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Riboswitch-Dependent Gene Regulation and Its Evolution in the Plant Kingdom

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Riboswitch-Dependent Gene Regulation and Its Evolution in the Plant Kingdom Downloaded from genesdev.cshlp.org on September 28, 2021 - Published by Cold Spring Harbor Laboratory Press RESEARCH COMMUNICATION related compounds (Sudarsan et al. 2005). In prokaryotes, Riboswitch-dependent gene genetic control mediated by riboswitches is a prevalent regulation and its evolution phenomenon, and the dozen riboswitches identified to date regulate >3% of all bacterial genes (Nudler 2006). in the plant kingdom Thiamin pyrophosphate (TPP) is a coenzyme derived from vitamin B1 (thiamin) that is synthesized by bacte- 1,2 1 1 Samuel Bocobza, Avital Adato, Tali Mandel, ria, fungi, and plants, and serves as a dietary requirement Michal Shapira,2 Evgeny Nudler,3 and for humans and other animals. It is an obligatory cofac- Asaph Aharoni1,4 tor that plays an important role in amino acid and car- bohydrate metabolism; thus, fine control of its levels is 1Department of Plant Sciences, Weizmann Institute of essential for the viability of all living cells. TPP-binding Science, Rehovot 76100, Israel; 2Department of Life Sciences, riboswitches, first identified in Bacillus subtilis and Ben Gurion University, Beer Sheva 84105, Israel; 3Department Escherichia coli (Mironov et al. 2002; Winkler et al. of Biochemistry, New York University School of Medicine, 2002), exist in the genomes of species belonging to most New York, New York 10016, USA bacterial phyla (Rodionov et al. 2002). Binding of TPP by the riboswitch down-regulates expression of thiamin Riboswitches are natural RNA sensors that affect gene biosynthesis genes by inducing either the formation of a control via their capacity to bind small molecules. Their transcription terminator hairpin or the formation of a prevalence in higher eukaryotes is unclear. We discov- Shine-Dalgarno sequester hairpin (Mironov et al. 2002; ered a post-transcriptional mechanism in plants that Rodionov et al. 2002; Winkler et al. 2002). Much less is uses a riboswitch to control a metabolic feedback loop known about riboswitch-mediated gene control in eu- through differential processing of the precursor RNA 3؅ karyotic organisms, and TPP-binding riboswitches are terminus. When cellular thiamin pyrophosphate (TPP) the only ones identified to date (Kubodera et al. 2003; Sudarsan et al. 2003). Very recently, Cheah et al. (2007) levels rise, metabolite sensing by the riboswitch located demonstrated that a TPP riboswitch controls expression in TPP biosynthesis genes directs formation of an un- of the THI4 and NMT1 genes in Neurospora crassa by stable splicing product, and consequently TPP levels directing the splicing of an intron located in the 5Ј un- drop. When transformed in plants, engineered TPP ribo- translated region (UTR). Intron retention results in the switches can act autonomously to modulate gene expres- appearance of upstream and out-of-frame initiation sion. In an evolutionary perspective, a TPP riboswitch is codons, whereas intron splicing generates a complete also present in ancient plant taxa, suggesting that this and correct open reading frame. While the prokaryotic mechanism is active since vascular plants emerged 400 and fungi riboswitches are located in the 5Ј UTR, a plant Ј million years ago. TPP riboswitch located in the 3 UTR of the Arabidopsis THIAMINE C SYNTHASE gene (AtTHIC) was recently Supplemental material is available at http://www.genesdev.org. identified (Sudarsan et al. 2003). This difference in loca- tion suggests a unique mode of action for the plant ri- Received June 5, 2007; revised version accepted September 24, boswitch. Yet, the mechanisms mediated by riboswitch 2007. activity in higher eukaryotes, particularly in the plant kingdom, remain to be elucidated. Here we propose a Riboswitches are regions in mRNA that act as metabo- unique prevalent model for TPP riboswitch-controlled lite sensors. These selectively bind small molecule tar- gene expression in all flowering plants, according to gets such as vitamins, amino acids, and nucleotides which TPP binding to THIC pre-mRNA engenders alter- through a conserved sensor domain. Upon substrate native splicing that leads to the generation of an unstable binding, the conformation of a variable “expression plat- transcript, which in turn lowers TPP biosynthesis. form” coupled to the sensor domain is changed, and this can affect different modes of gene control including tran- scription termination, translation initiation, or mRNA Results and Discussion processing (Mandal and Breaker 2004; Nudler and Miro- nov 2004; Coppins et al. 2007). Notably, riboswitches The concentration of TPP controls alternative splicing Ј exert their functions without the need for protein cofac- in the 3 UTR of flowering plant THIC genes tors (Mironov et al. 2002; Winkler et al. 2002). In most The conservation of the TPP-binding riboswitch in the cases, they act in feedback regulation mechanisms; once plant kingdom was examined by computational screen- the level of an end product in a metabolic pathway rises, ing of plant EST libraries (http://compbio.dfci.harvard. riboswitch binding occurs, triggering a repression of gene edu/tgi). We discovered that a TPP riboswitch is present expression in the same pathway. The substrate specific- in the 3Ј UTR of THIAMINE C SYNTHASE (THIC) genes ity of riboswitches is extremely high, allowing them to of 13 different plant species (Supplementary Table S2). perform their activity amid the presence of numerous Moreover, in eight of them, two alternatively spliced variants were detected in which the second intron of the 3Ј UTR (int2) was either retained or spliced out, without [Keywords: Thiamine pyrophosphate; riboswitch; alternative splicing; affecting the coding sequence (Fig. 1A; Supplementary higher eukaryotes; metabolite sensing] Table S2). In all cases, the 3Ј splice site of int2 was lo- 4Corresponding author. E-MAIL [email protected]; FAX 972-89344181. cated inside the riboswitch P2 box. Previous studies Article is online at http://www.genesdev.org/cgi/doi/10.1101/gad.443907. showed that RNA folding at 3Ј splice sites may alter the 2874 GENES & DEVELOPMENT 21:2874–2879 © 2007 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/07; www.genesdev.org Downloaded from genesdev.cshlp.org on September 28, 2021 - Published by Cold Spring Harbor Laboratory Press Riboswitch-dependent gene regulation in plants cant 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 auxo- troph mutant (Boynton 1966). Indeed, as detected for AtTHIC, the tomato THIC RNA splicing products showed a TPP concentration-dependent expression pat- tern (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. Figure 1. TPP-dependent alternative splicing in the 3Ј UTR of Ara- bidopsis and tomato THIC genes. (A) Schematic representation of The two RNA splicing variants of the THIC gene differ the THIC pre-mRNA and the two RNA splicing products, marking in their stability the TPP riboswitch, introns 1 and 2 (dark gray), and exons (light gray). Numbers indicate oligonucleotides used for experiments de- Elevated levels of TPP results in the generation of a tran- scribed in B (see Supplemental Material). (B) Transcript levels in script in which the second intron (int2) of the 3Ј UTR is TPP-treated Atthi1 mutant seedlings resolved by qPCR experiments spliced out. We found that the THIC intron-spliced vari- (n =3;P < 0.01; SEM; see Materials and Methods for details). (var.) ant is different from the intron-retention variant in two Variant. The oligonucleotides (see A) used were #21 and #22 (coding seq.), #23 and #24 (intron-retention var.), and #25 and #26 (intron- major features. The intron-retention variant has a single spliced var.), and AtACTIN2 was used as endogenous control (oli- consensus polyadenylation [poly(A) formation] signal, gonucleotides #34 and #35). (C) End-point analysis RT–PCR prod- and polyadenylation occurs at one accurate site (U370 in ucts that monitor the levels of the THIC RNA splicing products in the 3Ј UTR, detected by 3Ј Rapid Amplification of cDNA the tomato thiamin auxotroph mutant strain grown under increas- Ends [RACE] experiment) (Supplementary Fig. S2). How- ing TPP concentrations. The two splicing variants were amplified ever, in the intron-spliced variant, the consensus poly- with specific oligonucleotides (#8 and #9, and #8 and #10), and the coding region was amplified with oligonucleotides derived from adenylation signal is eliminated, and polyadenylation conserved regions (#8 and #11). The intron-retention variant, the occurs at a range of positions along the 3Ј UTR (from intron-spliced variant, and the coding sequence were amplified for U531 to C945, detected by 3Ј RACE experiments). An- 31, 35, and 33 cycles, respectively. The effect of TPP addition on other feature that distinguishes between the two RNA whole plants is shown at the bottom of each panel. (D) Stability of splicing products is that the 3Ј UTR transcript is longer the AtTHIC splice variants and coding sequence during variable when int2 is spliced out due to the absence of a consen- time points after the addition of the transcription inhibitor actino- mycin D to Arabidopsis cell suspension culture supplemented with sus poly(A) signal (Fig. 1A; Supplementary Fig. S2). It is 1 mM TPP, as resolved by qPCR using the same oligonucleotides as documented that the selection of polyadenylation sites in B (n =3;P < 0.01; SEM). AtUBIQUITINC was used as the endog- and 3Ј UTR length may result in different intrinsic sta- enous control (oligonucleotides #27 and #28). The decay of the in- bilities of a transcript (Maret et al. 2004). The observa- tron-spliced variant was on average 63% per hour. tion 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 el- splicing process (Buratti and Baralle 2004).
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