Differences in alternative splicing and their potential underlying factors between animals and plants

Graphical abstract

Highlights

• •AS dysregulation is involved in a vast repertoire of diseases, including many cancers.
• •AS is important for reprogramming stress-related genes in response to abiotic stresses.
• •Divergence in intron, UTR, and exon confers gene architecture differences in animals and plants.
• •Members (e.g. U2AF) of spliceosome components evolved differently in animals and plants.
• •The main AS mode is exon skipping in animals versus intron retention in plants.
• •Compared to plants, animals have a greater diversity of hnRNPs but fewer SR proteins.

Abstract

Background

Alternative splicing (AS), a posttranscriptional process, contributes to the complexity of transcripts from a limited number of genes in a genome, and AS is considered a great source of genetic and phenotypic diversity in eukaryotes. In animals, AS is tightly regulated during the processes of cell growth and differentiation, and its dysregulation is involved in many diseases, including cancers. Likewise, in plants, AS occurs in all stages of plant growth and development, and it seems to play important roles in the rapid reprogramming of genes in response to environmental stressors. To date, the prevalence and functional roles of AS have been extensively reviewed in animals and plants. However, AS differences between animals and plants, especially their underlying molecular mechanisms and impact factors, are anecdotal and rarely reviewed.

Aim of Review

This review aims to broaden our understanding of AS roles in a variety of biological processes and provide insights into the underlying mechanisms and impact factors likely leading to AS differences between animals and plants.

Key scientific concepts of review

We briefly summarize the roles of AS regulation in physiological and biochemical activities in animals and plants. Then, we underline the differences in the process of AS between plants and animals and especially analyze the potential impact factors, such as gene exon/intron architecture, 5′/3′ untranslated regions (UTRs), spliceosome components, chromatin dynamics and transcription speeds, splicing factors [serine/arginine-rich (SR) proteins and heterogeneous nuclear ribonucleoproteins (hnRNPs)], noncoding RNAs, and environmental stimuli, which might lead to the differences. Moreover, we compare the nonsense-mediated mRNA decay (NMD)-mediated turnover of the transcripts with a premature termination codon (PTC) in animals and plants. Finally, we summarize the current AS knowledge published in animals versus plants and discuss the potential development of disease therapies and superior crops in the future.

Introduction

Unlike unicellular prokaryotes, higher eukaryotes evolved elaborate and interconnected regulatory mechanisms to coordinate, supervise, and regulate cell differentiation and the development of multicellular organization. Among the regulatory mechanisms, RNA alternative splicing (AS) is essential for gene expression control. Over 95 % of human multiexon genes can be alternatively spliced to produce multiple transcripts [1], [2], and in plants, approximately 83 % and 73 % of intron-containing genes undergo AS in Arabidopsis thaliana and Oryza sativa, respectively [3], [4]. These studies have consistently demonstrated that AS greatly contributes to the diversity of transcriptomes in animals and plants.

The RNA splicing process, including constitutive splicing (CS) and AS, is basically the same in animals and plants [5], [6]. During the process, precursor mRNA (pre-mRNA) is bound by the spliceosome complex, introns are excised through transesterification reactions, and exon segments are reconnected to generate a mature mRNA. Typically, the spliceosome complex is composed of five core small nuclear ribonucleoprotein particles (snRNPs), including U1, U2, U4, U5, and U6, and other related splicing factors and RNA molecules [5], [6]. In the early stage of spliceosome assembly, recognition of the 5′ splice site is initiated by U1 snRNP, and almost simultaneously, splicing factor 1 (SF1) is localized to the branch point sequence. The U2 small nuclear ribonucleoprotein auxiliary factor (U2AF), a heterodimer containing (U2AF65) and small (U2AF35) subunits, binds to the downstream polypyrimidine tract and 3′ splice site [5], [6]. With the assistance of U2AF and/or other splicing factors, U2 snRNP replaces SF1 and binds to the branch site [5]. Then, U4/U6.U5 tri-snRNPs bind to U1 and U2 snRNPs, forming a precatalytic complex. After multiple molecular rearrangements, the U1 and U4 snRNPs are further dissociated from the precatalytic spliceosome, and the activated spliceosome is formed for RNA splicing reactions. During splicing, an exchange reaction leads to a rapid degradation of intron segments, and two nearby exon ends are reconnected, whereby the process is highly controlled by the recognition and binding of trans-splicing factors and cis-acting elements on the pre-mRNA [5], [7]. According to their functions and relative positions, cis-acting elements can be classified into exon splicing enhancers (ESEs), intron splicing enhancers (ISEs), exon splicing silencers (ESSs), and intron splicing silencers (ISSs) [8]. These elements can recruit trans-splicing factors to promote or inhibit snRNPs to recognize and bind to splice sites, thereby regulating occurrences of CS and/or AS [8], [9].

There are five main AS types: exon skipping (ES), intron retention (IR), mutually exclusive exons (MXE), alternative 5′ splice site (A5SS), and alternative 3′ splice site (A3SS) [10]. Different outcomes of AS events may result in different transcripts and proteins, contributing to different physiological and biochemical activities in animals and plants. In animals, mounting evidence suggests that AS is prevalent in the cell cycle, growth, and differentiation [11], and its dysregulation is involved in a vast repertoire of diseases, including cardiovascular, autoimmune and neurological disorders, diabetes, aging and longevity, and finally cancers and even deaths [12]. Likewise, in plants, AS plays an important role in plant growth and development [13], and it seems to be of more importance in developing a rapid and global reprogramming of genes associated with abiotic and biotic stressors [14], [15].

The prevalence, regulation, and functional role of AS have been actively studied and extensively reviewed in eukaryotes, especially in animals. Although similar AS mechanisms are generally shared among eukaryotes, different adjustments can be found in different phyla [16]. In animals, AS mostly occurs in genes encoding the cytoskeleton, cell adhesion, and binding proteins [17], while in plants, AS seems to mainly respond to environmental stressors [18], [19]. To our knowledge, the prevalence, similarities, and basic differences of AS, the regulation of cotranscriptional splicing and chromatin dynamics on AS, and AS-mediated transcript and protein diversity and nonsense-mediated mRNA decay (NMD) between plants and humans have been well reviewed and/or discussed previously [18], [19], [20], [21], [22]. However, the influencing factors underlying AS differences between animals and plants are far less clear and sometimes considered anecdotal.

In this review, we summarize our current understanding of AS roles in plants and animals, mainly focusing on AS differences and the potential underlying impact factors, and further compare NMD-mediated turnover of transcripts with a premature termination codon (PTC) partially introduced by AS. Finally, we conclude the reports of AS published in animals and plants and discuss that the knowledge of AS regulatory mechanisms can potentially be used for the development of disease therapies and the breeding of superior crops.

AS in animals

In animals, AS is tightly regulated in the expression of many genes and is involved in physiological and biochemical activities. For example, over 95 % of human multiexon genes undergo one or several AS events [2], among which more than 40 % are exon skipping (ES) events that contribute to cell differentiation, immune defense, and basic physiological activities [2], [23].

Cell differentiation and migration

Cell differentiation, tissue morphogenesis, and organ development in animals are controlled by a complex regulatory network of many genes. It has been determined that AS events are highly enriched in human neurons, hearts, and other organs, suggesting a role of AS in the gene regulatory network for cell differentiation and organ development [1], [24]. As cells progress along the neuronal lineage, splicing factors, such as polypyrimidine tract binding proteins (PTBPs) and serine/arginine repetitive matrix protein 4 (SRRM4; also known as nSR100), are interconnected and fine-regulated to adjust AS outcomes, which further affect neuronal differentiation and morphology [25]. For example, PTBP1 is abundant in neural stem cells and progenitors and can abolish the function of PTBP2 by promoting exon 10 skipping of PTBP2 to produce a short transcript. The downregulation of PTBP1 induces the expression of the long PTBP2 transcript encoding a functional splicing factor, which is essential for neural differentiation and maturation [26], [27]. Additionally, many PTBP-binding sites are enriched surrounding SRRM4-dependent exons, suggesting an interplay between splicing factor SRRM4 and PTBPs [25]. Moreover, SRRM4 promotes the splicing of the short isoform of repressor element 1-silencing transcription factor (REST4) to increase the expression of miR-124, which further targets PTBP1 mRNA and reduces its expression [25]. During neuronal differentiation and maturation, other splicing factors, such as NOVA1/2, Rbfox2, RBM4, and SLM1/2, are also involved in a coordinated or conjunctive manner to regulate AS of gene transcripts [27].

Some splicing factors may simultaneously regulate the commitment and differentiation of different cell types by adjusting AS events of gene transcripts. For example, in addition to a role in neural differentiation and maturation, PTBP1 can couple with RNA binding motif protein 20 (RBM20) to regulate the AS process and contribute to cardiomyocyte differentiation [28], [29]. Additionally, RBM20 controls two spliced variants of titin, N2A and N2BA, and their ratio is critical for normal myocardial wall stiffness [30]. Moreover, in the early stage of muscle cell differentiation, PTBP1 inhibits exon 5 inclusion of cardiac troponin T (cTNT) in primary embryonic skeletal muscle and induces exon skipping of α-actin 1 in smooth muscle cells, thereby controlling the differentiation of muscle cells [28].

AS also plays a crucial role in signal transduction during cell migration [31]. To enhance cell migration signaling, epidermal growth factors (EGFs) trigger hnRNPA1 ubiquitination, activate SR proteins, and regulate AS of GTPase Ras-related c3 botulinum toxin substrate 1 (Rac1), which are well-characterized factors in the migration of cancer cells [32]. Additionally, AS is involved in the remodeling of the F-actin cytoskeleton in lamellipodia upon cell migration. Normally, a functional transcript of cortactin is produced to bind to F-actin and control the deposition of extracellular matrix proteins, and in contrast, when alternatively spliced variants of cortactin are abnormally produced, they reduce the binding to F-actin and lead to cell migration [33].

Cell growth and metabolism

The expression of genes involved in cell growth and metabolism is also regulated by AS. For example, the mechanistic target of the rapamycin (mTOR) pathway is a key regulator of cell growth and metabolism by regulating protein synthesis, cell cycle, and lipid metabolism [34]. Hyperactivation of mTOR leads to loss of proteome function by upregulating the content of SRSF3, which in turn regulates downstream genes to generate more exon-skipping transcripts that often encode truncated proteins with loss of functional domains [35]. Moreover, mTOR-mediated AS regulates the expression of the long isoform of Sirtuin 2, which creates a unique phosphoserine site allowing cell cycle-dependent chromatin localization [35]. Alternative splicing is also involved in the expression of genes in other metabolic pathways. For example, the pyruvate kinase M gene (PK-M) undergoes AS regulated by hnRNPs to produce two isoforms with mutually exclusive exon fragments (M1 and M2), which impact the production of ATP in energy metabolism [36].

AS dysregulation and carcinogenesis

AS dysregulation and/or mutation may lead to the production of abnormal transcripts and protein isoforms, which may cause diseases such as neurological disorders, heart and skeletal muscle abnormalities, multiple genetic disorders, and even cancers [12]. In past years, many AS-initiated cancers have been extensively studied [37], [38]. Splicing factor 3b subunit 1 (SF3B1), an important part of the U2 snRNP, can recognize 3′ acceptor sequences in intron fragments and regulate the early splicing stage, and its dysregulation is widely found in cancers [39]. For example, RNA-seq analysis of chronic lymphocytic leukemia patients showed that mutation of SF3B1 could lead to extensive changes in 3′ splice site recognition and produce transcript frameshifts and/or introduction of premature termination codons (PTCs), which results in protein truncation and/or suppression of gene expression by NMD [40]. P53, also known as tumor protein 53 (TP53), acts as an important tumor suppressor to inhibit cancer development in various cellular functions, such as cellular senescence, apoptosis, and DNA repair [41], [42]. Stabilization of the p53 protein requires the participation of murine double minute 2 (MDM2). Mis-splicing of MDM2 produces a variety of splice variants, which directly lead to functional changes in p53 in transcriptional splicing, cellular localization, and protein stability [41]. Additionally, abnormal AS of the p53 gene could result in G1 cell cycle arrest and promote cell senescence and apoptosis [43].

AS in plants

Similar to AS events involved in cell growth, differentiation, migration, and carcinogenesis in animals, AS events are involved in various aspects of plant growth and development and environmental stress responses [13], [15]. In contrast to a dominant percentage of exon skipping (ES) in animals, intron retention (IR) generally shows the highest percentage in plants [44], despite a possible underestimation of non-IR events in plants suggested by a recent study [18].

Growth and development

Plant growth and development are controlled by a complex regulatory network of many genes involved in alternative splicing. For example, the MADS-box transcription factor FLOWERING LOCUS M (FLM) undergoes temperature-dependent AS to produce differential accumulation of FLM-β and FLM-δ transcripts in Arabidopsis [45]. The two transcripts encode antagonistic proteins and compete for interaction with the floral repressor SHORT VEGETATIVE PHASE (SVP) in controlling floral transition. At lower temperatures, FLM-β represses flowering, while FLM-δ is preferentially produced at higher temperatures to promote flowering [46]. In rice, grain size 3 (GS3) is a major protein that controls grain size and weight [47]. The GS3 gene can produce two major isoforms (GS3.1 and GS3.2), of which GS3.2 encodes a truncated protein containing only the organ size regulation (OSR) domain due to a PTC introduced by retention of a 14-bp intron sequence. This truncated protein competitively binds to the heterotrimeric G protein β-subunit (RGB1), disrupting the GS3.1 signaling pathway and abolishing the negative effect of GS3.1 on grain size [48].

Abiotic and biotic stress responses

Plants can control their life activities by regulating AS to limit the negative effects of environmental stressors [14], [15], [19]. For example, under low temperatures, some components of the circadian clock are alternatively spliced, including transcripts of CIRCADIAN CLOCK ASSOCIATED 1 (CCA1), LATE ELONGATED HYPOCOTYL (LHY), and PSEUDO RESPONSE REGULATORs (PRRs, such as PRR1, PRR3, PRR5, PRR7, and PRR9), contributing to the cold responsiveness of downstream gene expression [49], [50]. Recently, comparative transcriptomics revealed an ancient circadian component-launched cold-responsive network in rosids at the transcriptional and AS regulatory levels [49]. In addition to low temperature, high temperature is another important environmental factor that significantly affects AS in plants. For example, heat shock transcription factor A2 (HsfA2) is subjected to several IR events under high temperatures (42 °C–45 °C). Among the generated transcripts, HsfA2-Ⅲ encodes a truncated S-HsfA2 that acts as a positive transcriptional activator to enhance the expression of HsfA2 for heat tolerance [51]. In Solanum lycopersicum, the pre-mRNA splicing efficiency under nonextremal heat stress is also directly regulated by the intron polymorphism of HsfA2. Compared to wild-type lines, domesticated cultivated tomatoes accumulate more HsfA2-Ⅰ but less HsfA2-Ⅱ, and the accumulated HsfA1-I enhances tomato resistance to repeated heat stress [52].

Interestingly, increasing studies showed that plants have evolved “molecular memory” mechanisms to confer stress defense[53], [54]. For example, under heat stress, compared to “primary stressed plants”, “heat-shock memory plants” generate more H3K36me3, which in turn leads to lower levels of IR and more efficient splicing capacity when exposed to high temperatures again [55]. Given that PTCs are often introduced by retained intron segments, many IR-containing transcripts may undergo NMD to maintain the homeostasis of dynamic transcripts, which is essential for plant growth and development and stress responses [56]. For example, IR-containing transcripts of two homologous galactinol synthase (GolS1/2) genes contain a conserved PTC in rice photosynthetic tissue, and their premature transcripts could thus be degraded by the NMD pathway under drought and salt stresses [57].

Moreover, AS plays a role in resistance gene (R)-driven effector-triggered immunity (ETI) in plant immune responses [58]. For example, Arabidopsis resistance to pseudomonas syringae 4 (RPS4) generates multiple AS transcripts via retention of intron 2 and/or intron 3 [59]. In mos14-1 (a mutation suppressing the immune responses), the nuclear import of several SR splicing factors is disabled, which leads to an accumulation of the RPS4 transcript with intron 2 and 3 retention and an impaired immune response [60]. Recent studies have also demonstrated that external microorganisms can weaken plant immunity by modulating AS in plant hosts [61], [62]. For example, overexpression of Puccinia striiformis f. sp. tritici arginine-rich effector (Pst_A23) in wheat decreases functional transcripts of TaXa21-H and TaWRKY53, thereby interfering with wheat resistance to Pst [62].

Comparison of AS between animals and plants

Despite a common process of RNA splicing and AS after gene transcription in animals and plants, many different features have evolved, including gene exon–intron architecture, 5′ and 3′ untranslated regions (5′ UTRs and 3′ UTRs), spliceosome components, and predominant types of AS events.

Gene Exon-Intron architecture

The composition of eukaryotic genes evolved extensively by intron/exon loss or gain, and gene architecture is quite different in animals and plants. In animals, a large number of long introns have been detected, and some even reach hundreds of kilobase pairs (kb) in size [63], [64]. However, in 'higher' plants, the length of most introns is generally shorter than that in animals [65]. One possible explanation is that plant introns contain fewer transposable elements than animal introns [65]. To further compare gene architecture in animals and plants, we calculated and compared the exon/intron number of protein-coding genes and their length in 18 selected representative species (Fig. 1). Except for 'lower' animals (Caenorhabditis elegans in this review), the exon number in 'higher' animals is generally more than that in plants, but their exon length is far shorter than their counterpart in plants. Meanwhile, 'higher' animals seem to have longer introns. Thus, genes in 'higher' animals containing shorter but more exons interrupted by longer introns are more likely to be alternatively spliced than those in plants, which may be attributed to different evolutionary processes of animals and plants [66], [67].

Fig. 1.

Differences in gene architecture between animals and plants. The evolutionary tree of the 18 selected species was collected from the TimeTree website [169]. The boxplots show comparisons of exon number and lengths of exons, introns, 5′ untranslated regions (5′ UTRs), and 3′ UTRs of genes between animals and plants. Difference between animals and plants was assessed by a two-sided Mann-Whitney test in R programming. Gene architecture data was extracted from the reference gene annotations of the species.

Interestingly, the inferred most recent common ancestor (MRCA) in the evolutionary history of multicellular organisms contains ∼ 3.39 introns per kb, suggesting a greater density of introns than most extant fungi [67]. In primitive unicellular eukaryotes, AS has a low incidence and is dominated by IR, suggesting that IR may be the oldest type of AS [68]. However, the gain or loss of introns between and within plants and animals has changed extensively in subsequent evolution. The gain of more introns and/or the split of preexisting exons in animals in combination with the advent of exon-defining mechanisms probably make exon skipping the predominant AS event in animals [66]. In contrast, the occurrence of high IR frequency in plants may be caused by a slower evolution of the AS mechanism and a high level of polyploidy [69].

Untranslated regions (UTRs)

The 5′- and 3′-UTRs of transcripts play distinct and independent roles in gene expression [70]. In UTRs, there are conserved features and evolved differences between and within animals and plants. For example, the base composition in UTRs shows a conserved pattern of high uracil (U) and low cytosine (C) contents in animals and plants [71]. Regarding the evolved differences, compared to animals, many plants evolved longer 5′-UTRs (Fig. 1), in which the occurrence of IR events could enhance translation efficiency through the intron-mediated enhancement (IME) process [68]. In photorespiration regulation, the maximal abundance of the glu:glyoxylate aminotransferase 1 (GGT1) transcript is mediated by its 5′ UTR leader intron, and the intron enhances promoter activity by recruiting RNA polymerase Ⅱ [69]. Zinc-inducible promoter 2 (ZIF2) in Arabidopsis root cortex cells produces two transcripts (ZIF2.1 and ZIF2.2). Intron retention in the ZIF2.2 5′ UTR enhances translation in a zinc-sensitive manner, which promotes the expression of ZIF2 and enhances plant tolerance to metal ions [70]. In contrast, transcripts with short 5′ UTRs in animals (Fig. 1) are reported to be generally associated with the most highly expressed genes [72]. These studies suggest that the 5′ UTRs, at least some of them, may have evolved different roles in regulating gene expression and translation between plants and animals.

In addition to the regulatory role of the 5′ UTR, 3′ UTR-mediated regulation has been described in alternative splicing, resulting in different posttranscriptional processes, such as RNA stability, transport, translation, and even protein localization [73]. Interestingly, compared to that of the 5′ UTR, the length of the 3′ UTR shows a reversed pattern between animals and plants (Fig. 1). A longer length of the 3′ UTR explains a higher possibility of AS that is prevalent across the 3′ UTRs of oncogenes especially in tumorigenesis [74]. For example, the oncogene CTNNB1 exhibits preferential splicing in the 3′ UTR, which enhances its translation and promotes cell proliferation and migration [74]. In some plants, the presence of a full-length transcript including the 3′ UTR may enhance its effectiveness [73]. For example, transgenic lines containing the vacuolar Na(+)/H(+) antiporter gene (OsNHX1) with the 3′ UTR were significantly more tolerant to salt stress than transgenic lines of the same gene but without the 3′ UTR [73].

Spliceosome components

As described in the Introduction section, small nuclear ribonucleoproteins (snRNPs) are protein-RNA complexes composed of specific snRNP-related proteins and small nuclear RNAs (snRNAs) for RNA constitutive splicing and AS processes [5], [6]. Although the core components of snRNPs are conserved in the primary and secondary structures between animals and plants, there have evolved some different features in snRNAs or snRNPs that are not interchangeable in the recognition of introns and the splicing of pre-mRNA [75], [76], [77]. Among snRNPs, U2 snRNP is an important subcomplex that participates in the first step of spliceosome assembly together with U1 snRNP. The recognition of splicing sites by U2 snRNP in animals is highly dependent on U2AF, which is composed of U2AF65 and U2AF35 subunits. However, the number of U2AF coding genes is quite different in animals and plants. For example, in humans, there are two U2AF genes: U2AF65 and U2AF35, while in Arabidopsis, four members have been identified, including U2AF65a, U2AF65b, U2AF35a, and U2AF35b [76]. U2AF65 and U2AF35 are conserved to bind the polypyrimidine tract and 3′ splice site, facilitating U2 snRNP recruitment to 3′ splice site region in both animals and plants [76], [78]. However, in plants, such as Arabidopsis, introns often have noncanonical intron boundaries, and it may be more difficult for U2 snRNP to recognize 3′ splice sites than in animals, and splicing noncanonical introns in plants seems to be largely dependent on the assistance of U2AF [79], which explains well mutiple U2AF copies in plants. Thus, the differences in spliceosome components may also be a part of the factors that lead to differences in RNA constitutive splicing and AS between animals and plants.

Additionally, there are other factors associated with the remodeling of the spliceosome, such as the nineteen complex (NTC). The NTC is involved in the combination of U4/U6.U5 tri-snRNPs and plays a role in the assembly and disassembly of spliceosomes, excision of intron fragments, and molecular rearrangement of exon fragments [80]. Although the NTC is highly conserved with the same or similar functions across species, some components of the NTC may have evolved differently between plants and animals. For example, SYF2, a core component of NTC, has only one or two common interacting proteins between animals and plants, indicating that SYF2 might have evolved specific interacting networks in different phyla [81]. Additionally, SYF2 in animals has an average of two or three different transcript variants with an N-terminal truncated protein, while in most plants, SYF2 seems to be a single copy [82].

Predominant AS events

It has been reported that IR is the major AS type in plants, whereas ES is the predominant mode in animals [44]. To confirm this pattern, we compared the proportion of various AS events in representative animals and plants by computing AS events from publications [82], [83], [84], [85], [86], [87], [88], [89], [90], [91] (Fig. 2). The proportion of IR events in 10 plant species ranges from 29.0 % to 56.1 % significantly higher than that of the other four types of AS events, which is also supported by isoform sequencing (Iso-seq) analysis in Arabidopsis and rice [92], [93]. Interestingly, the gymnosperm Ginkgo biloba evolved a considerable number of ES events, which might be caused by the insertion of transposable elements across genes [94]. In contrast, the highest proportion of AS events is ES in eight animal species, also described by Iso-seq in humans and mice [95], although the 'lower' animal C. elegans shows comparative numbers of ES and IR events (Fig. 2). This phenomenon may be related to differences in gene exon/intron architecture and diversification of splicing factors between plants and animals. As described above, ES events in animals play important roles in cell growth, differentiation, and migration, and their dysregulation could lead to carcinogenesis. In contrast, IR events in plants contribute significantly to plant growth and development and stress responses [44], [56], [57]. Although a recent study suggests that non-IR events are likely underestimated, IR events are still the major sources of alternative splicing in plants [18]. Another recent large-scale study found that more than half of introns remain unspliced after Pol II transcribes past their 3′ splice sites, which suggests that chromatin-tethered posttranscriptional splicing might be an important contributor to the widespread IR events in plants [96]. However, the factors leading to the main difference in ES in animals versus IR in plants remain to be resolved.

Fig. 2.

Percentage comparison of five AS types in animals and plants. The percentage of AS types was obtained from previous publications mainly based on short-read sequencing [82], [83], [84], [85], [86], [87], [88], [89], [90], [91] and was roughly consistent with long-read sequencing analysis [92], [93], [95]. Alternative transcription starts and termination sites are not included in the AS analysis in this study. IR: intron retention; ES: exon skipping; A5SS: alternative 5′ splice site; A3SS: alternative 3′ splice site; Others include mutually exclusive exons (MXE) and complex AS events.

Other factors influencing AS differences in animals and plants

Moreover, many other factors could influence the course of AS and induce AS differences between animals and plants, such as chromatin dynamics, transcription speed, and the activities of splicing regulators (e.g., the well-known SR proteins and hnRNPs). Additionally, some noncoding RNAs (ncRNAs) and external stimuli of environmental conditions also play a role in AS outcomes.

Chromatin dynamics and transcription speed

Chromatin dynamics and transcription speed contribute to the regulation of cotranscriptional processes, including splicing, polyadenylation, and transcription termination, thus affecting the production of AS variants [97]. During the process, it seems that slow transcription allows weak splice sites to be recognized and leads to a higher inclusion of alternative exons. Unexpectedly, slow or fast transcription does not necessarily increase or decrease the inclusion of alternative exons or introns in opposite directions [97], [98]. Moreover, the slow and fast transcription mutants of Drosophila and Arabidopsis both enhance constitutive splicing [99], [100], whereas slow and fast transcription mutants of human cells inhibit constitutive splicing [101]. Therefore, there is a complex role of RNA Pol II speed in regulating constitutive and alternative splicing, which seems to depend on the species, genes and/or other contexts.

Recently, Agirre et al. identified eleven chromatin modifications that differentially mark AS exons in a combinatorial and position-dependent way, which creates splicing-associated chromatin signatures (SACS) [102]. Of the SACS, H3K36me3, a hallmark of transcription elongation, can mark the recognition splice site to recruit the splicing factor PTBPs and form a chromatin splicing complex [103]. In animals, this histone mark appears to be significantly associated with constitutive exons [104], while it is prevalent at the beginning of the gene body (or promoters) in plants [55]. The same histone modification but in different positional associations may trigger different AS effects in animals and plants. Moreover, a link between nucleosome positioning and exon–intron structure recognition has been shown [105]. Further comparative analysis of the gene architecture of animals and plants suggests that the promoters of Arabidopsis genes have a lower GC content and lack nucleosome binding [106]. Accordingly, plant Pol II elongation rates are higher at transcription initiation sites, and substantial Pol II accumulation is also detected at polyadenylation sites [107]. These findings indicate that plants may mainly regulate transcription at the initiation level, which reflects the difference in chromatin dynamics during cotranscriptional splicing [20].

SR proteins

SR proteins are a class of RNA-binding proteins that have an N-terminal RNA recognition motif (RRM) and a C-terminal serine/arginine-dipeptide rich (RS) domain [108]. SR proteins are ubiquitous in cells, some of which can participate in transcription-coupled splicing and mRNA processing in the nucleus, while some are nucleocytoplasmic shuttling factors (such as SRSF1, SRSF3, SRSF7, RSZ22, SR34, and SR34a), indicating that SR proteins may participate in multiple cellular activities [109]. SR proteins are highly conserved in regulating AS by binding to exon splicing enhancers (ESEs) in animals and plants. Regulatory elements and self-phosphorylation levels of SR proteins may affect different AS outcomes [110]. SR protein binding sites appear not only in alternatively spliced exons but also in constitutively spliced exons, which shows that SR proteins regulate AS and constitutive splicing [38], [111].

The number of SR genes varies greatly between animals and plants, with 12 in humans and 18 in Arabidopsis [112]. In animals, SR proteins such as SRSF11, SRSF2 and SRSF10 are widely conserved [113], among which SRSF11 has been reported to be correlated with human telomerase activity. Human telomerase reverse transcriptase (hTERT, a core component of telomerase) requires translation of hTERT mRNA, but overexpression of SRSF11 strongly increases the transcript level of the variant with hTERT exon 7–8 deletion, which in turn negatively regulates telomerase activity [114]. For another example, SRSF10 can regulate hundreds of AS events by binding to the GAAA-rich motif [115] and its knockout exhibits severe hypoplasia of subcutaneous white adipose tissue due to defective adipogenic differentiation, which is most likely due to the erroneous skipping of exon 7 in the lipin1 gene [116]. Compared to animals, 'higher' plants generally have more SR genes and unique subfamilies (Fig. 3). This may be explained by a rampant occurrence of whole-genome duplication events (WGDs) in higher plants [117], [118], despite a relatively low number of SR genes in certain plant species (e.g., Vitis vinifera and Chlamydomonas reinhardtii) due to fewer occurrences of WGDs [119]. Given successive WGDs in 'higher' plants, SR genes underwent several rounds of duplications to produce plant-specific SR genes. For example, in Arabidopsis, SR genes in the SR, RSZ and SC subfamilies are homologs of human SRSF1, SRSF7 and SRSF2 [120], but other SR genes in the RS, SCL, and RS2Z subfamilies are unique with novel structural features in plants [121]. Likewise, despite a shortage of SR genes, animals have evolved unique SR subfamily genes, such as SRSF4/5/6, SRSF11, and SRSF10 (Fig. 3).

Fig. 3.

Comparison of SR gene number and classification between animals and plants. The number and subfamily classification of SR family genes was obtained from previous studies [113], [170]. Differential analysis of SR gene number between animals and plants was assessed by a two-sided Mann-Whitney test.

Among RS, SCL, and RS2Z in plants, the RS2Z subfamily contains two zinc finger domains and is largely restricted to land plants [121]. RSZ33, a member of the RS2Z subfamily, can interact with the cyclin-dependent protein kinase cyclin-dependent kinase g1 (CDKG1) to regulate the splicing of callose synthase 5 (CalS5) for maintaining normal pollen wall formation [122]. SCL, the largest plant-specific subfamily, is present in a variety of plants, including eudicots, monocots, and mosses. Heterologous transformation of Populus trichocarpa PtSCL30 into Arabidopsis resulted in abnormal AS changes in cold tolerance-related genes (e.g., ICE2 and COR15A), thereby reducing the cold tolerance of the plants [112]. In addition to SR proteins, there are many SR-like proteins in plants that have similar functions to SR proteins [123]. For example, SR45 has an additional RS domain at the N-terminus and can directly bind to U1-70 K and U2AF35 in splicing regulation. It is speculated that the RS domain is a characteristic domain of SR-like proteins that performs splicing functions [108], which still needs experimental evidence. Compared to animals, plants generally have more SR and SR-like proteins, and thus, there might be more complex regulatory relationships and probably functional redundancy.

HnRNPs

Compared to SR proteins, hnRNPs form a more structurally diverse group of RNA-binding proteins that are involved in both constitutive and AS processes [5], [124]. The main domains of hnRNPs can be divided into two categories: RNA-binding domains and auxiliary domains. The RNA-binding domains mainly include RRM, K-homology (KH), and RGG (arg-gly-gly repeats). The auxiliary domains may include gly-rich or proline-rich acidic regions [124]. A challenge in hnRNP research is the high flexibility in the definition and classification of hnRNPs, and in humans, a total of 37 hnRNPs were identified and classified into 13 subfamilies [124]. Interestingly, these hnRNPs can bind to a great catalog of cis-regulatory motifs in pre-mRNAs. For example, hnRNP H1 binds to GAAGAG or G(G/A)₇, hnRNP M binds to GGUUGGUU, hnRNP A1 binds to GUAGUA, and hnRNP A2/B1 binds to GGUAGGUAG or AGG(A/U)U [125]. These different motif-binding preferences suggest functional divergences of hnRNPs in recognizing and regulating RNA splicing in different pre-mRNA loci. Compared to plants, animals have a higher number of hnRNPs, which suggests a greater diversity of hnRNPs. This shows a contrasting relationship of fewer SR genes in animals than in plants (see 5.2. SR Proteins). The increased number and diversity of hnRNPs in animals seem to originate from successive gene duplication events, which, together with contrasting differences in SR genes, may be related to the complexity of AS in the evolutionary process of plants and animals [126].

In animals, hnRNPs play important roles in stem cell differentiation, disease occurrence and treatment, and sustaining circadian rhythms [124]. HnRNP A1 and hnRNP H cooperate to modulate 5′ splice site selection [127]. hnRNP H/F regulate the splicing of T-cell factor 3 (TCF3), which is involved in embryonic stem cell differentiation [128]. HnRNP K cooperates with nuclear speck-related protein NS1-binding protein (NS1-BP) to regulate splicing of the M segment in influenza A virus (IAV), while mutation of hnRNP K and/or NS1-BP results in mis-splicing of the M segment [129]. In contrast, the functional analysis of plant hnRNPs is significantly lagging, although some progress has been made. Arabidopsis hnRNP glycine-rich RNA-binding protein 7 (GRP7) affects the splicing of glutathione s-transferase zeta 1 (GSTZ1), Arabidopsis pumilio protein 23 (APUM23), and other genes [130]. Overexpression of GRP7 and its close homolog GRP8 affect the AS of FLM, which increases the content of the flower inhibitory isoform FLM-β and affects the flowering time of Arabidopsis [131]. LHP1-interacting factor 2 (LIF2) in Arabidopsis, a homolog of human hnRNP-Q, interacts with like heterochromatin protein 1 (LHP1) to affect gene expression during flowering and flower-related tissue differentiation. In addition, LIF2 also participates in plant innate immune responses via salicylic acid- and jasmonic acid-dependent pathways [132]. However, the functions of hnRNPs in plants are still poorly understood and need to be further investigated, especially in the complex interactions of hnRNPs with other splicing factors and their regulation in AS.

NcRNAs

In humans, noncoding RNAs (ncRNAs) are involved in various biological activities and play important roles in promoting cell invasion, metastasis, antiapoptosis, and drug resistance in the process of cancer occurrence [133]. Many different types of ncRNAs have been identified in AS regulation, such as microRNAs (miRNAs), long noncoding RNAs (lncRNAs), and circular RNAs (circRNAs). miRNAs are short, approximately 19–25 nucleotides, and its maturation process is initiated by a microprocessor composed of RNase Ⅲ and its cofactor DiGeorge Critical Region 8 (DGCR8) [134]. A study found that miR-10b binds to U6 snRNP and alters U6 pseudo uracil content and conformational stability, thus affecting AS of GTPase cell division control 42 (CDC42) transcripts and promoting glial tumor differentiation (Fig. 4A) [135]. CircRNA 100,146 binds to miR-361-3p and miR-615-5p in non-small cell lung cancer (NSCLC) and releases the blockade of SF3B3 translation, which activates the processing of SF3B3-mediated AS to promote cancer cell growth and invasion (Fig. 4B) [136]. Additionally, circRNAs can directly affect the functions of splicing factors to regulate AS processes. For example, a circRNA called cTTN1, generated by RBM20-dependent splicing of human Titin (TTN), has a unique motif bound to the splicing factor SRSF10 to regulate the splicing of its target genes (e.g., MEF2A and CASQ2). Moreover, cTTN1 has a feedback regulatory role in localization of RBM20 to the nucleus and plays a role in the process of TTN splicing to produce cTTN1 (Fig. 4C) [137].

Fig. 4.

Examples of ncRNAs regulating AS events in animals. (A) miRNA regulates RNA splicing. Binding of miR-10b to U6 snRNA results in U6 N-6-A methylation and the formation of U4/U6.U5 tri-snRNPs to further promote exon inclusion of cell division control 42 (CDC42) mRNA [135]. (B) Combined effects of miRNA and circRNA on splicing factor activities. By binding miR-361-3P and miR-615-5P, circRNA 100,146 acts as a molecular sponge to maintain the translation of the SF3B3 transcript, which further regulates AS events of many genes in non-small cell lung cancer [136]. (C) CircRNAs control the localization of splicing factors and participate in the AS process. CircRNA cTTN1 promotes the nuclear localization of RBM20 to regulate the splicing of Titin (TNN). cTNN1 also binds to SRSF10 and inhibits exon 9 skipping of MEF2A in cardiomyocytes [137].

In plants, studies of lncRNAs in regulating AS are just beginning to emerge [138], and the main mechanisms can be roughly divided into three types: 1) interacting with splicing factors to change the structure and function of transcription; 2) forming a DNA-RNA or RNA-RNA duplex structure to mediate transcription and/or the binding of splicing factors; and 3) affecting chromatin structure and RNA transcription processes [139]. For example, early nodulin 40 (ENOD40) binds to RNA binding protein 1 (RBP1) and promotes its nucleocytoplasmic transport, thereby enhancing the ability of RBP1 to participate in AS regulation (Fig. 5A) [140]. Arabidopsis lncRNA alternative splicing competitor (ASCO) was identified to bind to nuclear speckle RNA binding proteins (NSRs) and spliceosome components (e.g., PRP8a and SmD1b), indicating that lncRNA ASCO may integrate a dynamic network including core spliceosome components to modulate transcriptome reprogramming through RNA splicing and AS regulation (Fig. 5B) [141]. For another example, the circRNA of Arabidopsis SEPALLATA 3 (SEP3) is generated from the splicing of SEP3 exon 6. By binding its parental DNA to remodel chromatin structure, this circRNA forms a DNA-RNA R-loop, which pauses transcription and promotes exon 6 skipping to produce the SPE3.3 transcript and leads to floral homeotic phenotypes (Fig. 5C) [142]. However, the conserved functional role of ncRNAs in regulating AS is still poorly understood, which might be largely due to the rapid evolution of ncRNAs between and within animals and plants.

Fig. 5.

Examples of ncRNAs regulating AS events in plants. (A) LncRNA influences nucleus-cytoplasmic trafficking. The lncRNA early nodulin 40 (ENOD40) promotes the nuclear-cytoplasmic trafficking of RNA binding protein 1 (RBP1) to modulate RBP1-dependent splicing [140]. (B) NcRNA regulates AS events by binding to splicing factors. The ncRNA alternative splicing competitor (ASCO) binds to components of the spliceosome, such as PRP8a, SmD1b, and NSR, and modulates AS regulation [141]. (C) CircRNA binds DNA to form an R-loop and affects AS regulation. The circRNA encoded by exon 6 of the SEPALLATA 3 (SEP3) gene forms an R-loop after binding to its cognate DNA locus, resulting in transcriptional pausing. This pausing promotes the skipping of exon 6 and increases the accumulation of the SEP3.3 transcript [142].

Environmental stimuli

Excessive ultraviolet (UV) irradiation may induce double-stranded DNA damage, including chromosomal defects in cyclobutane pyrimidine dimer (CPD) and pyrimidine 64-PP [143]. In the case of DNA damage and chromosome dissimilation, the elongation rate of Pol Ⅱ is greatly affected, and the Pol Ⅱ carboxyl-terminal domain (CTD) is also hyperphosphorylated, resulting in a reduced elongation rate that changes the recognition sites of the spliceosome and subsequent RNA splicing and AS regulation [144]. In addition to DNA damage, UV irradiation induces cells to produce specific RNA fragments, some of which are lncRNAs with specific functions. For example, under UV irradiation, activating signal co-integrator 1 complex subunit 3 (ASCC3, a DNA helicase) undergoes an AS event to produce a 25 kb lncRNA, which interferes with the normal function of ASCC3 (Fig. 6A) [145]. Moreover, the lack of carbohydrate intake inhibits the phosphorylation of SR proteins, which in turn represses the retention of glucose-6-phosphate dehydrogenase (G6pd) intron 11 involved in the pentose phosphate pathway [146]. Some viral invasions may trigger AS in host cells. For example, herpes simplex virus type 1 (HSV-1) infection increases intron retention events of infectious cell culture protein 34.5 (ICP34.5) by interacting with ICP27 (a splicing factor with antiviral effects) (Fig. 6A). By interfering with AS regulation, infection leads to more transcripts of ICP34.5 and promotes the spread of the virus [147].

Fig. 6.

Environmental stimuli regulate AS in animals and plants. (A) In animals, three environmental stressors were exampled to show AS changes under environmental stimuli. Excessive UV irradiation induces DNA damage and alternative last exon (ALE) of activating signal co-integrator 1 complex subunit 3 (ASCC3) to produce a lncRNA, which interferes with the normal function of ASCC3 [145]. Under low carbohydrate conditions, phosphorylation of the SR protein is inhibited to decrease the binding of SR to exons and leads to the retention of glucose-6-phosphate dehydrogenase (G6pd) intron 11 [146]. Herpes simplex virus type 1 (HSV-1) infection induces intron retention of infectious cell culture protein 34.5 (ICP34.5) through infected cell culture polypeptide 27 (ICP27), which promotes the spread of the virus [147]. (B) In plants, darkness decreases the Pol Ⅱ elongation rate and increases the recognition of weak 3′ splice sites to improve transcripts with alternative exons or introns [153]. Light activates phytochromes (Pfr) and the plastid-to-nucleus signaling system and modulates splicing factors and spliceosomes to further regulate the AS of downstream light-related genes [171]. Salt stress induces differential AS regulation of SR45 and SRAS1 to stabilize the transcripts of salt-responsive genes and decrease the accumulation of oxidants or reactive oxygen species (ROS). ABA or drought treatment induces a higher accumulation of CIPK3.1 and CIPK3.4. The former has a strong binding affinity for calcineurin B-like protein interactors (CBLs) and AP2-like ABA repressor 1 (ABR1) and induces the expression of ABA-responsive genes [152].

Unlike animals, plants are sessile organisms that are unable to avoid various environmental stimuli. It has been reported that different environmental stresses induce many different AS events in plants, suggesting that the regulation of many AS events may be environment-specific [148]. Heat stress triggers differential AS events in maize, and more half of them are ES and IR events [149]. Under cold stress, the number of AS events in plants such as Arabidopsis, Populus, and Carya is also significantly increased [49]. Under salt stress, both SR45a and salt-responsive alternatively spliced 1 (SRAS1) undergo differential IR events to produce two splice variants (Fig. 6B). For SR45a, the transcript SR45a-1b encodes a protein that enhances the combination of SR45a-1a and the cap-binding complex (CBC) and stabilizes the transcripts of salt-responsive genes [150]. Under salt stress, salt-responsive alternatively spliced gene 1 (SRAS1) is induced to produce more SRAS1.1 than SRAS1.2 [151]. The latter SRAS1.2 contains an intron with a PTC and encodes a truncated protein, while the former SRAS1.1 encodes a full-length protein, which shows a strong binding affinity to COP9 signalosome 5A (CSN5A). The binding promotes CSN5A degradation through the 26S proteasome to decrease the accumulation of oxidants or reactive oxygen species (ROS) [151]. Under ABA and drought treatments, CBL-interacting protein kinase 3 (CIPK3) generates several splice variants encoding different proteins that have different binding tendencies for ABA pathway-related proteins [152]. Among the variants, the full-length transcript CIPK3.4 encodes a protein that does not show a binding preference for AP2-like ABA repressor 1 (ABR1), whereas the shorter transcript CIPK3.1 encodes a protein that has a binding preference for ABR1 and calcineurin B-like protein interactors (CBLs), which further contributes to the expression of ABA responsive genes [152]. Light or dark is also an environmental stimulus that can regulate AS events. Dark decreases the Pol Ⅱ elongation rate (Fig. 6B), which allows recognition of weak 3′ splice sites, resulting in the inclusion of alternative exons or introns [153]. Light can induce AS of SR genes by improving the combination of phytochromes (Pfr) with other splicing factors (e.g., splicing factor for phytochrome signaling: SFPs). Moreover, ambient light and temperature signals can regulate the abundance of spliced isoforms of CCA1 in plant photomorphogenesis, thereby maintaining circadian rhythm and endowing plants with the capability to adapt to extreme environments [154].

The NMD pathway in animals and plants

The classical pathway of NMD has been well studied and can be divided into two main stages (Fig. 7): 1) Translation termination coupling recognition of PTC and the formation of the SMG1–UPF1–eRF1–eRF3 (SURF) complex; 2) The SURF complex triggering the rapid decay of target mRNA [155].

Fig. 7.

Nonsense-mediated mRNA decay (NMD) in plants and animals. At the early stage, the ribosome recognizes the premature termination codon (PTC) site and recruits eukaryotic translation termination factor 1 (eRF1) and eRF3, leading to the end of the transcription process [156]. The eRF3 interacts with up-frameshift suppressor 1 (UPF1) and integrates the suppressor of morphogenesis in genitalia 1 (SMG1) to form the SURF complex, which recruits SMG8 and SMG9. Almost simultaneously, the exon junction complex (EJC) recruits UPF3 and UPF2 [157]. Then, UPF1 is activated by SMG1-mediated phosphorylation through contact between the SURF complex and the EJC complex. At the degradation stage, the ribosome dissociates from mRNA, SMG8 and SMG9 are replaced by SMG5/SMG7 or SMG6 to send out a degradation signal, and decapping enzymes (DCPs) remove the m7G cap from the mRNA 5′-end. Then, the mRNA undergoes rapid decay by the 5′-to-3′ exoribonuclease (XRN) and/or 3′-to-5′ exosome [161]. The trans-acting factors marked in blue ovals indicate a well-documented role in animals, while those marked in green ovals indicate a well-documented role in plants. Other trans-acting factors in yellow indicate an important role of the factors in both animals and plants. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

At the early stage of the NMD pathway, normal phosphorylation of the eukaryotic conserved upframeshift suppressor 1 (UPF1) is critical for the formation of the SURF complex. In animals and plants, the homologs of SURF complex components, including UPF1, UPF2, UPF3 and exon junction complex (EJC) proteins, generally play conserved roles in the NMD pathway [156]. However, some other components of SURF may be different in animals and plants. The SMG1C complex, including suppressor of morphogenesis in genitalia 1 (SMG1), SMG8, and SMG9, is conserved in animals, but the SMG1 gene is lost in some algae and cruciferous plants and its function may be performed by other factors [157]. Additionally, homologs of animal SMGs, such as SMG5, SMG6, SMG8, and SMG9, have not been identified in plants [158]. At the late stage of the NMD pathway, the process by which EJC proteins degrade PTC-containing RNA is also somewhat different in animals and plants. In animals, there are EJC-dependent and EJC-independent NMD pathways that have redundant functions in mRNA degradation, and the EJC-independent NMD pathway is more sensitive to a decreased concentration of UPF2 and UPF3b. However, EJC proteins are indispensable for the intron-based NMD pathway in plants [159], [160]. At the final step of NMD in plants, exoribonuclease 4 (XRN4) performs a degradation pathway dependent on SMG7-mediated priming, while in animals, XRN1, a homolog of plant XRN4, performs degradation in an SMG6- or SMG5–SMG7-activated manner [161]. Moreover, the degradation of some mRNAs starts close to the PTC in animals [162], but it is unknown in plants.

Conclusions and outlook

Alternative splicing is an essential component of gene expression that contributes to the diversity of the transcriptome and proteome in both animals and plants [10], [13]. Despite similar molecular mechanisms underlying AS processing, different evolutionary histories led to divergent evolution of AS after the split of animals and plants c. 1.6 billion years ago. The divergent evolution of AS involves multiple levels, including gene architecture, UTRs, spliceosome components, AS types, chromatin dynamics and transcription speed, splicing factors, ncRNAs, and even postsplicing NMD pathways. Although the proposal of various sequencing methods and analysis schemes in recent decades have comprehensively promoted the research progress of whole-genome AS events in both animals and plants [14], [18], [44], [49], [66], the molecular mechanisms and/or influencing factors leading to AS differences, including but not limited to different predominant AS events, are still poorly understood in plants and animals. We reviewed reports of AS in genes involved in physiological and biochemical activities and underlined the similarities and differences between animals and plants. Notably, a literature search in PubMed (until April 07, 2023) showed a striking number of 26,255 reports based on the keywords 'alternative splicing AND humans' but only 2,585 reports based on 'alternative splicing AND (Arabidopsis OR plants)', suggesting that research on AS mechanisms in plants is significantly lagging behind that in animals. Therefore, the knowledge and understanding of AS in animals versus plants are severely asymmetric and must have been affected by a lack of comprehensive studies of AS regulation in plants. Fortunately, the asymmetry has been changing: in the past year (April 08, 2022 to April 07, 2023), a literature search in PubMed showed comparative numbers of reports (376 versus 230) in animals versus plants. Moreover, many of the cited references regarding plants in this review were published in the past five years. However, despite the rapid increase in AS research in plants, the influencing factors that lead to different AS regulation in plants compared to animals still need to be intensively studied and experimentally verified.

On the other hand, utilizing the regulatory mechanism of AS to develop new functional organic molecules is a current research hotspot. For example, applications of AS for cancer-targeted therapy have been intensively studied [163]. To alleviate cancer progression, a specially designed antisense oligonucleotide (AON) was used to enter cells of diseased tissue through a delivery system of lipid complexes, electroporation, and/or other means, to interact with pre-mRNA and interfere with the mistaken assembly of the spliceosome [164], [165]. AON-induced splice-switching and DM1 protein kinase (DMPK) pre-mRNA degradation are potential therapeutic approaches for type 1 myotonic dystrophy (DM1) [166]. In addition to RNA-targeted treatments, a variety of chemical modulators, e.g., spliceostatin A and E7107, have been found to bind to specific residues in splicing factors SF3B1 and PHD finger protein 5a (PHF5A) [37], [167]. In plants, potential AS for plant breeding has also been studied. For example, 20 AS transcription factors were identified to play roles in drought memory in rice, which may be useful for crop breeding [168]. Although these compounds have been shown to be associated with specific diseases or plant growth and stress by altering AS, more studies are required to demonstrate their safety and effectiveness. Moreover, restricted by the existence of genetic barriers between species, current research is limited to a few model species. Further analysis of AS differences between and within animals and plants has a positive guiding significance for the creation of new functional molecules in the future, and even for the development of disease therapy programs or breeding of superior crops.

Compliance with Ethics Requirements:

Ethical approval is not required in this review article.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Biographies

Yufei Du is a Master's student at Zhejiang A&F University. His study is to identify the factors that determine differences in alternative splicing between animals and plants.

Lu Cao is a Master's student at Zhejiang A&F University. His study is related to bioinformatic analysis of transcriptome changes under abiotic stresses.

Shuo Wang is a lecturer at Zhejiang A&F University. She obtained her Ph.D. degree from Northeast Forestry University in 2019. After two years of postdoctoral training, she joined Zhejiang A&F University in 2022. Her research interests include gene expression, alternative splicing, and molecular evolution of plants in response to abiotic stresses.

Liangyu Guo is a Master's student at Zhejiang A&F University. His study is mainly to reveal the evolutionary processes of plants in adaptation to environmental changes.

Lingling Tan is a Master graduated from Zhejiang A&F University. Her study was related to bioinformatic analysis of RNA-binding proteins (e.g., SR proteins) in plants and animals.

Hua Liu is a lecturer at Zhejiang A&F University. She obtained her Ph.D. degree from the University of Tokyo, Japan in 2013. After four years of postdoctoral training at the National Institute of Genetics, in Japan, she joined Zhejiang A&F University in 2017. Her research is mainly on the roles of RNA-binding proteins on plant flowering.

Ying Feng is a professor at the Shanghai Institute of Nutrition and Health, Chinese Academy of Sciences (CAS). She obtained her Ph.D. degree from the former Shanghai Institute of Biochemistry, CAS in 2001. After five years of postdoctoral training in the Department of Biological Sciences, Columbia University, USA, she was promoted to Associate Research Scientist in 2006. In 2009, Dr. Feng joined CAS, and her research focuses on molecular mechanisms of gene expression, alternative splicing, and their implications in cancers.

Wenwu Wu is a professor at Zhejiang A&F University. He obtained his Ph.D. degree from Northwest A&F University in 2012. After two years of postdoctoral training on animal alternative splicing at the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (CAS), he was promoted to associate professor in 2014 at Shanghai Center for Plant Stress Biology, and then he studied molecular mechanisms (including alternative splicing) of plants under abiotic stresses. In 2016, Dr. Wu joined Zhejiang A&F University, and his research interests include gene regulation, alternative splicing, and especially molecular evolution of plants in adaptation to environmental changes.