Chemical Modulation of Alternative Splicing for Molecular-Target

Review Cite This: J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Chemical Modulation of Alternative Splicing for Molecular-Target Identification by Potential Genetic Control in Agrochemical Research Mo-Xian Chen,†,§,∥,⊥,○ Boyagane D. I. K. Wijethunge,‡,○ Shao-Ming Zhou,§ Jing-Fang Yang,‡ Lei Dai,∥ Shan-Shan Wang,⊥ Chen Chen,¶ Li-Jun Fu,□ Jianhua Zhang,■ Ge-Fei Hao,*,† and Guang-Fu Yang*,‡ Downloaded via UNIV OF LOUISIANA AT LAFAYETTE on April 28, 2019 at 23:36:21 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

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State Key Laboratory Breeding Base of Green Pesticide and Agricultural Bioengineering; Key Laboratory of Green Pesticide and Agricultural Bioengineering, Ministry of Education; Research and Development Center for Fine Chemicals, Guizhou University, Guiyang 550025, PR China ‡ Key Laboratory of Pesticide & Chemical Biology, Ministry of Education, College of Chemistry, Central China Normal University, Wuhan 430079, PR China § Division of Gastroenterology, Shenzhen Children’s Hospital, Shenzhen 518038, PR China ∥ Institute of Synthetic Biology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, PR China ⊥ School of Life Sciences and Shenzhen Research Institute, The Chinese University of Hong Kong, Shenzhen 518063, PR China ¶ Department of Infectious Disease, Nanjing Second Hospital, Nanjing University of Chinese Medicine, Nanjing 210003, PR China □ Fujian Provincial Key Laboratory of Ecology-Toxicological Effects & Control for Emerging Contaminants, Putian University, Putian, Fujian 351100, PR China ■ Department of Biology, Hong Kong Baptist University and State Key Laboratory of Agrobiotechnology, The Chinese University of Hong Kong, Shatin, Hong Kong, PR China ABSTRACT: Alternative splicing (AS), the process of removing introns from pre-mRNA and the rearrangement of exons to produce several types of mature transcripts, is a remarkable step preceding protein synthesis. In particular, it has now been conclusively shown that up to ∼95% of genes are alternatively spliced to generate a complex and diverse proteome in eukaryotic organisms. Consequently, AS is one of the determinants of the functional repertoire of cells. Many studies have revealed that AS in plants can be regulated by cell type, developmental stage, environmental stress, and the circadian clock. Moreover, increasing amounts of evidence reveal that chemical compounds can affect various steps during splicing to induce major effects on plant physiology. Hence, the chemical modulation of AS can serve as a good strategy for molecular-target identification in attempts to potentially control plant genetics. However, the kind of mechanisms involved in the chemical modulation of AS that can be used in agrochemical research remain largely unknown. This review introduces recent studies describing the specific roles AS plays in plant adaptation to environmental stressors and in the regulation of development. We also discuss recent advances in small molecules that induce alterations of AS and the possibility of using this strategy in agrochemical-target identification, giving a new direction for potential genetic control in agrochemical research. KEYWORDS: alternative splicing, agrochemical, genetic control, molecular targets, spliceosome, synthetic biology

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INTRODUCTION Agricultural pests can cause crop losses of greater than 50% for some crops globally. Since the dawn of agriculture, agrochemicals have played an important role in protecting crops from pests, ensuring crop productivity, and reducing the risk of a food crisis.1 However, the rapid emergence of resistance is expanding to the existing commercial agrochemicals, the most important tool used in the control of insects, pathogens, and weeds.2 Such resistance has been continuously reported for numerous combinations of agrochemicals and pest species. Additionally, the future of agrochemical innovation and the market is changing: food production is driven by dietary change, increasing abiotic stresses, demand for better-quality food, a context of increasingly stringent pesticide regulations © XXXX American Chemical Society

resulting in minimal use of agrochemicals with maintained efficacy, various emerging novel crop-protection technologies, shifting pest spectra, and a desire for products with more friendly environmental and toxicological profiles, among others. Therefore, the introduction of a potential strategy to discover agrochemicals with new modes of action when compared with traditional methods continues to play a leading role in current agrochemical research, which can create Received: Revised: Accepted: Published: A

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3, 2019 13, 2019 15, 2019 15, 2019 DOI: 10.1021/acs.jafc.9b02086 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

exon skipping, which is caused by some abiotic-stress conditions, including salinity, drought, and cold treatments.11 Furthermore, the expression of the stabilized 1 (STA1) gene in Arabidopsis results in the production of splicing-related protein, which can be up-regulated by cold stress. In particular, note that STA1 plays crucial roles both as a pre-mRNA-splicing factor and as an abiotic-stress-responsive factor.12 Therefore, during the development of new chemicals that can be used to modulate AS variants of plants, it is essential to identify the potential agrochemical targets. If the effects of chemicals on AS are ignored, the drugtargeting process will only expose a fraction of the actual proteomic world and may miss many potential protein targets. Considering the importance and advantages of the aforementioned technologies, here we discuss the pervasive pathophysiological consequences of aberrant splicing in human- and plant-disease generation and therapy. We also discuss the potential targets and diverse mechanisms of the chemical manipulation of AS in plants. All these efforts are for the sake of defining the requirements of an innovative and promising strategy from the perspective of agrochemical discovery. Importantly, we hope to demonstrate that the chemical manipulation of AS can be used as a good strategy with different crop-protection needs and requirements. Moreover, an outlook on the potential advantages and challenges of this strategy as a target for agrochemical discovery are provided.

opportunities for innovation to address grower and consumer needs. Traditionally, the discovery of new agrochemicals involves testing a variety of chemicals directly on the whole organism, including fungi, weeds, or insects, to derive new leads. Currently, molecular target-based approaches using in vitro assays are becoming more and more common. Despite the use of various methods, recent analyses of previously developed but discontinued agrochemical products and the failures of field trials point to the fact that the number of new molecules introduced to the market place has declined in recent years. One possible reason for this decline is that the research and development costs required to find and develop a new agrochemical have increased from U.S. $184 million in 2000 to $286 million in 2014, which represents an increase of 55%.3 In addition, currently a lack of knowledge exists related to both the number of proteins that a modern agrochemical can act on and the number of potential targets. Although biologists have enlarged the space of protein targets, the number of appropriate agrochemical targets is still limited. Currently, fewer than 100 determined protein targets are known for approved agrochemicals. The increasing costs and lack of molecular targets for agrochemical development limit the companies and departments that are both able and willing to make the relatively long-term investment required for the discovery of new agrochemicals. When focusing on the druggable challenges, alternative splicing (AS) stands out because of its contribution to increasing the proteomic diversity of organisms and the consequences for pharmacogenomics.4 In recent studies, scientists have realized that some disease therapies exist as a result of small-molecule-induced splicing modifications. For example, sulfonamides can target some splicing factors, including U2 snRNP auxiliary factor (U2AF) as well as the coactivator of activating protein 1 and estrogen receptor α (CAPERα), which results in the selective degradation of those splicing factors. Theophylline can induce sequestration of branch points in pre-mRNA and lead to exon skipping. The chemical interference of AS is highly associated with physiological processes and cell-development programs, which has great potential in human disease diagnosis and treatment.5 Likewise, assessing potential drug abilities in the chemical interference of plant AS may identify novel, promising, and innovative agrochemical targets. Plant stress-related genes are particularly prone to AS events; these events often modulate the ratio between active and nonactive isoforms of abiotic- and biotic-stress regulators. Important roles for numerous splicing factors in the control of plant stress responses have been identified by recent genetic and transcriptomic analyses. Emerging evidence indicates that splicing factors modulate stress responses by targeting components in the plant immunity pathway, unveiling a novel regulatory layer in plant biotic-stress tolerance. For example, the nonexpressor of pathogenesis-related gene 1 (NPR1) is accountable for plant disease resistance.6,7 Hence, increasing the consistency of NPR1 in parallel through plantgenomic-engineering technology in economic crops can increase their productivity.8 In addition, WRKY is a transcription factor9 that produces two isoforms, WRKY62.1 and WRKY76.1, through AS. Transcripts of alternatively spliced isoforms in rice have the power to improve immunity.10 In addition, the gene for wheat dehydration-responsive binding protein (WDREB2) generates three splice variants through

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AS CONTRIBUTION TO PLANT DEVELOPMENT AND STRESS RESPONSES Splicing Process and Its Importance. Splicing is a posttranscriptional process in which introns are removed and the neighboring exons are joined, generating an uninterrupted open reading frame (ORF) for translation.13 The pre-mRNAsplicing mechanism takes place in the spliceosome in eukaryotes, a high-molecular-weight complex that is assembled at every intron and consists of large ribonuclear proteins (RNPs).14 Conventionally, each spliceosome is composed of five small nuclear RNAs (snRNAs) and a range of associated protein factors. Small nuclear ribonucleoproteins (snRNPs) are combinations of these small RNAs and the protein factors. The snRNAs that make up the major spliceosome are named U1, U2, U4, U5, and U6 snRNAs. The core particles of the U1, U2, U4, and U5 snRNPs are formed by Sm proteins, whereas the U6 snRNP contains the related like-Sm2 (Lsm2) to Lsm8 proteins. The initial step of splice-site recognition comprises the U1 snRNP binding to the 5′ splice site and the U2 auxiliary factor (U2AF), which is very important for the recognition of the correct splice site for pre-mRNA,15 binding to the 3′ splice site. U2AF35, the small subunit of U2AF, binds to the intron− exon border, whereas the large subunit, U2AF65, binds to a region rich in pyrimidines designated as the polypyrimidine tract (Figure 1). Subsequently, U2 snRNP binds to the branch point, and a preformed complex of U4, U5, and U6 snRNPs is recruited to the intron. After major rearrangements and release of the U1 and U4 snRNPs, the splicing reaction takes place. Pre-mRNA splicing consists of two sequential transesterification reactions. Three functional sites on pre-mRNA are involved in this splicing process: the 5′ splice site (5′ss), the 3′ splice site (3′ss), and the branch point (BP).16 In the first transesterification, a 2′OH of a special adenosine residue at the BP commences a nucleophilic attack on the phosphodiester moiety at the 5′ss. In the second transesterification, the 3′OH B

DOI: 10.1021/acs.jafc.9b02086 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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introns are not fully removed from pre-mRNA, which is one form of aberrant splicing called intron retention, the mature mRNA may lead to some diseases. Furthermore, the physical properties of the sarcomere are carefully orchestrated through AS to fit the varying demands of the heart. Recently, tissuespecific RBM20 was discovered to be a major heart- and skeletal-muscle-restricted splicing factor; it was found that RBM20 can regulate the AS of sarcomeric genes by acting as a splicing silencer. However, aberrant splicing of sarcomeric genes may cause heart failure.23 Moreover, some proteins are produced through AS and are used for the maintenance of heart functions. Titin is this kind of protein, and high expression ratios of Titin isoforms can reduce stiffness of the myofibrillar protein in the heart. However, heart failure may easily occur as a result of the incorrect splicing of the heart’s pre-mRNA.23 Sometimes mRNA isoforms with retained introns can create harmful protein isoforms; this condition is considered a hallmark of cancer and has been commonly reported in various cancers.24 Reported examples, including some lung,25 breast,26 and prostate cancers,27 have arisen because of intron retention in genes.28 In particular, Bcl-x is a critical apoptotic regulator in human tissue,29 and two splicing isoforms conferring opposite functions can be generated, namely, anti-apoptotic Bcl-xL and pro-apoptotic Bcl-xS. Multiple studies have indicated that several small molecules or drugs, including D-e-C6 ceramide, emetine, and homoharringtonine, could regulate the AS pattern of Bcl-x through a protein-phosphatase-1 (PP1)-dependent mechanism.30−33 Furthermore, mutations of splicing factor 3B subunit 1 (SF3B1) are closely associated with a disease called chronic lymphocytic leukemia, which is a lymphoproliferative disorder in adults.34 Mutations of SF3B1 alter the pre-mRNA-splicing process.35 In addition to SF3B1, other splicing machinery that can cause cancer include mutations of U2AF1 and SRSF2, which cause breast and pancreatic cancers because of their aberrant splicing patterns.36,37 Activation of splicing factors is abnormal in the presence of cancer in that it will drive aberrant splicing. In addition, more than 20 AS isoforms exist for the CD44 gene in humans, and some of them are common in cancers.38 Therefore, alternatively spliced variants are potential biomarkers for cancer diagnosis and prognosis and may be targets for cancer therapy. Unlike in humans, where aberrant splicing has a level of prominence in causing disease, AS obviously serves as the basis of evolution and natural selection in plants. Recent RNA sequencing studies have suggested that over 80% of introncontaining genes could generate transcript isoforms in model plant Arabidopsis.39 An increasing amount of evidence has implicated the responses to developmental and stress cues in plants in altered splicing.40 Here, we will summarize keystone research studies in the area of plant splicing regulation. AS as a Regulator of Plant Development. A variety of plant development processes are strictly regulated by AS.40 For example, AS acts as an endogenous timer, extensively affecting the circadian rhythm of plants by introducing rhythmic introns on core clock genes.41,42 Specifically, an intron-retention (IR) was found in one of the isoforms of the core clock MYB transcription factor circadian-clock-associated 1 (CCA1). The IR at the fourth intron resulted in a nonfunctional transcript, which was induced in high-light conditions but repressed under low temperatures,43 in turn affecting the abundance and function of its primary transcript.44 Similar cases have been reported for transcription factors late elongated hypocotyl

Figure 1. Mechanism of pre-mRNA splicing. Pre-mRNA splicing is accomplished by a multiprotein complex. Pre-mRNA splicing is completed with two transesterification reactions. In the first transesterification, a 2′ hydroxyl of an adenosine at the branch point (BP) attacks a phosphodiester moiety at the 5′ splice site (5′ss) resulting in the free form of the 5′ exon and the intermediate lariat with a 3′ exon−intron moiety. In the second transesterification, the 3′ hydroxyl of the 5′ exon attacks the phosphodiester moiety at the 3′ splice site (3′ss), resulting in ligation of two flanking exons and release of the lariat, which thus results in different splicing isoforms. U1, U2, U4, U5, and U6 designate different snRNPs. U2AF, U2-snRNP auxiliary factor; 5′ss, 5′ splice site; 3′ss, 3′ splice site; NTC, 19 complex; NTR, 19 complex related.

of the 5′ exon commences a nucleophilic attack on the phosphodiester moiety at the 3′ss, resulting in spliced exons and an intron lariat13 (Figure 1). Eukaryotic cells also have non-snRNP splicing factors, including splicing regulators.17 Although core proteins play a major role in splicing, other regulatory splicing proteins fulfill the remaining requirements needed for correct splicing. Splicing enhancers, including the RNA recognition motif (RRM) domain, contain proteins that facilitate splicing, whereas silencers include some hnRNPs, which repress splicing.16 Furthermore, a major spliceosome (U2) and minor spliceosome (U12) exist that support the splicing function. 18 Some pre-mRNA select their splice sites themselves, but other pre-mRNA molecules do not. In those cases, the splice sites are chosen by splicing factors.19 Generally, splicing factors can be categorized into several different groups as follows: small nuclear ribonucleoproteins, splicing factors, splicing regulators, novel spliceosome proteins, and possible splicing-related proteins.20 Some splicing proteins are common to all eukaryotes, whereas others are specific to particular species. Rice and Arabidopsis were predicted to have 108 and 111 splicing factors, respectively,21 whereas 71 human-related splicing factors are known to exist.22 In humans, the importance of AS is clearly seen in hereditary genetic diseases caused by aberrant splicing; for example, if C


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factors, SNW/Ski-interacting protein (SKIP), resulted in hypersensitivity of this mutant during salt and osmotic stresses.61 In drought-stress-induced ABA signaling, one wellknown example is the group A protein type 2C phosphatases (PP2Cs), which serve as negative regulators of abscisic acid (ABA) signaling and plant adaptation to stress. AS of regulator genes is essential for plants because it allows plants to cope with a changing environment. Two splice variants of the PP2C hypersensitive to ABA1 (HAB1), HAB1.1 and HAB1.2, have been demonstrated to have antagonistic roles in ABA-signal transduction. During seed germination, HAB1.2 competitively interacts with open stomata 1 (OST1), but it cannot inhibit OST1-kinase activity to modulate the ABA-signaling pathways.62 Furthermore, the highly orchestrated spliceosome assembly at each intron involves snRNAs and hundreds of proteins, which play important roles in plant growth and stress responses. Polypyrimidine-tract-binding-protein homologues (PTBs, PTB1, and PTB2), involved in early spliceosome assembly, have been documented to play crucial roles in the response to environmental stress by regulating the AS of hundreds of genes.63 Detailed analyses of many mutants with altered splicing have identified mutations that directly disrupt splice sites or splicing signals, as well as some that affect nearby sequences not predicted to alter splicing, illustrating how subtle sequence changes can determine splicing outcomes. Variations affecting splicing outcomes can provide flexibility in the transcriptome and proteome, which can contribute to the ability of plants to adapt to their environment.

(LHY), pseudoresponse regulator 5 (PRR5), and timing of cab expression 1 (TOC1).41 Furthermore, splicing factors have been identified that are responsible for circadian control. For instance, loss of function of a gene encoding the human-TFP11 homologue RBP spliceosomal timekeeper locus 1 (STIPL1) was observed to lengthen the circadian period.45 In addition, a mutant of SNW/Ski-interacting-protein (SKIP)-domain splicing factors was found to induce a long circadian period, and this long-period phenotype was dependent on alternating temperatures.46 Besides influencing the circadian clock, AS regulates flowering time and pivotal signaling pathways to initiate reproductive growth of plants in parallel with traditional transcriptional control. In particular, altered expression of splicing-related proteins has led to a change of time of flowering.47 Moreover, AS regulation of flowering converged to the famous flowering-control factor flowering locus C (FLC). Studies have indicated that the proportion of functional FLC transcripts could be affected by the up-regulation of a nonfunctional AS isoform, and this was regulated by an SR splicing regulator, SR45, in Arabidopsis.48 Intriguingly, AS regulation was also found in a natural antisense gene encoding an lncRNA.48 Furthermore, two flowering-related proteins, known as MADS associated flowering 1 (MAF1)/flowering locus M (FLM) and MAF2 were also affected by AS. For example, antagonistic-FLM-splicing variants have been reported to control flowering in a temperature-dependent manner.49,50 Meanwhile, low-temperature treatment induced one splicing isoform of MAF2 (MAF2-var1) and repressed MAF2-var2, potentially encoding a full-length repressor protein and a truncated protein, respectively.42 In addition, differential protein-interaction networks caused by the incorporation of various splicing isoforms of short vegetative phase (SVP) and MAF1/FLM have been demonstrated by using transgenic plant materials.51 Despite the aforementioned two developmental processes, a keystone study published recently has demonstrated that light induces extensive changes in AS via phytochrome-mediated signaling by affecting the 5′-end AS pattern and subsequent protein subcellular localization of the corresponding splicing isoforms.52 AS as a Regulator Used to Enhance Plant Stress Resistance. Similar to the facts that have been demonstrated in plant development, increasing evidence from both highthroughput sequencing and functional studies suggests that AS plays a crucial role in stress perception, signal transduction, and responsive mechanisms.39,53−56 For example, a group of conserved regulators known as heat-shock transcription factors (HSFs) have been found to undergo substantial AS modification under high-temperature stress.57 Particularly, one splicing isoform produced a truncated HsfA2 protein, generating a self-activated transcriptional regulatory loop by binding to its own promoter.57 Furthermore, a nuclear-targeted isoform of basic leucine zipper 60 (bZIP60) was produced under heat stress, resulting in the activation of genes involved in an unfolded-protein response.58 Emerging evidence has suggested that crucial splicing regulators, such as SR proteins, undergo extensive AS modifications under various stress treatments.59 However, the molecular functions of these identified splicing isoforms need to be validated in further investigations. Furthermore, genetic studies using knockout mutants have indicated that sr34 is hypersensitive to Cd stress, possibly resulting from the mis-splicing of Cd-transporter genes.60 In addition, a loss of function in one of the splicing

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CHEMICAL MODULATION OF AS FOR POTENTIAL GENETIC THERAPY AND CONTROL The AS process is known to be achieved through the precise assembly of the spliceosome, which is a dynamic supercomplex with snRNAs and the associated protein components. AS can be regulated by additional factors that interact with the spliceosome, making the situation complicated. Various disorders emerging from AS can be divided into two categories: external factors, including chemicals and environmental-cue-oriented alterations, and internal factors, including splicing-related proteins and splicing-site-selection-oriented alterations. Understanding the specific functions of the participating proteins and monitoring spliceosome assembly will help researchers determine how the alteration of this process can contribute to disease pathophysiology and plant adaptation to environment changes, which may provide new opportunities related to gene therapy and control. According to the recent advances, various chemical compounds may affect the different steps of splicing. For example, some compounds have been shown to directly target a specific factor of the spliceosome, but most have the possibility of functioning through acting on multiple factors. Moreover, because splicing factors can bind to short consensus cis-elements, compounds targeting a splicing factor have the potential to regulate the AS of multiple transcripts. Basically, those small molecules can influence every aspect of splicing, including snRNA backbones, splicing-related proteins, splicing regulators, and downstream effector proteins, by cutting at an exact location of the splice site, degrading splicing proteins, or binding to splicing-related proteins, as summarized and shown in Figure 2.64 Despite having some negative effects, small molecules that interfere with different aspects of the spliceosome assembly may provide valuable tools in uncovering novel mechanisms D


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proteins could unravel the role of the chemical modulation of spliceosomes for potential genetic therapy and control.

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STRATEGY AND MECHANISM USED FOR CHEMICAL MODULATION OF AS Molecules Targeting snRNAs. snRNAs are considered the backbone structure of each snRNP. An elegant approach to modulating AS is to screen for chemicals targeting snRNAs, which is a class of small RNA molecules that are found within the splicing speckles and are core components of the catalytic centers of spliceosomes. Extensive modifications, such as 2′-Omethylation and pseudouridylation, have been reported on snRNA in animals, and over 10% of vertebrate U2 snRNA is post-transcriptionally modified.72 In particular, the modification from uridine to pseudouridine (ψ) has been detected in eukaryotic rRNAs and snRNAs.73,74 In the first transesterification reaction of splicing, the 2′OH of adenosine at the branch site initiates a nucleophilic attack toward the phosphodiester moiety at the 5′ss of the pre-mRNA. In this reaction, the modified base ψ at the conserved site of the U2 snRNA interacts with the branch site, which refines the stability of the pre-mRNA and facilitates the first step in the process of splicing. In addition, this conversion potentially contributes to creating additional sites for RNA−RNA or protein−RNA interactions.75,76 The molecular mechanism of 5-fluorouracil (5FU, Table 1), a potent drug widely used in cancer therapy, have been studied for more than five decades77.78 Previous results led to hypotheses that propose that 5FU is able to interfere with DNA metabolism.79 However, the latest research proposed that 5FU can inhibit snRNA pseudouridylation and splicing.77 These researchers examined the inhibitory effect of 5FU on pre-mRNA splicing in HeLa cells and found that 5FU could incorporate U2 snRNA at several functional pseudouridylation sites.80 Interestingly, it also served as a potent inhibitor that affects U2 snRNA synthesis by blocking its site-specific pseudouridylation.81 Thus, the mechanism may contribute substantially to 5FU-mediated cell death, which can take effect during cancer therapy. Aside from use in cancer therapy, experiments carried out in several independent experimental systems have shown that the pseudouridines in spliceosomal snRNAs are functionally important for pre-mRNA splicing in plants. For example, inhibition of chloroplast-localized pseudouridine synthase can reduce plant sensitivity to phosphate starvation and thus trigger a suite of biochemical, physiological, and developmental changes, which can increase plant survival and growth.82 However, small molecules affecting snRNA pseudouridylation during splicing remain to be discovered. In addition, studies addressing the potential of snRNA targeting ranges from elimination of the mRNA in question to modification of the mature mRNA product and to repair the mRNA transcript should also be conducted. Molecules Targeting Core Spliceosomal Components. In comparison with chemicals that affect snRNA, small molecules targeting splicing factors are the most frequently found examples over the last two decades (Table 1). However, molecular targets of these drugs have not been reported until recently. For instance, sulfonamides and sulfonylated derivatives (e.g., indisulam, E7820, and chloroquinoxaline or CQS) target the 3′-splice-site-recognition complex U2AF and are used as anticancer agents for the treatment of different types of cancer.83 Indisulam can affect

Figure 2. Small-molecule-induced splicing regulation. Multiple components of pre-mRNA splicing could be targeted by small molecules, including structural-backbone snRNAs, core splicing components, and splicing regulators. As a post-transcriptional mechanism, alternative-splicing generates splice isoforms, which are responsible for the proteoforms (downstream effectors). In addition, some small molecules have been reported to affect the spliceosome assembly with unknown mechanisms. Thus, these small molecules could shed light on drug design of agrochemicals for plant-growth regulation, stress resistance, and pesticide development. U1, U2, U4, U5, and U6 designate different snRNPs. hnRNP, heterogeneous nuclear ribonucleoprotein; U2AF, U2-snRNP auxiliary factor; 5′ss, 5′ splice site; 3′ss, 3′ splice site; SF3b, splicing factor 3b; SR, serine− arginine-rich splicing factor; SRPK, SR-protein kinase; ESE, exonic splicing enhancer; ISE, intronic splicing enhancer; ISS, intronic splicing silencer; ESS, exonic splicing silencer.

that can be used to treat disease.64 Thus, chemical modulation of AS would provide a more straightforward method of identifying new drugs. For example, splicing factor 3B (SF3B) is one of integral parts of U2, which is one of the essential snRNPs and determines of correct excision of introns.65 A number of fermentation products with distinct chemical structures from Pseudomonas spp. (FR901463, FR901464, and FR901465)66 and Streptomyces spp.67 have been observed to target the human spliceosomal SF3B complex and have potential anticancer activities.66,68 These compounds have thus been synthesized and can serve as derivatives of new drugs to study their molecular mechanisms under cancer therapy. A flurry of studies has demonstrated that these drugs are able to affect both the expression levels and splicing patterns of cancer-promoting genes,69 which demonstrates the crucial role of the splicing machinery in cancer patients. Although some of these spliceosomes show potential for use as drug targets in cancer therapy, spliceosome-targeting small molecules can also trigger a developmental response in plants. For example, two SF3B-complex-targeting molecules, namely, Pladienolide B and Herboxidiene, have been demonstrated to inhibit both constitutive splicing and AS in Arabidopsis, thereby serving as good herbicide candidates.70,71 Although inhibitors targeted to the spliceosomal SF3B complex have been extensively investigated,64 the mechanisms involved in the actions of most of these chemical compounds remain largely unknown. Thus, understanding the molecular mechanisms of these small molecules that target spliceosomal E


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Journal of Agricultural and Food Chemistry Table 1. Effects of Small Molecules on Pre-mRNA Splicing

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Journal of Agricultural and Food Chemistry Table 1. continued

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Antitumor compound. bNatural product. cSmall molecule. dDrug. eHerbicide. fPesticide.

In the molecular aspect, the tri-snRNPs (U4−U5−U6) play pivotal roles in the early assembly of spliceosomes. Biflavonoid isoginkgetin (IsoG), which belongs to a subcategory of plant flavonoids, is a natural product initially isolated from leaf extract of Ginkgo biloba.88 Flavonoids have been demonstrated to elicit a variety of pharmacological activities against bacterial infection, inflammation, and cancer cells. In particular, this compound acts as a universal splicing inhibitor on both major and minor spliceosomes and was identified using a cell-based mammalian screening system.89 IsoG is also one of the few documented splicing inhibitors that does not target the U2associated SF3b subunit or other U2 snRNPs.90 In vivo, IsoG is capable of inhibiting pre-mRNA splicing by accumulating unspliced pre-mRNAs in the nucleus.89 Further in vitro experiments demonstrated that the inhibition of pre-mRNA splicing via uneven recruitment of U4−U5−U6 tri-snRNP

cancer-cell proliferation but was initially identified to be only effective on a small subset of tumors.84,85 However, no obvious toxicities were documented in cancer patients during clinical trials. Thus, a deeper insight of its molecular mechanism of antitumor activity has been elucidated, which might enhance its efficacy and spectrum in cancer therapy.86 Indisulam can induce protein ubiquitination and proteasomal degradation of some target proteins, including U2AF-related splicing factor and estrogen receptors (CAPERα). The selective degradation of these splicing factors has a great importance in reducing cancer risk by preventing the aberrant splicing of cancer cells.86 In plants, mutations of U2AF components, such as U2AF35a and U2AF35b, led to pleiotropic development impairment when treated plants were compared with wild-type plants,87 suggesting the potential role of this complex in plant development. G


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accumulate shorter isoforms of MDM2 with impaired function, thus leading to the activation of its downstream target, p53.102,103 It appears that this group of chemicals is able to alter chromatin structures by affecting the H3K36 methylation level.104 Although the same pharmacophore is used in classic SF3B inhibitors such as pladienolide and spliceostatin A, the potential interaction between sudemycins and SF3B components remains to be elucidated. Jerantinines (A−G) are indole alkaloids that were isolated from the leaf of Malayan Tabernaemontana corymbosa Roxb. Ex.105,106 These indole alkaloids showed multiple functions, including the inhibition of tubulin polymerization, the induction of cell-cycle arrest at G2/M, and the disruption of microtubules in multiple human cancer cell lines106.107 Intriguingly, jerantinine A (JA) has been demonstrated to target the spliceosome by upregulating SF3B components, such as SF3B1 and SF3B3.108 Furthermore, JA has been observed to stabilize SF3B1 protein in cells and influence the dissociation of the nucleosome complex.108 Herboxidiene (GEX1) is composed of six structurally related antibiotics isolated from Streptomyces spp. and has special potential for use as a potent herbicide.68 According to a recent study, GEX1A has been shown to have herbicidal activity and can inhibit pre-mRNA splicing by influencing SR proteins.70 This inhibition leads to abiotic stress in plants. In addition, GEX1A can delay seed germination of Arabidopsis. Furthermore, GEX1A can also cause negative effects on the growth and development of rice and tomato. It can act on abscisic acid inducible promoters of Arabidopsis in the abiotic-stress pathways to control stomatal closure.70 Furthermore, GEX1A was found to affect SF3B1/SAP155, an important splicing factor in the U2 complex, which has been identified by photoaffinity-labeling methods.68 Aside from the several aforementioned small molecules, most splicing modulators described to date, both those naturally occurring those synthesized (e.g., pladienolides, spliceostatin A, and herboxidine), target SF3B,100,109 a 450 kDa subcomplex of U2 snRNP containing seven proteins, including SF3B1−5, SF3B14, and PHF5A.110 The common molecular targets of these splicing regulators are concentrated to the largest subunit, SF3B1 (∼155 kDa), and its protein partner, PHF5A.111 Interestingly, two plant SF3B splicing factors have been demonstrated to play a crucial function in plant reproduction,112 suggesting that this splicing complex may have valuable candidates for designing agrochemicals. Molecules Targeting Splicing Regulators. Aside from those targeting core splicing components, several small molecules have been demonstrated to target splicing regulators and attenuate splicing outcomes (Table 1). The first example is the superoxide-producing, neurotoxic herbicide paraquat (PQ, 1,19-dimethyl-4,49-bipyridinium).113 PQ promotes the phosphorylation and nuclear accumulation of SRPK2, where SRPKs are activators of the SR protein.114,115 Hence, the changes of the aforementioned protein lead to modifications of premRNA splicing and then are involved in DNA repair, cell-cycle control, and apoptosis.116 To support the above explanation with previous findings, human neuroblastoma (SH-SY5Y) cells were incubated with 0.75 mM PQ for 18 h and examined by immunofluorescence microscopy. The intracellular distributions of several splicing-regulatory proteins were observed.113 Then, the formation of brighter nuclear speckles was seen as a result of hyperphosphorylation of SR proteins.113 For further elaboration, the PQ treatment increased the phosphorylation

results in a buildup of the prespliceosomal A complex, thus preventing spliceosome assembly from the A complex to the active spliceosome.89 Intriguingly, plant counterparts of trisnRNP have been reported to be involved in the process of seed development as well as in salt and osmotic resistance,91−93 showing themselves to be valuable targets in the development of agrochemicals. The U2-snRNP is responsible for the recognition of branch sites during splicing and coordinates early spliceosome assembly. The discovery of splicing-targeting small molecules brought together studies on this snRNP, especially at the SF3B complex. The following small molecules introduced here belong to this category (Table 1). Hinokiflavone is a biflavonoid compound that is characterized as an inhibitor of splicing through the obstruction of B-complex formation in the spliceosome.94 Furthermore, hinokiflavone was found to induce SUMOylation of cellular proteins and promote colocalization of spliceosomal-A-complex-associated proteins and SUMO enzymes both in vitro and in vivo.94 Mammalian splicing factors have been demonstrated to undergo substantial post-translational modifications (e.g., SUMOylation and phosphorylation) in attenuating spliceosome assembly and splicing efficacy.95 Pladienolides are 12-membered metabolic products isolated from a bacterial species, Streptomyces platensis Mer-11107, in a screen of repressors of hypoxia-induced genes.67,96 The unique pharmacological mechanisms of representatives of pladienolides (e.g., pladienolide B (PB), pladienolide D (PD), E7107, etc.) have been proposed by multiple research groups.97,98 Further experimental validation that employed photoaffinity labeling indicated that SF3B1 or SF3B3 could be the target of pladienolides.69 Another mechanistic study using point mutations suggested that the R1074H mutation of SF3B1 confers pladienolide resistance in cell lines.99 Interestingly, in plants, PB acts as a splicing inhibitor, which leads to the mimicking of abiotic signals such as salt, drought, and abscisic acid (ABA) in Arabidopsis. This plant stress hormone leads to stomatal-aperture adjustment.71 The effects of PB are very similar to the effects of GEX1A in plants, indicating that both of them inhibit pre-mRNA splicing.70 Naturally occurring FR901464 was initially isolated from Pseudomonas spp. as a putative antitumor reagent that had potent efficacy on a number of animal models. 66,100 Spliceostatin A (SSA) is a methylated and chemically stable derivative of FR901464. It has the ability to bind SF3B and inhibit in vitro pre-mRNA splicing, which results in unsplicedmRNA leakage into the cytoplasm and truncated proteins in yeast and mammalian cells; this occurs because introns are rich in stop codons in the same open reading frame following the upstream coding sequences.100,101 Similar phenotypes of unspliced-mRNA leakage have been observed in an SF3Bknockdown mutant, suggesting that the SF3B complex plays a crucial role in preventing unspliced-RNA leakage from the nucleus and is conserved from yeast to mammals. Therefore, FR901464 and its derivatives showed strong potential for controlling splicing. Sudemycins (C1, E, and F), are a group of synthesized small molecules with a consensus pharmacophore and are commonly present in pladienolide and spliceostatin A. These chemicals have been reported to have enhanced efficacy in tumor-cell models, resulting from their selective effects on the splicing patterns of their target genes, including caspase2/9 and BclX.102 Furthermore, sudemycin C1 has been proposed to H


Review


receptors only found in invertebrates such as Drosophila melanogaster.133 AS of GluCls has produced six different splice variants in small brown planthopper (Laodelphax striatellus).134 These new splice variants can be used as targets for discovering new insecticides because of the low efficacy of the previously available splice variants. Molecules with Unknown Mechanisms. The last group of small modulators are somehow found to affect proteins in AS, but their underlying mechanisms are unknown (Table 1). For example, amiprophos methyls (APMs) are herbicides that act as a kind of mitotic disrupter. They belong to the group of phosphoric amides that are known to inhibit the formation of microtubules, resulting in abnormal cellular morphology and mitotic disruption.135 A recent study revealed spindle-assembly and proteomic changes that were seriously disturbed in treated cells (10 μM APM for 12 h) analyzed by immunofluorescence and a proteomic technique. Moreover, the activity of DNA endonuclease, an RNA-splicing protein, is required for intron mobilization. DNA endonuclease has been identified as an APM-responsive protein and can induce mRNA folding and splicing in maize.135 Boric acid has some properties of biological resistance, such as the inhibition of mold and the disruption of termite growth. For example, boric acid and sodium borate can be used as insecticides to control various kinds of pests and are also used as ingredients in medicines and cosmetics.136 A novel mechanism was reported showing that boric acid can elicit a dose-dependent and reversible inhibition on the second step of pre-mRNA splicing.137 Then, the accumulation of B/C complexes and the inhibition of complex-M formation cause negative effects in organisms. In summary, the in vitro addition of 18 mM boric acid to a pre-mRNA-splicing reaction can efficiently and reversibly inhibit the second transesterification step of splicing.137

(hyperphosphorylation) of SR proteins. The results indicate that phosphorylation of serine 581 is required for nuclear accumulation of SRPK2. Because PQ treatment leads to the translocation of SRPK2 from the cytoplasm to the nucleus, hyperphosphorylation and accumulation of SR proteins results in nuclear speckles. These events may affect splice siteselection by modifying the balance between SR proteins and other splicing-regulatory proteins, such as hnRNP proteins. PQ can also increase intramitochondrial superoxide production to cause oxidative damage. However, in-depth analysis needs to be carried out for further confirmation of underlying mechanisms. SR proteins are also a conserved protein family that plays crucial roles in both AS and constitutive splicing in eukaryotes. According to a previous study, atSR45a, a homologue of SR protein in Arabidopsis, has six different splice variants, according to the AS of atSR45a pre-mRNA. Notably, SR proteins are sensitive to various types of stress conditions, including irradiation and salinity; as a result, alterations of AS occur.117 A representative small molecule, known as amiloride, has been demonstrated to participate in the regulation of AS in cancer cells.118 In particular, recent advancement indicates that amiloride can either affect the phosphorylation level of the splicing regulator SRSF1 or regulate transcript levels of SRSF3 and some other hnRNPs,118,119 which play roles in attenuating the splicing process. In addition, two compounds, chlorhexidine and TG003, were discovered as a result of a highthroughput cell-based screening of splicing inhibitors,120 and were observed to inhibit the SR-protein kinase Clks.121,122 Given that SR proteins have been demonstrated to play an essential role in various plant developmental processes and stress responses,123−125 SR proteins and their activating kinases are promising candidates for agrochemical design. Molecules Targeting Downstream Effector Proteins. In the natural environment, eukaryotic transcript isoforms resulting from AS could be translated into corresponding protein isoforms, namely, proteoforms, and serve as good candidates for agrochemical development in a variety of aspects. In the model plant Arabidopsis, approximately 83% of intron-containing genes were found to be alternatively spliced, giving a substantial number of targets for the development of agrochemicals.39 According to recent findings, some splice variants and their resulting proteoforms account for the resistance of some pests to agrochemicals (Table 1). Spinosad, a bioinsecticide isolated from a soil bacterium, is used to control leaf miners, fruit flies, and other insects. Meanwhile, the tomato-leaf miner (Tuta absoluta) is a common pest in tomato-growing regions.126 The insect nicotinic acetylcholine receptor (nAChR) α6 subunit is the target of spinosad. Resistance to spinosad is formed by exon skipping of the nAChRα6 gene.127 The Silkworm (Bombyx mori) shows differential AS of the nAChR gene, which has the maximum number of nAChRs subunits.128 Moreover, organophosphates are frequently used in agrochemicals targeting acetylcholinesterase (AChE). However, some splice variants of AChE do not have any sensitivity to organophosphates.129,130 γ-Aminobutyric acid (GABA) receptors are also targets of commonly used insecticides.131 The diversity of GABA receptors increases as a result of AS, which has resulted in insecticide resistance in Drosophila melanogaster.132 Glutamate-gated chloride channels (GluCls) are the targets of the insecticide fibronil. GluCls are neurotransmitter

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FUTURE PERSPECTIVES Alternative splicing is the starting point of proteome diversity in eukaryotes. The modifications of AS by small molecules can give rise to splice variants. Hence, genes that undergo AS are vital for plant biotic- and abiotic-stress resistance (Figure 2). Moreover, some splice variants of targets could cause major impacts on resistance and assist in target identification for agrochemicals. Gaining an understanding of new isoforms of AS that result from different small molecules is important when developing potential agrochemicals. Although many examples of splicing-targeting small molecules listed here are used as medicines and limited examples could be found for plants, we believe that the molecular targets of AS could serve as good candidates when developing agrochemicals in plants because of the conservation of splicing machinery among eukaryotic organisms. Several lines of evidence could support this hypothesis. First, the depletion of plant homologues of the molecular targets reported in disease therapy showed various developmental or stress-responsive phenotypes in plants, including in terms of SF3b components112 and SRSF1 splicing factors.124,125 Moreover, as SF3b-complex-targeting and antitumor compounds, pladienolides and herboxidiene were found to trigger abiotic-stress responses in the model plant Arabidopsis.70,71 Therefore, new strategies and technologies should be developed for the analysis of proteoforms, which may be used as valuable tools in this potential research area. I


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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (G.-F.Y.). *E-mail: [email protected] (G.-F.H.). ORCID

Ge-Fei Hao: 0000-0003-4090-8411 Guang-Fu Yang: 0000-0003-4384-2593 Author Contributions ○

M.-X.C. and B.D.I.K.W. contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported in part by the National Key Research and Development Program (Grant No. 2017YFD0200501), the Natural Science Foundation of Guangdong Province (Grant No. 2018A030313030), the National Natural Science Foundation of China (Grant Nos. 91853127 and 21772059), Shenzhen Virtual University Park Support Scheme to CUHK Shenzhen Research Institute, and the Hong Kong Research Grant Council (Grant Nos. AoE/M-05/12, AoE/M-403/16, and GRF12100318).

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Chemical Modulation of Alternative Splicing for Molecular-Target

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