Lessons from non-canonical splicing

Journal name:
Nature Reviews Genetics
Volume:
17,
Pages:
407–421
Year published:
DOI:
doi:10.1038/nrg.2016.46
Published online

Abstract

Recent improvements in experimental and computational techniques that are used to study the transcriptome have enabled an unprecedented view of RNA processing, revealing many previously unknown non-canonical splicing events. This includes cryptic events located far from the currently annotated exons and unconventional splicing mechanisms that have important roles in regulating gene expression. These non-canonical splicing events are a major source of newly emerging transcripts during evolution, especially when they involve sequences derived from transposable elements. They are therefore under precise regulation and quality control, which minimizes their potential to disrupt gene expression. We explain how non-canonical splicing can lead to aberrant transcripts that cause many diseases, and also how it can be exploited for new therapeutic strategies.

At a glance

Figures

  1. Cryptic exons and microexons.
    Figure 1: Cryptic exons and microexons.

    a | Many introns contain proximally spaced sequences that resemble splice sites (such as GU for 5′ splice sites and YAG for 3′ splice sites), which can in some cases lead to splicing of 'cryptic' exons. Cryptic exons often introduce premature termination codons, which may target the resulting transcripts for nonsense-mediated decay (NMD). Such NMD exons are common within transcripts that encode splicing activators and function as part of autoregulatory mechanisms33, 34, 35. In this example, a serine/arginine-rich (SR) protein enhances the inclusion of an NMD exon within its own mRNA as part of a negative autoregulatory feedback that maintains appropriate steady-state abundance. b | An Alu element is normally composed of two arms, which contain an adenine-rich linker (A-linker; left arm) and a poly(A) tail (right arm). The Alu element can become retrotransposed into the antisense strand relative to the gene, so that transcription of the gene produces an antisense Alu sequence that contains two poly(U) tracts (U tracts) at the beginning of each arm. Many such antisense Alu elements are capable of forming cryptic exons owing to the presence of splice-site-like motifs37. However, they are normally repressed by a heterogeneous nuclear ribonucleoprotein C (HNRNPC) tetramer (C, green circle), possibly because each U tract can bind the two RNA recognition motif domains that are present on the opposite surfaces of the tetramer (as indicated by the double arrow)8, 190. The example provided here shows the U tracts around the Alu exon from the CD55 gene (encoding CD55 molecule). Mutations in the U tracts can decrease the binding of HNRNPC, allowing the binding of U2 small nuclear RNA auxiliary factor (U2AF2) and T cell-restricted intracellular antigen 1 (TIA1), which initiate splicing of a cryptic Alu exon8, 37, 39. c | Microexons (denoted here by μ) can be detected from gapped regions in sequencing reads11, 13, 44. After mapping of multiple parts of the sequence read to flanking exons, unmapped intervening sequences are aligned to the intronic sequence present between the two exons, with preference given to those that are flanked by conserved splice site motifs. Inclusion of microexons can be enhanced by RNA-binding proteins (RBPs), such as serine/arginine repetitive matrix 4 (SRRM4), an SR protein that binds upstream of microexons and promotes microexon splicing. Inclusion of microexons typically leads to the modulation of overlapping or adjacent protein domains and a change in protein activity. SRRM4 is reduced in patients with autism spectrum disorder (ASD), leading to decreased inclusion of microexons12.

  2. Recursive splicing of long introns.
    Figure 2: Recursive splicing of long introns.

    a | Total RNA sequencing (RNA-seq) read counts display a characteristic pattern of depletion from the start to the end of long introns, which can be used to infer exon positions and splicing events47, 48. The 'saw-tooth' patterns that overlap novel junction reads indicate splicing at deep intronic loci and are candidates for recursive splicing14, 15. Here, the upstream exon first uses a 3′ splice site (3ss) to remove the first part of the intron. This process reconstitutes a 5′ splice site (5ss) that can then be used to remove the next section of the intron. This special type of splice site is referred to as a recursive splice site (RS site). b | Recursive splicing in vertebrates requires the RS site to overlap a cryptic 'RS exon', which initiates the exon definition mechanism that is required for the recognition of the 3′ splice site (YAG) of the RS site14. After the first splicing step, the 5′ splice site (GURAG) of the RS site competes with the 5′ splice site of the RS exon. In the second step, the outcome of this competition determines whether the RS exon is skipped, owing to recursive splicing, or included as a nonsense-mediated decay (NMD) exon. Whereas the preceding exons from major isoforms end in sequences that favour RS exon skipping, the minor isoforms and cryptic elements end in sequences that favour RS exon inclusion.

  3. Intron retention and exitrons.
    Figure 3: Intron retention and exitrons.

    a | Intron retention events are detected as an accumulation of reads across intronic regions or increases in the ratio of exon–intron reads to exon–exon reads21, 22, 23, 24, 25. Intron retention events are characterized by numerous features including weak splice sites, high GC content and short intron lengths. Trans-acting factors such as RNA-binding proteins (RBPs), the spliceosome and the exon junction complex (EJC) can also regulate specific intron retention events. The resulting transcripts are typically either retained in the nucleus or targeted for nonsense-mediated decay (NMD) in the cytoplasm or may result in truncated proteins21, 53, 55. b | Exonic introns (exitrons) are introns within annotated protein-coding exons that can be removed owing to the presence of internal splice site motifs within the exon25, 26. Exitron-containing exons are longer than typical exons, and removal of the exitron can lead to changes in protein structure or degradation through NMD.

  4. Formation of circularRNAs and chimeric transcripts.
    Figure 4: Formation of circularRNAs and chimeric transcripts.

    a | Circular RNAs (circRNAs) are produced by head-to-tail splicing and can be either mono- or multi-exonic. In this multi-exonic example, the 3′ splice site of an upstream exon becomes spliced to the 5′ splice site of a downstream exon to generate a circular transcript in which the intervening intron is either removed (exonic circRNA) or retained between the two circularized exons (exon–intron circRNA)20. circRNA formation is promoted when the pre-mRNA regions flanking the exon termini are brought into close proximity. This can be due to the action of RNA-binding proteins (RBPs) such as quaking (QKI) or muscleblind-like (MBNL), which bind to the flanking regions74, 75. Alternatively, this can be due to RNA hybridization of the flanking regions, which can be caused by Alu elements in primates70. b | CircRNAs are resistant to RNase R exoribonuclease activity, which can be used for their enrichment during preparation of cDNA libraries. They can then be detected in RNA sequencing (RNA-seq) data by junction reads that are in a head-to-tail orientation16, 17, 18, 19. c | Chimeric RNA products can also be produced by cis-splicing when transcript termination is deficient76. This process results in read-through of one gene into its neighbouring gene, before splicing occurs between the penultimate exon of gene 1 and the second exon of gene 2, which is seen in the chimeric cathepsin C (CTSC)–RAB38 gene in some cancers. d |Trans-splicing occurs when exons of two different transcripts become spliced together80, 81, 82, 83, 84, 85, 86, 87. Alternatively, the same chimeric transcripts can be produced when genes become fused, such as in JAZF1–SUZ12 gene fusion in some cancers, which leads to the same chimeric transcript being produced by a linear splicing reaction.

  5. A summary of human splice site DNA consensus motifs.
    Figure 5: A summary of human splice site DNA consensus motifs.

    Summarized splice site sequences are classified using the nucleotides marked by the grey boxes. All borders of human exons within Ensembl v83 multi-exon transcripts that overlap with Reference Sequence (RefSeq) database mRNA IDs were used. Identical coordinates from overlapping transcripts were collapsed into a single occurrence so that junctions were not counted multiple times. The first exons had only their exon–intron junction evaluated, whereas terminal exons had only their intron–exon junction evaluated. This led to a total of 189,255 5′ splice sites (left panels, with the black line marking the exon–intron border) and 187,091 3′ splice sites (right panels, with the black line marking the intron–exon border). Splice site sequences of U12-type introns were obtained from the U12 Intron Database191. After identifying the 5′ and 3′ sites that overlap with the respective U11-type and U12-type splice sites, the remaining U2-type intron splice site sequences were examined. 5′ and 3′ splice sites were classified independently and sequentially based on the indicated nucleotides. For example, 53.58% of unique U1-type exon–intron junctions contain GTRAG, and the remaining U1-type junctions were classified on the basis of the first two intronic nucleotides, GT. The percentage of unique junctions containing each motif is indicated. Weblogo 3 was used to show the relative frequency of nucleotides at each position192. a | Consensus motifs of the U1-type 5′ splice sites with GT at the border and the U2-type 3′ splice sites with AG at the border. b | Consensus motifs of the U11-type 5′ splice sites and U12-type 3′ splice sites. c | Consensus motifs of U1-type 5′ splice sites with GC at the border, or with TN or VN at the border (in which N stands for any nucleotide, and V stands for any nucleotide except T), and U2-type 3′ splice sites with BG or W at the border (in which B stands for any nucleotide except A, and W stands for T or A).

  6. Cryptic splicing in disease and therapeutic strategies.
    Figure 6: Cryptic splicing in disease and therapeutic strategies.

    a | Cryptic exons are normally repressed by RNA-binding proteins (RBPs) such as heterogeneous nuclear ribonucleoprotein C (HNRNPC) or by U1 small nuclear ribonucleoproteins (snRNP). b | Examples of mutations (α, β, γ and δ) in deep intronic regions that can activate cryptic splicing events in disease-associated genes. Mutation α: HNRNPC binding to a poly(U) tract (U tract) upstream of an antisense Alu element represses recognition of the cryptic 3′ splice site within the element. Intronic deletions or point mutations that shorten the U tract can impede HNRNPC recruitment but allow U2 small nuclear RNA auxiliary factor (U2AF2) binding, leading to Alu exonization. A deletion within an Alu in the PTS gene (encoding 6-pyruvoyltetrahydropterin synthase) leads to splicing of an Alu exon that introduces a frameshift, thereby causing the neurologic disease hyperphenylalaninaemia8, 141. Mutation β: in ataxia telangiectasia mutated (ATM), U1 snRNP binding to an intronic element within a cryptic exon inhibits its recognition as a splicing competent exon. Patients with ataxia telangiectasia present a 4 nt deletion that abolishes U1 snRNP interaction, causing cryptic exon activation110. Mutation γ: a point mutation within a deep intronic sequence of cystic fibrosis transmembrane conductance regulator (CFTR) generates an active 5′ splice site that allows insertion of a cryptic exon within the CFTR transcripts, which causes cystic fibrosis135. Mutation δ: in breast cancer 2 (BRCA2), a point mutation that disrupts a canonical 3′ splice site activates an upstream cryptic exon (grey arrow)136. Disrupted BRCA2 expression causes breast, ovarian and other cancer types. c | New therapeutic strategies in cancer involve spliceosome targeting156, 161, 162. In MYC-driven tumours, oncogenic MYC causes transcriptional amplification, which overloads the splicing machinery and makes these cells more sensitive to alterations in splicing fidelity. Genetic knockdown or pharmacological inhibition of spliceosomal components leads to the accumulation of retained introns, which results in increased apoptosis and reduced tumorigenic and metastatic potential of MYC-driven tumours.

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Author information

  1. These authors contributed equally to this work.

    • Christopher R. Sibley &
    • Lorea Blazquez

Affiliations

  1. Department of Molecular Neuroscience, University College London Institute of Neurology, Russell Square House, Russell Square, London WC1B 5EH, UK.

    • Christopher R. Sibley,
    • Lorea Blazquez &
    • Jernej Ule
  2. Department of Medicine, Division of Brain Sciences, Imperial College London, Burlington Danes, DuCane Road, London W12 0NN, UK.

    • Christopher R. Sibley

Competing interests statement

The authors declare no competing interests.

Corresponding author

Correspondence to:

Author details

  • Christopher R. Sibley

    Christopher R. Sibley is a junior group leader in the Division of Brain Sciences, Imperial College London, UK. During his postdoctoral work with Jernej Ule he studied post-transcriptional processing of RNA in the nervous system using the functional genomics approaches of individual-nucleotide-resolution crosslinking and immunoprecipitation (iCLIP) and RNA sequencing. This led to the discovery of the first reported mammalian recursive splice sites. He continues to research the numerous unannotated and non-canonical features of the transcriptome discussed in this Review. In addition, he uses functional genomics and systems biology approaches to study master regulators in neurological diseases.

  • Lorea Blazquez

    Lorea Blazquez completed her Ph.D. at the Biodonostia Institute, San Sebastian, Spain, where she worked on identification, functional characterization and therapeutics of splicing mutations. She then studied strategies to inhibit gene expression with small RNA molecules at Center for Applied Medical Research (CIMA), Pamplona, Spain. She joined the Ule laboratory to study the function and regulation of recursive splicing.

  • Jernej Ule

    Jernej Ule is Professor of Molecular Neuroscience at the University College London Institute of Neurology, London, UK. He has a long-standing interest in mechanisms of ribonucleoprotein (RNP) complexes and RNA structure in the regulation of pre-mRNA processing and mRNA translation. His group developed iCLIP (individual-nucleotide-resolution crosslinking and immunoprecipitation) and hiCLIP (RNA hybrid and iCLIP), which are methods to study in vivo protein–RNA and RNA–RNA contacts in a transcriptome-wide manner. Recently, the group has begun to focus on the regulation and functions of cryptic splicing events, such as those generated by transposable elements or recursive splice sites. Jernej Ule's homepage.

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