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Gene regulation by structured mRNA elements Andreas Wachter Center for Plant Molecular Biology (ZMBP), University of Tu¨bingen, Auf der Morgenstelle 32, 72076 Tu¨bingen, Germany

The precise temporal and spatial coordination of gene activity, based on the integration of internal and external signals, is crucial for the accurate functioning of all biological processes. Although the basic principles of gene expression were established some 60 years ago, recent research has revealed a surprising complexity in the control of gene activity. Many of these gene regulatory mechanisms occur at the level of the mRNA, including sophisticated gene control tasks mediated by structured mRNA elements. We now know that mRNA folds can serve as highly specific receptors for various types of molecules, as exemplified by metabolite-binding riboswitches, and interfere with pro- and eukaryotic gene expression at the level of transcription, translation, and RNA processing. Gene regulation by structured mRNA elements comprises versatile strategies including self-cleaving ribozymes, RNA-folding-mediated occlusion or presentation of cis-regulatory sequences, and sequestration of trans-acting factors including other RNAs and proteins. Structure–function relationship of RNA mRNAs represent the most complex group of cellular RNAs, with the diversity originating not only from transcription but also from numerous modification reactions such as precursor mRNA (pre-mRNA) splicing, polyadenylation, and 30 end processing. Furthermore, mRNAs are usually associated with numerous protein factors and complexes which play an essential role in translation and mRNA metabolism. For several classes of non-coding RNAs, such as transfer RNAs (tRNAs), ribosomal RNAs (rRNAs), and catalytically active RNAs, so-called ribozymes (see Glossary), the formation of complex structures and their functions have been established. Owing to its identical chemical nature, mRNA has the same potential to form intricate folds; nevertheless, its implications in gene regulation have only been recognized quite recently. For example, riboswitches represent mRNA domains that sense small molecules and regulate gene activity by means of alternative RNA conformations [1,2]. RNA folds are typically assembled from canonical basepairing elements, but also other, conserved structural motifs as well as tertiary interactions [3], and are stabilized by different forces, including hydrogen bonds, stacking, Corresponding author: Wachter, A. ([email protected]). Keywords: mRNA; structure; riboswitch; gene regulation. 0168-9525/ ß 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tig.2014.03.001

electrostatic, and van der Waals interactions. In particular, structural studies of riboswitches have provided important insight into the molecular basis of ligand recognition and gene regulation by mRNA folds [2,4,5]. In contrast to the highly conserved, metabolite-binding aptamer domains of riboswitches, the discovery of structured motifs in the context of other large mRNAs is challenging because a variety of alternative folds are often predicted. Nonetheless, the combination of bioinformatics, such as exploiting the potential of comparative analyses, with biochemical and biophysical structure-probing experiments and genetics, has resulted in a substantial progress in our understanding of the functional relevance of structured mRNA motifs. Furthermore, transcriptome-wide profiling of in vivo RNA structures [6,7] is expected to accelerate the discovery of functionally relevant mRNA folds. This review discusses common principles of gene control by structured mRNA elements in pro- and eukaryotes. Although many intriguing discoveries of gene regulation based on the interaction of mRNAs with other macromolecules such as RNAs and proteins have been reported, the focus here is on cases where the mRNA structure itself or a more complex mRNA fold regulates gene activity. This review is limited to natural examples of regulatory mRNA elements; however, their potential is becoming increasingly used in artificial gene control [8,9]. Gene regulation by ribozyme-containing mRNA domains Ribozymes possess intrinsic catalytic activity and often modify RNA molecules in cis or trans. Many ribozymes are part of viral or non-coding RNAs; however, the glmS (glutamine-fructose-6-phosphate amidotransferase) ribozyme [10] and an allosteric self-splicing intron [11] were

Glossary Aptamer: ligand-binding domain of a riboswitch. Dissociation constant (KD): a measure of the affinity between a ligand and the ligand-binding molecule under equilibrium conditions. Expression platform: gene-regulatory domain of a riboswitch. Internal ribosomal entry sites (IRESs): allow cap-independent translation initiation in eukaryotes. Nonsense-mediated decay (NMD): eukaryotic RNA surveillance mechanism targeting transcripts with particular cis-elements such as premature termination codons (PTCs) or long 30 untranslated regions (UTRs) for degradation. Riboswitch: mRNA domain that senses small molecules and regulates gene activity by means of alternative RNA conformations. Ribozyme: RNA with catalytic function. Shine–Dalgarno (SD) sequence: sequence upstream of the start codon in bacterial mRNAs, being involved in ribosomal binding and translation initiation.

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the absence of GlcN6P [14], enabling a rapid response to elevated levels of its ligand. Further instances of selfcleaving ribozymes have been reported (Box 1); however, in most cases the implications for the regulation of associated genes were not revealed. Group I self-splicing introns are another common class of ribozymes that have been identified in viruses, bacteria, organelles, and eukaryotic microorganisms [15]. Although generally assumed to represent selfish genetic elements, group I ribozymes can have functions in RNA processing [15] and in gene regulation, as described for a representative from Clostridium difficile [11,16]. In the latter example, the group I intron is interconnected with a cyclic di-guanosine monophosphate (c-di-GMP)-sensing riboswitch, controlling the formation of alternative RNA structures in the 50 UTR of the mRNA from a putative virulence gene [11]. In the

identified as domains within bacterial mRNAs. The glmS ribozyme is positioned in the 50 untranslated region (UTR) of the mRNA transcribed from the glmS gene in Grampositive bacteria and catalyzes auto-cleavage in the presence of glucosamine-6-phosphate (GlcN6P) [10]. Interestingly, the glmS ribozyme is responsive to different hexose metabolites in vivo, acting as an integrator of positive and negative chemical signals [12]. Self-cleavage produces a 50 fragment with a cyclic 20 ,30 -phosphate and a 30 RNA containing a free 50 hydroxyl group (Figure 1A). Ribozyme cleavage in response to elevated GlcN6P levels results in gene repression because transcripts with a 50 hydroxyl group are degraded via RNase J1 in Bacillus subtilis [13]. This mechanism allows feedback regulation of the GlmS enzyme, which catalyzes GlcN6P formation. Interestingly, the glmS ribozyme is already completely folded in

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Figure 1. Gene regulation by metabolite-responsive ribozymes. (A) The glmS (glutamine-fructose-6-phosphate amidotransferase) mRNA shows self-cleavage in the presence of glucosamine-6-phosphate (GlcN6P), generating a free 50 hydroxyl group that allows mRNA degradation by RNase J1. Schematic representation of the glmS ribozyme is based on the previously reported consensus secondary structure model [77]. Light grey lines (left side) indicate connections between adjacent nucleobases that are distantly located in this display mode. (B) Mechanism of gene regulation by an allosteric self-splicing ribozyme. In the middle, the 50 region of the mRNA precursor, consisting of a cyclic di-guanosine monophosphate (c-di-GMP) aptamer and a group I self-splicing ribozyme, is displayed. This structure is assumed to be prevalent in the presence of c-di-GMP, whereas under conditions of low ligand levels, occurrence of alternate RNA base pairing (depicted in light and dark green boxes) has been demonstrated. Dashed lines indicate only partial display of the RNA in these regions. Splicing in the presence of both GTP and c-di-GMP (left side, top) results in a translation-competent mRNA, with a Shine–Dalgarno (SD) sequence positioned immediately upstream of the UUG translational start-site. Upon c-di-GMP dissociation, translation of this splice variant is blocked due to formation of alternative RNA structures (left side, bottom). In the absence of c-di-GMP (right side), GTP attack at a downstream site generates a 50 truncated mRNA, lacking the SD sequence, and that therefore is not efficiently translated. Abbreviation: SS, splice site.

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Box 1. Self-cleaving ribozymes in eukaryotes Self-cleaving ribozymes are not restricted to bacterial mRNAs and have also been found in protein-coding RNAs from eukaryotes. In the myxomycetes Physarum polycephalum and Didymium iridis, investigation of group I introns located within rRNA precursors revealed twin-ribozyme functions [78,79], involving self-splicing and internal processing as part of the maturation process of the ORFcontaining mRNAs. Autocatalytic mRNA cleavage has also been detected for the mRNA transcribed from the human b-globin (HBB) gene, and a dependency of efficient transcription termination on ribozyme function was demonstrated [80]. Using in vitro selection, additional instances of self-cleaving ribozymes in the human genome were identified [81]. One of these ribozymes occurs within an intron of the pre-mRNA for the cytoplasmic polyadenylation element-binding protein 3 (CPEB3), and experimental support for expression and in vivo activity of this ribozyme was provided. The CPEB3 ribozyme is structurally related to the human hepatitis delta virus (HDV) ribozyme, both of which are characterized by a nested double pseudoknot [81]. A structure-based approach aiming at computational identification of HDV-related motifs followed by in vitro self-scission studies led to the discovery of novel ribozymes in various eukaryotes, one bacterium, and one virus [82]. Interestingly, an analysis of steady-state transcript levels for one representative ribozyme from Anopheles gambiae revealed varying amounts of total and uncleaved ribozyme RNAs at different developmental stages, suggesting that ribozyme activity might be regulated. Bioinformatics also resulted in the discovery of a discontinuous hammerhead ribozyme in the 30 UTR of rodent Ctype lectin type II (Clec2) mRNAs [83]. Despite its discontinuous nature, this ribozyme was found to self-cleave in vitro as well as in vivo, where ribozyme action diminished gene expression.

unprocessed RNA, the Shine–Dalgarno (SD) sequence, required for ribosome binding, is masked by a stem structure, causing translation suppression (Figure 1B) [16]. Binding of c-di-GMP to the aptamer results in the attack of GTP at an upstream site and intron removal, thus bringing the SD sequence into close proximity with the start codon and allowing translation. Interestingly, owing to the presence of a functional aptamer, c-di-GMP-dependent regulation of translation can still occur [16]. Alternative precursor RNA folding in the absence of c-di-GMP promotes GTP attack at a downstream site and the generation of a truncated mRNA lacking the ribosome binding site, leading to poor translation. Because ribozyme action also requires GTP as a cofactor, this tandem RNA element might be responsive to two chemical inputs. Transcriptional and translational control by noncatalytic, structured mRNA elements in prokaryotes Alternative 50 UTR structures frequently contribute to the regulation of gene activity in bacteria. The two most common mechanisms are regulated premature transcription termination, which prevents transcription of the complete open reading frame (ORF), and translation control via SD availability (Figure 2A). The formation of mutually exclusive RNA structures, linked to different degrees of gene activity, can be controlled by various trans- and cis-acting factors, including RNA polymerase (RNAP) processivity,

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Figure 2. Mechanisms of transcriptional and translational gene control based on alternative mRNA folds in prokaryotes. (A) Gene regulation by structured mRNA elements in bacteria most commonly exploits transcriptional (left) or translational (right) attenuation. For each box, the upper and lower schemes represent ON and OFF stages of gene expression, respectively. In the case of transcriptional control, formation of an anti-terminator structure allows transcriptional elongation and expression of the gene. By contrast, alternative mRNA folding into a terminator structure followed by a uridine-rich sequence triggers premature transcription termination and release of the RNA polymerase (RNAP). Similarly, translational regulation is achieved by mutually exclusive mRNA folds, either allowing ribosome access or sequestering the Shine–Dalgarno (SD) sequence. (B) Overview of cis- and trans-acting factors as well as further parameters that are known to modulate gene expression-relevant mRNA structures. In addition to transcriptional and translational attenuation, structured mRNA elements can also contribute to gene regulation by altering mRNA stability.

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Review translation rate, temperature, and interaction with various types of molecules (Figure 2B) [17]. RNAP processivity RNAP pausing can affect folding of the nascent RNA and its interaction with trans factors. A direct effect of RNAP pausing on gene activity has been described for the pyrG (CTP synthetase) gene from B. subtilis [18]. The first nucleotides of the pyrG transcript include a cytosine residue, resulting in pausing at this site when CTP levels are low. Pausing triggers template-independent, iterative addition of G residues, preventing the formation of a premature terminator hairpin. By contrast, high cellular CTP levels allow fast read-through of the RNAP, resulting in terminator formation and negative feedback regulation of CTP synthetase expression. Similarly, pH-responsive expression of the alx (ygjT, integral membrane protein) locus from Escherichia coli is mediated by RNAP pausing-dependent, alternative mRNA structures [19]. While alkaline conditions cause RNAP pausing at distinct sites, triggering folding into a translation-competent structure, no pausing and formation of a translation-suppressing RNA conformation is observed at low pH. Importantly, the two structures could not be converted into each other by a pH shift, but were dependent on transcription at different pH values. Translation rate Co-occurrence of transcription and translation in bacteria is utilized to sense metabolite levels, as in the well-known example of the trp (tryptophan) operon in E. coli [20]. Rapid translation of a tryptophan-rich leader region gives rise to a premature termination structure, whereas low tryptophan levels cause ribosome stalling, thus preventing terminator folding. Similar mechanisms are used to sense other amino acids and translation-interfering antibiotics [21]. Cis-acting mRNA elements with dual sensory and regulatory functions Thermosensors exploit the temperature dependency of RNA folding, with a temperature shift altering the equilibrium between two alternative mRNA folds linked to different degrees of gene activity. Most thermosensors are positioned in the 50 UTRs of bacterial mRNAs and suppress translation at low temperature by trapping the SD sequence in a structured element [22]. Upon a sudden temperature increase, such as a heat shock or upon host invasion by a pathogen, thermosensor-mediated gene control facilitates a rapid translational response. Interestingly, thermosensors can also activate gene expression in response to cold, as has been demonstrated for the cspA (cold-shock protein A) mRNA from E. coli [23]. Thermosensor function is only based on structural modulation within a particular temperature interval. Given the low extent of sequence and structural constraints, thermosensors might be more widespread than reflected by the number of currently known motifs. A second type of cis-acting mRNA element with sensory and regulatory properties are riboswitches. Riboswitches are composed of a metabolite-sensing aptamer domain that 4

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is functionally connected to the gene-regulatory expression platform. The aptamer domains directly and specifically bind their ligands, including coenzymes, nucleobases, amino acids, second messengers, and ions (Table 1) [1,2]. The discovery of more than 20 distinct riboswitch classes has provided unprecedented evidence for the functional capacity of mRNA motifs, which can form specific binding pockets and display in vitro dissociation constants (KD) down to the picomolar range (Box 2). Ligand binding to the aptamer is transmitted into a structural modulation of the expression platform, most frequently controlling transcription terminator folding or translation via the accessibility of the SD sequence. Further modes of action include premature, Rho-helicase-dependent termination [24], regulated RNA degradation via accessibility of RNase E cleavage sites [25], and control of alternative splicing (AS) in eukaryotes (see below). Primarily, riboswitches act in cis; however, further transregulatory functions have been described. On the one hand, riboswitch control of regulatory antisense RNAs was reported [26,27]. On the other hand, prematurely terminated transcripts resulting from riboswitch control can act

Table 1. Riboswitch classes Ligand Enzyme cofactors Thiamine pyrophosphate (TPP) Adenosylcobalamin (B12) S-adenosylmethionine (SAM-I) Flavin mononucleotide (FMN) Molybdenum cofactor (Moco) Tetrahydrofolate Nucleobases, nucleoside, nucleotides Guanine Prequeuosine-1 (preQ1) Cyclic di-guanosine monophosphate (c-di-GMP-I) Cyclic di-adenosine monophosphate (c-di-AMP) b Amino acids Lysine Glycine Glutamine c Ions Mg2+ F– Others Glucosamine-6-phosphate S-adenosylhomocysteine (SAH) Aminoglycoside d

Structural variants

SAM-II, -III, SAM/SAH Tungsten cofactor

Adenine, 20 deoxyguanosine preQ2 c-di-GMP-II

Refs a [84,85] [86] [87–90] [84,91] [92] [93]

[94–96] [97] [30] [31]

[98,99] [100] [101] [102,103] [104] [10] [105] [106]

a

References refer to the first descriptions of the respective riboswitch classes. Additional references for structural variants are only provided in cases of altered ligand specificity.

b

A representative motif of this class has been reported to sense ATP [107]; however, c-di-AMP is bound in vitro several orders of magnitude more strongly than ATP and can interfere with in vitro transcription termination [31].

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No genetic evidence for riboswitch function was provided.

d

Based on the association of the DNA sequence corresponding to this motif with integron recombination sites, the biological relevance of this riboswitch class has been discussed [108,109].

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Review Box 2. In vitro binding affinities of riboswitch aptamers Biochemical analyses of aptamer–ligand interactions in vitro revealed KD values that are frequently far below the expected cellular level of the respective metabolite [1]. Applying these binding parameters to the in vivo situation, no or only weak alterations of gene expression would be achieved by the fluctuations of ligand concentrations expected to occur under physiological conditions. However, the dynamic range of in vivo riboswitch regulation might not be fully reflected by the dissociation constants determined in vitro. First, the KD values of aptamers measured in vitro might not match the strength of the RNA–ligand interaction in vivo, where a nascent transcript with full aptamer flanking sequences is exposed to a completely different chemical environment. Comparing the in vitro and in vivo structures of the adenine riboswitch aptamer has indeed revealed a major impact of the intracellular environment on RNA structure formation [110]. Second, dissociation constants are measured under conditions of a chemical equilibrium that might not be reached in vivo. Accordingly, several studies [1,111,112] support the idea of a kinetic instead of a thermodynamic control for at least some riboswitches. In this case, the speed of ligand binding would be a limiting factor, allowing modulation of gene expression only within a critical time-window. This time interval could, for example, span the duration between transcription and folding of the aptamer and a terminator structure, with important implications for the RNA polymerase speed. Accordingly, RNAP pausing at strategic locations has been demonstrated to influence folding of the E. coli btuB (vitamin B12/ cobalamin outer membrane transporter) riboswitch and to suppress the formation of alternative RNA structures [113]. Finally, studies of the tetrahydrofolate riboswitch have revealed that the affinity of an aptamer for a ligand does not necessarily correlate with the effectiveness of this compound in riboswitch regulation because alternative binding mechanisms can occur [114,115].

in trans by binding to a complementary mRNA and thereby inhibiting its translation [28]. The distribution of different riboswitch classes varies considerably [1], with thiamine pyrophosphate (TPP)-binding riboswitches being most widespread and also the only class present not only in eubacteria but also in archaea and eukaryotes [29]. Many riboswitches mediate feedback control of the enzymes involved in ligand metabolism; however, the identification of riboswitches responsive to the second messengers c-di-GMP [30] and c-di-AMP [31] has widely expanded the implications of this gene regulatory mechanism. Conserved variation patterns within the purine aptamer [32] and the occurrence of an extra stem-loop in a preQ1 riboswitch [33] have been linked to riboswitch activity, revealing that not only the expression platform but also the aptamer can contribute to the dynamic range of this gene regulatory system. Interestingly, the TPP riboswitch regulating THIC (thiamine C) expression in Chlamydomonas reinhardtii is also responsive to hydroxymethylpyrimidine pyrophosphate (HMP-PP) [34], one of the two precursors required for TPP synthesis. Because THIC acts in HMP-PP synthesis, HMP-PP-responsive riboswitch regulation contributes to balancing the production of TPP precursors. More complex riboswitches are based on tandem arrangements of aptamers, allowing tighter gene control or integration of different chemical signals [1,35]. Finally, riboswitch control can also integrate chemical and physical signals, as revealed by a three-state mechanism for an adenine-sensing riboswitch from Vibrio vulnificus [36].

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Trans-acting tRNAs and small RNAs T-box motifs in the 50 UTR sense the tRNA charging status and control expression of bacterial genes typically involved in amino acid metabolism [37]. T-box specificity is based on the interaction of the anticodon region of a tRNA with a complementary ‘specifier’ sequence in the mRNA, the detailed interaction of which has recently been revealed [38]. Binding of an uncharged tRNA allows additional basepairing and anti-terminator stabilization, which results in gene expression. Because T-boxes typically are associated with genes involved in the metabolism of the corresponding amino acids, tRNA charging serves as an indicator for the cellular demand for the respective amino acid. T-boxes specific for tRNAs for almost all amino acids have been described, including dual specificity [39]. Besides transcriptional control, T-boxes can also perform gene regulation on the level of translation. Gene regulation by trans-acting small RNAs is widespread in bacteria [40], and typically involves the formation of duplexes that inhibit translation or trigger mRNA degradation. Interestingly, small RNAs can also activate gene expression by disrupting translation-inhibiting structures. Additional modes of action have been described; however, these are beyond the scope of this review. Trans-acting proteins Protein binding can also alter mRNA folds, thereby affecting transcription termination or translation initiation. For example, upon tryptophan binding, TRAP (tryptophan operon RNA-binding attenuation protein) from B. subtilis interacts with the 50 leader of the trp operon mRNA [41], facilitating the formation of a transcriptional terminator. Analogously, translation can be suppressed by a TRAPtriggered mRNA fold that sequesters the SD sequence. Equivalent regulatory mechanisms have been reported for other RNA-binding proteins, often as part of negative autoregulation by association with their corresponding mRNAs [21,42]. Interestingly, structural mimicry between their original binding site and a related element in the mRNA encoding those protein factors has been observed, as for example for ribosomal proteins [43] and the threonyltRNA synthetase [42]. Similarly, mimicry-based regulation has also been described for eukaryotic mRNAs (see below). Eukaryotic translation and mRNA stability regulation by structured mRNA motifs Eukaryotic translation initiation typically involves scanning of the 40S ribosomal subunit in complex with additional factors along the mRNA from the 50 cap to the first start codon, followed by assembly of the two major ribosomal subunits and translation initiation. In the case of the ferritin mRNA, iron-responsive elements in the 50 UTR can be bound by iron-regulatory proteins in an iron-dependent manner, resulting in translation inhibition (Figure 3A) [44]. Several initiation factors have been reported to possess strong helicase activity [45], suggesting that structured 50 UTR elements typically are unwound in the process of ribosome scanning. However, in yeast the insertion of a tetracycline-responsive aptamer into the 50 UTR of a reporter resulted in ligand-dependent gene repression 5

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Figure 3. Eukaryotic gene-regulatory mechanisms involving structured 5 and 3 untranslated regions. Black lines and orange shapes correspond to schematic representations of RNAs and proteins or protein complexes, respectively. Dotted lines indicate that the transcript sequence continues in this direction. The translation-active conformation is indicated by its association with a schematic ribosome (A–C). Alternatively, mRNA structures can affect transcript degradation (D). Abbreviations: CAT1, cationic amino acid transporter; IRES, internal ribosomal entry site; TNF, tumor necrosis factor; UTR, untranslated region; VEGFA, vascular endothelial growth factor-A.

[46], and stalling of translation elongation by an artificially inserted, stable stem-loop structure triggered endonucleolytic mRNA cleavage [47]. In addition to the cap-dependent scanning mechanism, eukaryotic translation from internal ribosomal entry sites (IRESs), found on particular viral and endogenous mRNAs, has been described [48]. IRESs do not seem to exhibit universally conserved structural motifs and vary in the number and types of proteins required for translation initiation from those sites [48]. Even though it is under debate how widespread translation of cellular mRNAs from IRES is [49], this process might allow translational control similar to bacterial mechanisms. In support of this idea, the mRNA of the cationic amino acid transporter (CAT1) contains a small upstream ORF (uORF), the translation of which causes structural reorganization and activation of the IRES responsible for translation of the main ORF (Figure 3B) [50]. Because ribosome stalling in the uORF resulted in IRES activation, the speed of uORF translation might be crucial in this regulatory mechanism [51]. An alternative mechanism of translational regulation has been described for the human vascular endothelial growth factor-A (VEGFA) mRNA, which can integrate signal inputs via alterations in the 30 UTR structure (Figure 3C) [52]. Under hypoxia, heterogeneous nuclear RNP L (hnRNPL) binds to the VEGFA 30 UTR and stabilizes a translationally active mRNA fold. Low levels of translation, however, are linked to an alternative 30 UTR structure, which is induced by binding of the interferon-g-activated inhibitor of translation complex under normoxia. Finally, 30 UTR-positioned structures have been found to trigger mRNA decay (Figure 3D). Examples include 6

mRNAs for replication-dependent histones [53,54], transferrin receptor [44], interleukin-6 [55], and tumor necrosis factor-a (TNF-a) [56]. Interestingly, TNF-a mRNA is destabilized by binding of the protein Roquin to a stemloop motif, which is conserved between vertebrates and is found in many mRNAs [56], suggesting a more widespread gene regulatory function. AS control by structured mRNA motifs The majority of genes from higher eukaryotes contain introns which need to be removed from the pre-mRNA during mRNA maturation. Usage of different splice sites allows the formation of AS variants, a common process in higher eukaryotes that contributes to proteome expansion and gene regulation [57,58]. Interestingly, previous studies have provided evidence that structured mRNA regions can affect AS regulation at different levels, including the availability of cis-regulatory sequences, interaction of splicing factors, and variations in the critical distances between binding motifs [59]. These mechanisms are supported by single case studies [59] as well as bioinformatic approaches and phylogenetic comparisons, suggesting a more widespread correlation between mRNA structures and AS [60,61]. Interestingly, a major function of mRNA structure in global 30 splice site selection has been described in yeast [62,63], including one instance of an RNA thermosensor regulating AS [62]. One impressive example for AS diversity mediated by structured mRNA elements is constituted by the splicing of the exon 6 cluster from the insect Dscam (Down syndrome cell adhesion molecule) pre-mRNA [64], which results in selection of one of 48 mutually exclusive exons. In most cases, however, the factors triggering formation of alternative mRNA folds, being linked to different

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splicing outcomes, remained unknown. In one scenario the mRNA spontaneously folds into alternate conformations with only the binding of particular protein factors locking it in one or the other structure. This type of mechanism might, for example, apply to AS of the cardiac troponin T type 2 (TNNT2) pre-mRNA, which depends on competition between the protein factors MBNL1 (muscleblind-like 1) and U2AF65 (U2 snRNP auxiliary factor 65-kDa subunit) that bind to mutually exclusive RNA structures [65]. A different mechanism for the promotion of alternative mRNA structures has been revealed by the analysis of TPP-responsive riboswitches in filamentous fungi [66,67], higher plants [68,69], and green algae [34,70]. In these cases metabolite binding to the aptamer triggers structural changes, which alter the availability of key splice sites and cause AS changes (Figure 4A). In the case of NMT1 (no message in thiamine 1) from filamentous fungi and THIC from higher plants [66,69], an aptamerproximal 50 splice site can be concealed, whereas in green algae AS modulation by occlusion of the branch point has been proposed [70]. The splice site-masking mRNA folds

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involve nucleobases of the TPP-binding aptamer, which are only available to form the inhibitory structures in the absence of the ligand, and therefore become disrupted upon TPP-binding. Interestingly, a different mechanism has been found for the TPP riboswitch associated with the gene NCU01977 from fungi [67], where the ligand-unbound aptamer supports the formation of a long-range RNA structure. This structure brings a distal 50 splice site and the 30 splice site into close proximity, resulting in preferential usage of this 50 splice site. TPP binding disrupts the long RNA structure and triggers usage of alternative, more proximal 50 splice sites. The splicing variants formed in the presence of TPP correspond in all investigated examples of eukaryotic riboswitches to the OFF stage and mediate negative feedback control in TPP synthesis. Expression of the main ORF is suppressed either by translation of uORFs, the presence of premature termination codons (PTCs), or the PTC-independent formation of a long 30 UTR, which are expected to result in nonsense-mediated decay (NMD) of these splicing variants. Based on their enormous potential, it seems

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Figure 4. Regulation of alternative pre-mRNA splicing by structured mRNA motifs. (A) Mechanisms of splicing control by thiamine pyrophosphate (TPP)-binding riboswitches for representative genes from filamentous fungi (Neurospora crassa, NcNMT1), higher plants (Arabidopsis thaliana, AtTHIC), and green algae (Chlamydomonas reinhardtii, CrTHIC). The cartoons depict the ligand-binding aptamer region as a stem-loop structure and extend to the regulation-relevant flanking sequences. Light-green boxes represent stretches of complementary nucleotides, which allow base-pairing between the aptamer and regions containing key splice sites in the absence of TPP. Splice sites in white boxes are constitutively used, whereas sites represented by light-green and turquoise boxes are selected in the absence and presence of TPP, respectively. Alternative splicing (AS) decisions in the presence of TPP result in diminished gene expression due to the presence of upstream open reading frames (uORFs) or a premature termination codon (PTC) for NcNMT1 and CrTHIC, respectively. In higher plants, splicing under conditions of elevated TPP levels removes a 30 end processing site, resulting in 30 extended transcripts of reduced stability. P1–P5 indicate base-paired elements within the TPP aptamer. (B) Model of AS control of transcription factor IIIA (TFIIIA) pre-mRNA (middle) by a plant 5S rRNA mimic (P5SM). Blue boxes represent flanking, constitutive exons and turquoise lines depict the cassette exon including the P5SM element. Mutually exclusive binding of L5 protein and a putative, exon-defining splicing factor (SF) influences the splicing outcome. In the presence of L5, the cassette exon is skipped and an mRNA variant encoding the full-length protein is generated. By contrast, SF binding leads to cassette exon inclusion, thereby introducing a PTC and resulting in nonsense-mediated decay (NMD) of this splicing variant.

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Review plausible that, in addition to TPP-responsive riboswitches, other similar regulatory mechanisms may exist in eukaryotes. For example, pyrvinium pamoate was found to alter the structure of the serotonin receptor 2C (HTR2C) pre-mRNA and to promote inclusion of an alternative exon [71]. Eukaryotic riboswitches form alternative RNA folds, whereas other structured mRNA motifs serve as binding modules for AS regulatory factors. In the example of a 5S rRNA mimic [72,73], this motif, which occurs within the plant transcription factor IIIA (TFIIIA) pre-mRNA, is capable of binding the L5 protein. L5 is an interaction partner of 5S rRNA in the ribosome and binding to the TFIIIA pre-mRNA affects its AS and adjusts TFIIIA production to the level of free L5 protein (Figure 4B). Unbound L5 protein signalizes a shortage of its natural interaction partner 5S rRNA, and triggers splicing of the TFIIIA premRNA to a variant resulting in TFIIIA protein production. Because TFIIIA is responsible for the transcription of 5S rRNA genes, the lack in this rRNA component can be compensated by increasing TFIIIA protein levels. Once sufficient 5S rRNA levels are reached, no free L5 protein is available and the TFIIIA pre-mRNA is spliced to an mRNA containing a PTC, resulting in its degradation by NMD. Mutational analysis of the 5S rRNA mimic suggested that L5 binding results in displacement of an unknown factor, causing splicing of the TFIIIA pre-mRNA to the NMD target [73]. Earlier studies in bacteria (e.g., [43]) and yeast (e.g., [74]) revealed feedback control of ribosomal proteins by mRNA folds that are structurally related to their rRNA binding sites. By contrast, the regulatory mechanism in the case of plant TFIIIA splicing links two different ribosomal components in a positive regulatory loop. Furthermore, while feedback control of ribosomal proteins typically involves mimicry of only short elements, representing the protein binding site on the mRNA and rRNA [75], a high level of conservation between 5S rRNA and the corresponding motif in the TFIIIA pre-mRNA has been found in diverse plant species [72,73]. One explanation for this high extent of similarity might be its possible origin by retrotransposition of a 5S rRNA copy, which is further supported by the finding that plant-specific Cassandra retrotransposons also carry 5S rRNA genes [76]. Thus, AS control of plant TFIIIA pre-mRNAs might represent an example of how novel regulatory functions can be assigned to structured RNAs with roles in distinct cellular processes. Concluding remarks Based on the regulatory principles discussed above, it is evident that both pro- and eukaryotes extensively utilize the potential of structured mRNA elements to perform sophisticated gene control functions in diverse cellular processes. A plethora of mechanisms have been uncovered so far, including transcriptional, translational, and splicing control, and it can be anticipated that this list will be further expanded with the characterization of novel mRNA motifs. Currently, the majority of investigated examples are derived from bacteria; however, several recent studies in eukaryotes substantiate the universality 8

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of gene regulation by mRNA folds. Although it seems plausible that some of those motifs, such as metabolitebinding riboswitches, have originated from ancestors of an RNA world [1], they now have adapted to the complex regulatory needs and processes of modern cells. Preservation during evolution, or, in other instances, the novel occurrence of mRNA folds, proves their suitability for the associated gene-regulatory tasks that represent novel, exciting additions to the steadily growing list of functions fulfilled by the complex interplay of proteins and RNAs in all living organisms. Acknowledgments The author would like to acknowledge the work in the field of mRNA structure-mediated gene control that could not be cited in this review due to size restrictions. Furthermore, the author is thankful to Christina Ru¨hl for proofreading the manuscript and to the German Research Foundation for financial support by an Emmy Noether fellowship (WA 2167/2-1).

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