Alternative splicing of mRNA in colorectal cancer: new strategies for tumor diagnosis and treatment

Abstract

Alternative splicing (AS) is an important event that contributes to posttranscriptional gene regulation. This process leads to several mature transcript variants with diverse physiological functions. Indeed, disruption of various aspects of this multistep process, such as cis- or trans- factor alteration, promotes the progression of colorectal cancer. Therefore, targeting some specific processes of AS may be an effective therapeutic strategy for treating cancer. Here, we provide an overview of the AS events related to colorectal cancer based on research done in the past 5 years. We focus on the mechanisms and functions of variant products of AS that are relevant to malignant hallmarks, with an emphasis on variants with clinical significance. In addition, novel strategies for exploiting the therapeutic value of AS events are discussed.

Fig. 1. Components of the splicesome machinery and the common types of alternative splicing.

The figure shows an outline diagram of splicesome components and clinical treatment targets with experimental evidence. A The elements that participate in pre-mRNA splicing. The U1, U2, U3, U4, and U5 are small nuclear ribonucleoprotein (snRNP) complexes that directly bind to splicing sites by recognization between snRNAs and pre-mRNA. The 5’ splice site (5’ SS), the branch point sequence (BPS) and the 3’ splice site (3’ SS) are relatively conserved sequences recognized by snRNPs. The major spliceosome splices introns containing GU at the 5’ SS and AG at the 3’ SS. The typical sequence of BPS is YNYYRAY (Y: pyrimidine; N: any nucleotide; R: purine; A: adenine). The classical/canonical hnRNPs (Heterogeneous nuclear ribonucleoproteins) and SR proteins (serine/arginine amino acid-rich proteins) regulate splicing by binding to the splicing cis-regulatory elements, including exonic splicing enhancer (ESE), exonic splicing silencer (ESS), intronic splicing enhancer (ISE), and intronic splicing silencer (ISS) sequences. B The major splicing process is accompanied by the interaction between cis-elements and trans-factors with spliceosome assembly cycle. The gray box shows the stepwise interaction of the small nuclear ribonucleoprotein (snRNP) particles changes with the removal of an intron from a pre-mRNA. The first step of splicing is trans-elements bind to the conserved sequence of introns, including U1 binding to 5’SS, SF1 binding to BPS and U2AF2 binding to Py-tract and U2AD1 binding to 3’SS, which forms the first spliceosome complex E. Then U2 will replace SF1 and interact with BPS, forming the spliceosome complex A. And U4/U5/U6 tri-snRNP substitutes U1, with U5 binding to 5’SS and U6 binding to U2. After that, U4 dissociates from the B complex and some regulatory splicing proteins are recruited, forming the early B act complex (B*). Two steps of transesterification complete splicing progress. U6/U2 catalyzes transesterification reactions by making the BPS ligate to 5’-end of the intron and form a lariat, and the 5’ site is cleaved, resulting in the formation of the lariat. This is followed by a 5ʹSS-mediated attack on the 3ʹSS, leading to the removal of the intron lariat and the formation of the spliced RNA product. The proteins are recycled and used in the next splicing process (showed as dotted lines). C Common models of alternative splicing and the corresponding transcript variants. The solid and dashed lines denote different alternative splicing models. Cassette exon skipping: an intervening exon between two other exons can be either included or skipped. Intron retention: an intron remains in the mature mRNA instead of being spliced. Mutually exclusive exon: only one out of two exons (or one group out of two exon groups) is retained with the other one is spliced out. Alternative 5’SS: a potential 5’SS replaces the consensus 5’SS and is joined to 3’SS. Alternative 3’SS: a potential 3’SS replaces the consensus 3’SS and is joined to 5’SS. Alternative first exon: the first exon is replaced by the identical boundaries in the second exon and is exclusive. Alternative last exon: the last exon is substituted by the penultimate with a similar splicing site and exclusive. Alternative promoter: alternative transcription initiation sites also affect the splicing pattern of downstream exons.

Fig. 2. The mechanism of aberrant splicing in colorectal cancer.

The diagram shows the classifiable explanation of abnormal splicing in CRC. Splicing occurs co-transcriptionally on nascent RNA, which is attached to chromatin by RNA polymerase II (showed in the left figure). Both alterations of cis-elements and trans-regulatory factors would cause abnormal splicing events and products. New recognized sites are created by mutation of cis-elements like single base substitutions, translocation, and alternative promoters (the first diagram on the right). Alteration of chromatin would influence affinity between splicing factors to splicing sites by conformational change or speed of transcription elongation with changed time of splicing factors loading on cis-elements (the second diagram on the right). In addition, the expression and post-modification of trans-regulatory alter the splicing by infecting recognization between splicing factors and splicing sites.

Fig. 3. Alternative splicing is associated with tumor hallmarks.

Five common tumor hallmarks related to alternative splicing include proliferation, invasion and migration, apoptosis, angiogenesis, and drug resistance. The figure shows the hallmarks and the associated genes. A The representative gene and their splicing variants show different functions in cell proliferation. B The representative gene and their splicing variants show different functions in cell apoptosis. C The representative gene and their splicing variants show different functions in angiogenesis. D The representative gene and their splicing variants show different functions in invasion and metastasis. E The representative gene and their splicing variants show different functions in drug resistance. More details about the mechanism of splicing and clinical application are listed in Supplementary Table 1.

Fig. 4. The potential therapeutic strategies for treating patients with CRC by splicing alterations.

Therapeutic strategies of splicing alterations include both the direct nucleic acid sites and splicing regulatory factor. A An ideogram shows splice-switching oligonucleotides (SSOs) targeting direct splicing site (5’SS or 3’SS), exon splicing enhancer (ESE) or inhibitor (ESI) and potential splicing sites. SSOs with experimental evidence were shown in the diagram. See text and Table 3 for detail. B Trans-regulatory factors are also targeted by small molecule inhibitors as treatment strategies through the splicing mechanism. Small molecule inhibitors targeting trans-regulatory factors and spliceosome are shown in the diagram. See text and Table 3 for detail. C Some special splicing variants related to carcinogenesis are effect targets for inhibitor and colorectal treatment. Inhibitors targeting oncogenic variants or signaling are shown in the diagram. See text and Table 3 for detail.