, without affecting the coding sequence (Fig. 1A; Supplementary Table S2). In all cases, the 3' splice site of int2 was located inside the riboswitch P2 box. Previous studies showed that RNA folding at 3' splice sites may alter the splicing process (Buratti and Baralle 2004). We therefore predicted that a conformation change due to ligand binding by the riboswitch would alter the splicing of this intron.
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) that contains very low levels of endogenous TPP was used. The results showed a fourfold decrease in the expression of the intron-retention variant in the presence of rising TPP concentrations (from 0 to 10 µM), while expression of the intron-spliced variant increased sevenfold (Fig. 1B). Furthermore, testing the expression of the total THIC mRNA population (using oligonucleotides driven from the coding sequence) revealed a 3.75-fold decrease with the same rising TPP concentrations (Fig. 1B). For all three transcripts, the statistical significant change in expression was detected between 100 nM and 1 µM TPP (Fig. 1B). This finding is in correspondence with the Kd of the Arabidopsis TPP riboswitch observed in our in-line probing assay (
500 nM) (Fig. 4C, below). In order to assess the universality of the TPP effect on alternative splicing, similar experiments were performed using a tomato (Solanum lycopersicon) thiamin auxotroph mutant (Boynton 1966). Indeed, as detected for AtTHIC, the tomato THIC RNA splicing products showed a TPP concentration-dependent expression pattern (Fig. 1C). These results, from two different plant species, provide evidence that the TPP level determines the pattern of RNA splicing of the THIC precursor mRNA.
The two RNA splicing variants of the THIC gene differ in their stability
Elevated levels of TPP results in the generation of a transcript in which the second intron (int2) of the 3' UTR is spliced out. We found that the THIC intron-spliced variant is different from the intron-retention variant in two major features. The intron-retention variant has a single consensus polyadenylation [poly(A) formation] signal, and polyadenylation occurs at one accurate site (U370 in the 3' UTR, detected by 3' Rapid Amplification of cDNA Ends [RACE] experiment) (Supplementary Fig. S2). However, in the intron-spliced variant, the consensus polyadenylation signal is eliminated, and polyadenylation occurs at a range of positions along the 3' UTR (from U531 to C945, detected by 3' RACE experiments). Another feature that distinguishes between the two RNA splicing products is that the 3' UTR transcript is longer when int2 is spliced out due to the absence of a consensus poly(A) signal (Fig. 1A; Supplementary Fig. S2). It is documented that the selection of polyadenylation sites and 3' UTR length may result in different intrinsic stabilities of a transcript (Maret et al. 2004). The observation that the overall transcript level of the THIC gene (the sum of RNA splicing products) is reduced while the level of the intron-spliced variant is increased (under elevated TPP concentrations) suggests reduced stability of the intron-spliced variant.
To evaluate the difference in stability between the two RNA splicing products, we examined their decay rate following incubation of Arabidopsis cell suspension cultures supplemented with the transcription inhibitor actinomycin D (Fig. 1D; Supplementary Fig. S1C). The results showed that the relative stability of the intron-spliced variant is much lower than that of the intron-retention variant. During the 2 h following the actinomycin D treatment, the intron-spliced variant exhibited an average decay rate of 63% per hour, while the intron-retention variant and the overall transcript level of the AtTHIC gene did not show a significant decay (Fig. 1D). The rapid decay of the intron-spliced variant detected here suggests that our analysis of its expression levels (e.g., Fig. 1B), particularly under high TPP concentration, is an underestimation of its genuine production rate.
The results show that the two alternatively spliced transcripts formed as a result of riboswitch activity differ in their stability, the intron-spliced variant retaining a much higher decay rate (compared with the intron-retention variant).
Processing of THIC pre-mRNA is dependent on TPP riboswitch activity, and engineered riboswitches can act autonomously to modulate gene expression
We further investigated whether the alteration in splicing under high TPP levels is independently mediated by the TPP riboswitch activity. These experiments were performed in planta, with the following constructs (Fig. 2A): (i) the YELLOW FLUORESCENCE PROTEIN (YFP) transcriptionally fused to the native AtTHIC 3' UTR (35S:YFP-THIC3'); (ii) the same construct but with a single point mutation (A515G relative to the stop codon) in the 3' UTR that prevents the riboswitch from binding TPP (35S:YFP-THIC-Mu3') (Sudarsan et al. 2003, 2005; Thore et al. 2006); (iii) the same construct but with a mutated 5' splice site of int2 (Fig. 1A) that prevents splicing of this intron (35S:YFP-THIC-NoSpl3'); and (iv) the same construct but with the YFP fused to the NOPALINE SYNTHASE terminator (35S:YFP-NOS3').
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). Expression of the retention variant in transgenic plants harboring the 35S:YFP-THIC-NoSpl3' was not affected by additional TPP (Fig. 2C). We also examined the level of YFP gene expression in the different transformed lines (using oligonucleotides specific to its coding sequence). Addition of TPP reduced the expression of YFP in 35S:YFP-THIC3' plants but had no such effect in plants or protoplasts harboring 35S:YFP-THIC-Mu3' or 35S:YFP-THIC-NoSpl3', as well as in the control 35S:YFP-NOS3' plants (Fig. 2B,C). The fact that disabling splicing of int2 prevented the decrease in overall YFP expression demonstrated that alternative splicing is essential for riboswitch controlled expression. At the protein level, YFP accumulation (measured either by Western blotting or by YFP fluorescence analysis) was greatly reduced in 35S:YFP-THIC3' plants supplemented with TPP, while its expression was not altered in 35S:YFP-THIC-Mu3' as well as in 35S:YFP-NOS3' (Fig. 2D).
Taken together, these experiments show that when transformed in plants, engineered TPP riboswitch can act autonomously to modulate gene expression. They also demonstrate that alternative splicing in the 3' UTR of the transgene is dependent on riboswitch activity. Furthermore, under high TPP concentration, alternative splicing is essential for the reduction in mRNA levels.
The TPP riboswitch is present in the THIC gene of all plant taxa, and an additional TPP riboswitch, lost during gymnosperm evolution, is present in the THI1 gene of ancient plants
To trace the evolutionary origin of plant riboswitches, we searched for homologous sequences of the TPP riboswitch across the plant kingdom. Homologous sequences were identified in the THIC 3' UTR of all examined species, ranging from the liverwort Marchantia polymorpha (bryophytes) to higher plants (angiosperms) (Fig. 3; Supplementary Fig. S3). However, when we examined the THIC gene of Physcomitrella patens (P. patens or Pp), we could not find evidence for splicing in the second intron of its 3' UTR, as detected for flowering plants (int2) (see Fig. 1A). Therefore, it seems that the PpTHIC gene is regulated by a different mechanism than the one used in flowering plants, which does not involve alternative splicing in its 3' UTR. This is in accordance with previous studies reporting that intron-containing genes and genes undergoing alternative splicing have increased considerably from ancestral to higher eukaryotes and that alternative splicing is a relatively advanced evolutionary process (Reddy 2007).
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). The PpTHI1 RNA binds TPP at a KD of 400 nM (Fig. 4C), which is comparable with the KD value (500 nM) obtained by our in-line probing assay using the AtTHIC riboswitch.
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In this study, we report on a novel post-transcriptional regulatory mechanism for gene regulation in plants (Fig. 5). The findings reveal that a TPP riboswitch mediates feedback regulation in the thiamin biosynthesis pathway through modulation of alternative splicing in the 3' UTR region of a gene of this pathway. High levels of TPP in the cell promote ligand binding and a change in the riboswitch structural conformation. Consequently, an unstable intron-spliced variant is formed, which in turn results in a decrease of TPP levels. Alternatively, under low cellular TPP levels, the conformation of the riboswitch dictates intron retention that generates a stable transcript, which in turn promotes an increase in TPP biosynthesis. An intriguing question that arises is: How does riboswitch conformational change direct alternative splicing? The structure of the Arabidopsis riboswitch–TPP complex was recently resolved by Thore et al. (2006). Examination of this complex structure reveals that in the ligand-bound state, the 3' splice site of the intron is exposed (Supplementary Fig. S6). We therefore hypothesize that ligand binding brings about conformational changes in the 3' region of the intron that promote splicing. On the other hand, riboswitch conformation in the unbound state could therefore hinder intron processing by preventing the splicing machinery from accessing this region (Buratti and Baralle 2004).
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Materials and methods |
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All oligonucleotides used in our experiments are listed in Supplementary Table S1.
Plant material
See the Supplemental Material for all details regarding plant material.
Plant transformation
For the generation of transgenic Arabidopsis, the AtTHIC 3' UTR was amplified by PCR using Arabidopsis genomic DNA (ColO) with oligonucleotides #1 and #2 (Supplementary Table S1). The mutation (A515G, starting from the stop codon) was introduced using the megaprimer-based mutagenesis strategy (Kammann et al. 1989) with oligonucleotides #1, #2, and #20. The AtTHIC 3' UTR was then fused to the YFP reporter gene, and the fusion fragment was subsequently inserted downstream from the double 35S promoter of the Cauliflower Mosaic Virus (CaMV). The 5' splice site mutation was obtained by amplifying the cassette-containing plasmid with oligonucleotides #29 and #30, followed by self-ligation of the PCR product and transformation of the resulting plasmid into Escherichia coli. This resulted in one construct containing the following mutations: T263A, T267A, T276A, T279A, T287A, and T292A. The cassettes were then inserted into the pBinPlus binary vector containing the kanamycin resistance gene for the selection of transformants (van Engelen et al. 1995). Arabidopsis plants were transformed using the floral-dip method (Clough and Bent 1998), and kanamycin-resistant seedlings were then transferred to soil.
Molecular biology and microscopy
Unless specified, all molecular biology manipulations were performed as described in Sambrook et al. (1989). RNA extractions were all performed using the RNeasy kit (Qiagen) and DNA extractions were performed using the CTAB method (Doyle and Doyle 1987).
Quantitative real-time PCR (qPCR) gene expression analysis was performed with three biological replicates using gene/variant-specific qPCR oligonucleotide pairs, designed with Primer Express software (Applied Biosystems). Specific oligonucleotides sequences are provided in Supplementary Table S1 (#21–#26), and their efficiency standard curves are depicted in Supplementary Figure S1D. AtACTIN2 was used as an endogenous control for the expression analysis of each AtTHIC variant, and AtUBIQUITIN C was used as an endogenous control for the relative stability experiment (with oligonucleotides #34–#35 and #27–#28, respectively). A fixed amount of DNase-treated total RNA was reverse-transcribed using AMV Reverse Transcriptase (EurX Ltd.). RT–PCR reactions were tracked on an ABI 7300 instrument (Applied Biosystems) using the PlatinumR SYBR SuperMix (Invitrogen). Each sample was PCR-amplified from the same amount of cDNA template in triplicate reactions. Following an initial step in the thermal cycler for 15 min at 95°C, PCR amplification proceeded for 45 cycles of 15 sec at 95°C and 30 sec at 60°C, and was completed by melting curve analysis to confirm specificity of the PCR products. The baseline and threshold values were adjusted according to the manufacturers’ instructions.
RT–PCR end-point analysis was performed as described in Aharoni et al. (2004a) with oligonucleotides #3–#7 (Supplementary Table S1). In tomato, the intron-retention variant was detected with oligonucleotides #8 and #9, the intron-spliced variant was detected with #8 and #10, and the SlTHIC coding region transcripts were detected with #8 and #11 (Supplementary Table S1). The TUBULIN or UBIQUITIN C genes (for Arabidopsis) and ACTIN gene (for tomato) were used for calibration with oligonucleotides #17–#18, #28–#33, and #12–#13, respectively. The YFP mRNA transcript levels were determined using oligonucleotides #16 and #4 for the intron-retention variant and #16 and #5 for the intron-spliced variant, and the YFP-coding region transcripts were determined with #14 and #15. For all other plant species, DNA or RNA fragments corresponding to the 3' region of the THIC and THI1 genes were amplified with the forward oligonucleotides #8 and #10, respectively, and reverse #9. All splicing products were confirmed by sequencing.
For RNA stability determination, actinomycin D (Sigma, catalog no. A1410) was added to Arabidopsis suspension cell culture to a final concentration of 50 µg/mL. Cells supplemented with 1 mM TPP were harvested at three or four time points (0, 1 h, 2 h, 4 h), followed by RNA extraction and quantitative real-time experiments with the specific oligonucleotides #21–#28 as described above (Fig. 1D). RNA stability was also determined by RT–PCR end-point analysis with the variant-specific oligonucleotides (#3–#7) (Supplementary Fig. S1C). All splicing products were confirmed by sequencing. 3' RACE experiments were performed as described by Aharoni et al. (2004b), with oligonucleotides #8, #19, #31, or #32. The YFP signal was detected by a standard fluorescence microscope.
In-line probing
The in-line probing experiment was conducted as described previously by Sudarsan et al. (2003). Briefly, RNA was synthesized in vitro from DNA templates by T7 polymerase, 5'-end-labeled with 32P, and gel-purified. The radiolabeled RNA was subjected to in-line probing by incubation for 40 h at 25°C in 20 mM MgCl2, 100 mM KCl, and 50 mM Tris·HCl (pH 8.3), in the presence of increasing TPP concentrations from 0 to 10 µM. The partially cleaved fragments were resolved on a denaturing gel (8 M urea–8% polyacrylamide), which was further subjected to a PhosphorImager analysis. Kd values were estimated as described in Cheah et al. (2007).
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E-MAIL asaph.aharoni{at}weizmann.ac.il; FAX 972-89344181. 
Supplemental material is available at http://www.genesdev.org.
Article is online at http://www.genesdev.org/cgi/doi/10.1101/gad.443907
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