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Defects in splicing, especially alternative splicing have been frequently found in cancers. Mutations in the splicing regulatory elements of important genes involved in cancers or the genes encoding regulatory splicing machinery could play a key role in carcinogenesis. Alterations in regulator factors in splicing have emerged as a new class of oncoproteins and tumor suppressor genes. Understanding the molecular mechanism of how defects in splicing and in particular alternative splicing are involved in carcinogenesis, could lead to new strategies to cancer therapy. Here, we review the molecular mechanism of splicing and regulatory factors involved in alternative splicing, as well as the aberrant splicing that affects cancer hallmarks. Finally, we summarize new approaches in cancer therapy based on splicing.
Department of Genetics, School of Medicine, Kazerun Branch, Islamic Azad University, Iran
Parisa Mohamadynejad
Department of Biology, Faculty of Basic Sciences, Shahrekord Branch, Islamic Azad University, Iran
*Address all correspondence to: mehdimoghani@yahoo.com
1. Introduction
Split genes in eukaryotes were discovered in 1977 for the first time. It was later indicated that the intron sequences should be removed by the spliceosome complex in the splicing process in the nucleus [1].
There are 20,687 protein-coding genes with introns (93%) and 1713 (7%) intron-less protein-coding genes in human genome [2]. It is interesting to note that only ∼4% of genes in the schizosaccharomyces cerevisiae contain introns and most of them are single intron genes [3]. The number of introns per gene varies in eukaryotic genomes so that in the human genome is 8.94, and in the fungi (0.05–3.43 introns per gene), plants (0.33–7.30 introns per gene), invertebrates (2.92–7.42 introns per gene) and vertebrates (7.35–10.09 introns per gene) [2].
The length of introns is less than 100 up to 100,000 bp in different genes of various organisms [3]. Unlike lower eukaryotes, there are relatively long introns and short exons in vertebrates [4] but in general, fungal introns are relatively short (93% of the introns in fungi are shorter than 250 nt). However, in invertebrates and plants, the average percentage of short introns (<250 nt) is 48% and 59%, respectively. In contrast, 48% of vertebrate introns are longer than 1000 nt [2].
In the present chapter, we explain the various types of splicing, the molecular mechanism of splicing, regulatory elements and factors in splicing, and finally the alternative splicing and the role of aberrant splicing in cancer are discussed.
The classic and ubiquitous of exons joining from the same pre-mRNA is called cis-splicing. Highly conserved splicing machinery in all eukaryotes shows that splicing is pivotal for all eukaryotic organisms [3]. Five small RNAs (snRNAs), including U1, U2, U4, U5, and U6 (because they are uracil-rich) and approximately 300 related proteins including hnRNPs and SR proteins, make up the spliceosome complexes and are involved in the splicing regulation.
The junction of introns and exons is identified by spliceosome and the “GU-AG” rule is mainly used. Four regions play a key role in splicing, including (a) the GU sequences at the beginning of the introns as 5′ splice-site (5′-SS or donor site), (b) AG dinucleotides at the end of intron referred to 3′ splice-site (3′-SS or acceptor site), (c) the sequences without high conservation in the upstream of the 3′SS called branch point sequence (BPS), and (d) polypyrimidine-rich sequences which is locating between the BPS and the 3′ SS [1].
For conventional splicing, first of all, the complex E (early) is formed by ATP-independent binding of U1 snRNP (small nuclear ribonucleoprotein) to 5′-SS via base-pairing, SF1 (splicing factor 1) to BPS, U2AF2 to polypyrimidine tract, and U2AF1 to 3′-SS. Second, SF1 is replaced by U2 snRNP (with the assistance of U2AF) through the base-pairing to BPS to form complex A. This step is ATP-dependent and reinforced by the SF3a and SF3b protein complexes, as well as U2AF2 and U2AF1. Third, complex B (inactive) is formed by rearranging complex A using recruiting U5/U4/U6 in such a way that U4 and U6 snRNPs bind strongly to each other due to complementary pairing of their RNA components, but U5 snRNP is weakly bound to them (through protein–protein interaction). Following several conformational changes, U1 snRNP leaves mRNA, U6 snRNP binds to 5′-SS, and simultaneously, U4 snRNP leaves mRNA so that U6 and U2 snRNPs pair together through their snRNA, leading to the formation of pre-catalytic (active) spliceosome complex B* (Figure 1). Finally, removal of introns and joining of exons are performed using two transesterification reactions by complex C [1] (Figures 1 and 2).
Figure 1.
Various steps of splicing are performed by different snRNAs and snRNPs [5].
Cis-acting elements in RNA constitute a set of rules called “splicing code” that acts as an anchor for trans-acting factors to produce functional transcripts [7]. There is a competition between various splicing factors for the binding to the splicing regulatory elements and in turn the increasing or decreasing the recruitment of spliceosome complexes to premature mRNA.
Generally, the serine/arginine (SR)-rich protein family bind preferentially to exonic splicing enhancer (ESEs) and intronic splicing enhancer (ISEs) sites and play a positive role in the splicing (exon inclusion). However, heterogeneous nuclear ribonucleoproteins (hnRNPs) bind to exonic splicing silencer (ESSs) and intronic splicing silencer (ISSs) elements and generally suppress exon inclusion (Figure 3) [1, 5].
Figure 3.
Control of splicing by cis-acting elements and splicing factors [5, 7, 8].
In addition to the characteristic domain of RS (arginine/serine dipeptides) in SR proteins, there is an RNA recognition motif (RRM) which is the basis for the classification of SR proteins. In humans, there are 12 types of SR proteins, now called serine/arginine-rich splicing factor (SRSF) 1–12. RS domain of different SR proteins binds to ESE, could directly interact with each other and facilitate the recruitment of spliceosome machinery components including U1 snRNP or U2AF to mRNA, and more importantly, it has been suggested that the SR proteins bound to ESEs play a key role in exon definition in constitutive and alternative splicing. There are no canonical SR proteins in Saccharomyces cerevisiae and three SR-like proteins including Npl3, regulate splicing efficiency [3].
RRM is also the most common domain in the hnRNPs for the interactions with the premRNA, but hnRNPs also contain another domain called the RGG boxes including Arg-Gly-Gly tripeptides repeats. hnRNP proteins as same as SR proteins, bind directly to a specific site in the target premRNA, but inhibit splicing via blocking the binding of snRNPs to targets or looping out exons [3, 9].
Some introns are spliced without requiring to spliceosome complex (self-splicing), including group I introns (found in bacteria, bacteriophages and eukaryotes including organelle and nuclear genomes) and group II introns (found in bacteria, archaea, and eukaryotic organelles). In both, trans-esterification reactions need to excise the intron and ligate the exons.
The difference between them is that the splicing reaction is initiated by a guanosine cofactor and an internal adenosine in group I and group II introns, respectively. Also, during splicing, group II introns form a lariat like structure, whereas group I introns do not (Figure 4) [10].
Figure 4.
Comparison of a self-splicing group I, group II introns and spliceosome-catalyzed splicing [10].
In addition to cis-splicing, there is another type that joins exons on the separate pre-mRNAs and results in the chimeric mRNAs originating from two different genes so-called trans-splicing and commonly occurs in unicellular organisms and caenorhabditis elegans. Interestingly, acyl-CoA-cholesterol acyltransferase 1, RGS12 and CYP3A genes produce mRNA that derived from different genes in human [11].
Different combination of exons in a special type of splicing, called alternative splicing, which lead to the formation of multiple protein isoforms from a single pre-mRNA and promotes the transcriptome and proteome complexity. A recent analysis of the Encyclopedia of DNA Elements (ENCODE) project 1 has been revealed that the human genome consists of approximately 21,306 protein-coding genes [12, 13] and it is estimated that 92–94% of human genes harbor alternative splicing [3, 14, 15].
The most common type of alternative splicing mechanism (40% of events) is exon skipping (also called cassette exon) [4] in which one or several exons are not included in the final mRNA (Figure 5a) but the rarest mechanism of AS is mutually exclusive exons (Figure 5d) [17]. Changing in the selection of either 3′- or 5′-splice sites are other mechanisms of AS (Figure 5b and c). The fifth type of alternative splicing is intron retention (Figure 5e), in which an intron remains in the mature mRNA (the most common after the exon skipping). Applying alternative promoters and unusual polyadenylation are other alternative splicing mechanisms (Figure 5f and g) [16]. Various trans-acting factors, as well as specific cis-acting elements, play a key role in alternative splicing.
Figure 5.
Schematic representation of different types of alternative splicing [16].
Expression alteration in splicing trans-acting factors and mutations (also, polymorphisms) including single-base substitutions, deletion, insertion, and translocation in cis-elements and trans-regulatory factors that encodes genes involved in splicing, would cause abnormal splicing and lead to diseases and cancer. In addition, the post-modification of trans-regulatory factors and chromatin remodeling could affect splicing by altering the recognition of splicing factors and splicing sites (Figure 6) [5].
Gene duplication and overexpression of SRSF1 were the first evidences that alternative splicing plays an important role in tumor growth [18]. Recently, it has been reported that splicing may be correlated with the various types of cancers, and in particular, alternative splicing can affect various aspects of cancer hallmarks [8, 19] (Figure 7) and splicing modulators have emerged as a new class of oncoproteins and tumor suppressor genes. Importantly, RNA sequencing analysis has shown that one of the common mechanisms of tumor suppressor genes inactivation in cancers is intron retention [20].
Figure 7.
Effects of aberrant alternative splicing on different hallmarks of cancer. Arrows up: Most contributor, arrows down: Least contributor, EMT (epithelial–mesenchymal transition) [8].
Recently, RNA-seq studies have been reported that one of the major differences between transcriptome of normal and cancerous tissues are caused by aberrant splicing with AML and hepatocellular carcinoma show the highest and lowest frequency of alternative splicing alteration compared to the corresponding normal tissues, respectively. The most extensive study ever performed on the splicing profile of various types of cancers involving more than 8700 patients from The Cancer Genome Atlas (TCGA) data, shows that alternatively splicing in cancerous tissues is 30% more than normal tissues [7]. Expression dysregulation and frequent somatic mutations in the splicing factors coding genes have been identified in more than 2% of tumors in several cohorts of patients, including TCGA data (Figure 8) [21].
Figure 8.
Frequently alteration of splicing-factors in human cancers. MDS: Myelodysplastic syndrome; CMML: Chronic myelomonocytic leukemia; HN: Head and neck; w/o RS: With or without ringed sideroblasts; RARS/RCMD: Refractory anemia with ringed sideroblasts and refractory cytopenia with multilineage dysplasia and ringed sideroblasts; MPN, myeloproliferative neoplasm [21].
7.1 Deregulation of splicing factors expression
A wide range of factors involved in splicing and regulation of alternative splicing has been shown to be dysregulated in gene expression level. For example, serine and arginine-rich splicing factor 1 (SRSF1), a key player of alternative splicing, is involved in tumorigenesis promotion by splicing of RAC1, Tyrosine-protein kinase (SYK), MKI67 and HNRPLL genes. Specifically, in colorectal cancer, SRSF1 and other members of the SR family, including SRSF3, SRSF6, SRSF7 and SRSF10, are overexpressed and referred to as oncogenes [5, 22].
Bcl-x protein, which plays a key role in the regulation of intrinsic pathway of apoptosis, harbor alternative splicing and can obtain two different isoforms Bcl-xl (involved in apoptosis inhibition) and Bcl-xs (participated in apoptosis promotion) [23]. PTB protein 1 preferentially promote Bcl-xs isoform that leads to apoptosis, but down-regulation of PTB protein 1 could produce Bcl-xl, which inhibits apoptosis and result in tumorigenesis [24].
Some aspects of the oncogenic role of C-Myc in tumors, are changing splicing patterns. For example, it can overexpress the ITGA6A variant [the pro-proliferative isoform of integrin subunit α6 gene (ITGA6)] compared to ITGA6B isoform in the colon cancer, which is mediated by epithelial splicing regulatory protein 2 (ESRP2) [22].
Tumor cells preferentially metabolize glucose via the aerobic glycolysis pathway compared to normal cells. Conversion of phosphoenolpyruvate (PEP) to pyruvate is performed by PKM (Pyruvate kinase) enzyme in the last step of glycolysis. PKM is encoded by PKLR (expressed mainly in the liver and hematopoietic cells) and PKM (expressed in most tissues) genes in which both of them harbor alternative splicing [25, 26]. Alternatively, splicing of the PKM gene results in PKM1 isoforms (mainly in normal cells) and PKM2 isoforms (mainly in tumor cells) [27, 28]. The polypyrimidine tract-binding protein (PTB), hnRNPA1 and hnRNPA2 genes are overexpressed by C-Myc and lead to upregulation of PKM2 isoform of pyruvate kinase (PKM) through splicing changing and lead to cell proliferation (Figure 9) [29, 30].
Figure 9.
Regulation of metabolic shift between oxidative phosphorylation and aerobic glycolysis through alternative splicing of PKM gene. In tumors, c-Myc and possibly other factors are involved in the upregulation of the hnRNPA1, A2, and PTB genes which result in exon 9 exclusion and exon 10 inclusion and PKM2 generation. PEP to pyruvate conversion is catalyzed less efficiently by PKM2 compared to PKM1, resulting in enhancement of metabolites for anabolic metabolism [28].
Vascular endothelial growth factor A (VEGF-A) is one of the key proteins in the angiogenesis process in tumor progression. The VEGF- A encoding gene produces various isoforms (angiogenic and antiangiogenic) using alternative 3′ and 5′-SSs in exons 6, 7, and 8 that can regulate angiogenesis (Figure 10) [31]. Interestingly, angiogenic VEGF isoforms are induced by Serine/arginine-rich protein-specific splicing factor kinase (Srpk1) and SRSF1 protein, which are activated by Wilms’ tumor suppressor 1 (Wt1) protein [32].
Figure 10.
The structure of the VEGF-A gene includes eight exons (a) and producing of various isoforms by alternative splicing (b). Two mRNA isoform families, pro-angiogenic (VEGF-Axxx, b. left) and anti-angiogenic (VEGF-Axxxb, b. right) are generated by alternative splicing. (xxx shows the amino acid number of the mature protein). The transcriptional start site (TSS), translational start site (ATG) in exon 1, and alternative stop codons within exon 8 (TGA1 and TGA2) are indicated [31].
7.2 Mutation in cis-acting elements and genes encode for trans-acting factors
It has been suggested that various mutations, including silent or synonymous mutations, maybe results in intron retention (often in tumor suppressor genes and lead to premature stop codon) or exon skipping through create and loss of exonic splicing enhancers, silencers, or create novel splice sites [7]. It has been reported that 40% of patients with hereditary non-polyposis colorectal cancer carry the MLH1 mutation, and interestingly, the most common mutation in this gene is located at splicing sites [33] which was in line with bioinformatics analysis results [34]. The most somatic mutations in the splicing factors encoding genes have been observed in bladder cancer and uveal melanoma [7].
Recently, in several cancers, a recurrent mutation in the U1 snRNA encoding gene has been found, which mainly affects the third base of U1 snRNA at the splice site recognition sequence that pairs directly with 5′SS. In addition, mutated SF3B1 (Table 1) recognizes different BPS and results in cryptic 3′ splice sites selection. Also, the SRSF2 and U2AF1 mutations alter splice site recognition in a sequence-dependent manner and results in alternative 3′ splice site selection and altered exon inclusion, respectively (Figure 11 and Table 1) [7, 8]. It has been suggested that these mutations may also affect the protein-protein interaction domains and lead to alteration of splicing factors interaction with nucleosomes, which plays an important role in driving splice site definition [7].
Mutation type
Gene
Spliceosome component
Top 3 commonly mutated cancer types*
SF3B1
UVM, UCEC, BLCA
U2AF1
U2 snRNP
UCS, LAML, UCEC
PHF5A
KICH, SKCM, LUSC
Hotspot
SRSF2
SR protein
LAML, UVM, UCEC
HNRNPCL1
hnRNP
SKCM, UCEC, STAD
PCBP1
hnRNP
READ, COAD, BLCA
HNRNPD
UCEC, DLBC, UCS
HNRNPDL
UCEC, COAD, STAD
HNRNPL
hnRNP
COAD, UCEC, SKCM
PCBP2
UCEC, SKCM, READ
CCAR1
UCEC, SKCM, BLCA
Loss of function
RBM10
A complex
UCEC, LUAD, BLCA
TCERG1
UCEC, COAD, SKCM
DDX50
Bact complex
UCEC, STAD, LUAD
FUBP3
UCEC, COAD, SKCM
FRA10AC1
C complex
UCEC, READ, UCS
CDC5L
PRP19
UCEC, STAD, SKCM
Table 1.
Mutation of splicing factors encoding genes in cancers [7].
Effects of cancer-associated mutations in splicing factors on alternative splice site selection. Processing patterns favored by the mutations are shown by thicker lines. WT: Wild type [8].
Response of the tumors to therapy may be affected by splicing and resistance to treatment may be reinforced by alternative splicing [35]. Alternative splicing could induce resistance to drugs in cancer through changing factors that are involved in the metabolism of drugs including uptake, transportation and inactivation (Figure 12). One of the most mutated genes in melanoma cancer is BRAF that V600E mutation is the most prevalence mutation in this gene and result in a variant without exons 4–8 (give rise to lack of the RAS-binding domain of BRAF rendered cells insensitive to RAF inhibition) and lead to resistance to vemurafenib in 30% patients of melanoma. One of the strategies for prostate cancer therapy is using antiandrogen specially enzalutamide (antagonist of the interaction of androgens with AR) and abiraterone (blocker of androgen biosynthesis) to inhibit the activity of the androgen receptor (AR). HNRNPA1 could drive a splice variant of AR called ARv7 arises due to the inclusion of a cryptic exon CE3 and result in antiandrogen treatment [7, 36].
Figure 12.
Aberrant alternative splicing in some oncogenes and resistance to therapy in cancers. ATG1 and ATG2: Start codon for full-length and short mRNA. TGA: Stop codon [36].
In addition to chemo and hormone therapy in cancers, radiotherapies are used for treatment and several factors involved in tumor sensitivity to ionizing radiation that some of them affected by alternative splicing. Interestingly, p73 (a transcription factor) is one of the most important proteins that could act as a marker for the sensitivity of tumor cells to radiation. There are two isoforms of the p73 including the full-length TAp73 and the N-terminally truncated ΔNp73 which truncated isoform has been indicated that strongly associated with poor survival and negative response to irradiation [16].
Recently, new strategies have been used to correct splicing errors and overcome drug resistance. In different levels could be applying various molecules to reverse aberrant splicing. Blocking of splicing factors and kinases which phosphorylated them is one of the strategies for correct splicing (Figure 13) [36].
Figure 13.
Various potential strategies for therapeutic approaches to correct splicing errors [36].
However, using splice switching oligonucleotides (SSOs) is the best-known method for therapy based on splicing [36]. For example, masking the proximal 5′ splice site by an antisense oligonucleotide (ASO) in BCL-X could shift splicing and induce apoptosis in tumor cells. The same strategy is used for reverse aberrant splicing of MDM4 and FAS mRNAs and inhibit p53 degradation and enhanced-cell death, respectively (Figure 14) [18]. In addition, ESEs and ESSs which are binding sites for SRSF and HNRNP proteins could be blocked by SSOs and affect splicing events [36].
Figure 14.
Cancer therapies based on antisense oligonucleotide-mediated splicing modulation [18].
In conclusion, aberrant splicing, especially alternative splicing, can play an important role in carcinogenesis in all hallmarks of cancer (Figure 15). Recently, data from TCGA have been reported that many of the key player genes involved in cancers are affected by alternative splicing (Figure 16). It has recently been suggested that some misspliced RNA transcripts of non-coding RNAs (ncRNAs), including long ncRNAs (lncRNAs) and circular RNAs (circRNAs), may also contribute to tumorigenesis [38, 39]. One of the isoforms of protein phosphatase 1 regulatory subunit 10 (PPP1R10, also called PNUTS) gene generated by alternative splicing is lncRNA-PNUTS, which act as a competitive sponge for miR-205 in breast cancer [40]. In addition, PCGEM1 and BC200 lncRNAs interact with splicing factors such as hnRNPA1, hnRNPA2/B1 and U2AF65 and regulate the alternative splicing of the AR and BCL-x genes, respectively. Moreover, MALAT1 as an LncRNA, regulates alternative splicing by affecting the sub nuclear localization of SR proteins [21]. The role of alternative splicing in carcinogenesis and new strategies for cancer therapy based on reverse abnormal splicing or blocking aberrant splicing are emerging areas of cancer research.
Figure 15.
The implication of alternative splicing of important genes (blue background) in the regulation of hallmarks of cancer (red background) [37].
Figure 16.
Tumor-associated isoforms of the important genes in cancers. ES, exon skipping; MXE, mutually exclusive exons; 5’ASS, 5’ alternative splice site selection; IR, intron; retention.OE, overexpression; KD, knockdown. Other tumors include: adrenal, gallbladder, ampullary, bone, and brain; gynecological tumors include: ovarian, cervical, and uterine; head and neck tumors include: Oral, head and neck, tongue, esophageal, and thyroid.. ES, exon skipping; MXE, mutually exclusive exons; 5’ASS, 5′ alternative splice site selection; IR, intron retention [21].
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Written By
Mehdi Moghanibashi and Parisa Mohamadynejad
Submitted: 12 January 2022Reviewed: 17 January 2022Published: 15 March 2022
Open access peer-reviewed chapter
