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Lessons from non-canonical splicing

Key Points

  • The development of new methods for preparing and sequencing RNA sequencing libraries, as well as new alignment algorithms, have revealed many thousands of previously unknown non-canonical splicing events.

  • Non-canonical splicing events are often tissue-specific and are particularly enriched in the central nervous system, thereby increasing proteome diversity or regulating gene expression.

  • Cryptic exons, microexons and recursive splice sites often require unconventional exon definition mechanisms.

  • Other non-canonical splicing events result from lower or higher splicing efficiency than normal (such as retained introns and exonic introns), changes in the usual order of splicing (circular RNAs and chimeric RNAs) or changes in the consensus sequence (atypical splice sites).

  • Transposable elements are a rich source of newly emerging cryptic exons, which can contribute to the evolution of gene regulatory networks.

  • Mutations that perturb functionally important non-canonical splicing events, or strongly increase the recognition of cryptic splice sites, can cause numerous diseases.

  • Non-canonical splicing mechanisms offer new therapeutic opportunities to treat disease.

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.

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Figure 1: Cryptic exons and microexons.
Figure 2: Recursive splicing of long introns.
Figure 3: Intron retention and exitrons.
Figure 4: Formation of circularRNAs and chimeric transcripts.
Figure 5: A summary of human splice site DNA consensus motifs.
Figure 6: Cryptic splicing in disease and therapeutic strategies.

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Acknowledgements

The authors thank K. Zarnack for comments on the manuscript. This work was supported by the European Research Council (617837-Translate) and a Marie Curie Post-doctoral Research Fellowship (627783-NeuroCRYSP) to L.B., and an Edmond and Lily Safra Fellowship to C.R.S.

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Glossary

Splice sites

Sequences at the boundary of exons and introns, which contain motifs that recruit small nuclear ribonucleoproteins and RNA-binding proteins to initiate the splicing reaction. 3′ and 5′ splice sites are located upstream and downstream of exons, respectively.

Cryptic exons

Exons that are not annotated by current genomic databases, such as ENSEMBL, and are often only revealed after removing a repressive RNA-binding protein or after a genomic mutation that increases their splicing efficiency.

Microexons

Exons that are shorter than 30 nt.

Recursive splicing

A mechanism that allows an intron to be spliced in two or more steps.

Circular RNAs

(circRNAs). RNA molecules that have become circularized owing to intramolecular ligation of their 5′ and 3′ ends.

Exonic introns

(Exitrons). Introns located within annotated exons.

Exon definition mechanism

The process by which exons are recognized and defined as functional units through interactions between multiple small nuclear ribonucleoproteins (snRNPs) and RNA-binding proteins (RBPs), especially U1 and U2 snRNPs and serine/arginine-rich proteins.

Small nuclear ribonucleoproteins

(snRNPs). Ribonucleoprotein complexes assembled around the small nuclear RNAs that interact with splice sites or the branch point on pre-mRNA and thereby coordinate and catalyse the splicing reaction.

U2 auxiliary factor complex

(U2AF complex). A complex of two U2AF RNA-binding proteins (RBPs) that bind the 3′ splice site and facilitate the recruitment of the U2 small nuclear ribonucleoprotein (snRNP) to the branch point.

Serine/arginine-rich proteins

(SR proteins). A family of RNA-binding proteins (RBPs) containing a protein domain with long repeats of serine and arginine that generally promote exon definition when binding to exons.

Recursive splice sites

(RS sites). The sites of recursive splicing, which consists of a 3′ splice site that is followed by a sequence that reconstitutes a 5′ splice site after the first splicing event.

Seed sequence

The section of a sequencing read that is used to align the read to the genome or transcriptome

Nonsense-mediated decay

(NMD). A pathway that initiates decay of certain transcripts, especially those containing a premature termination codon.

Alu elements

Retrotransposons, ~300 nt long, belonging to the family of short interspersed elements (SINEs), which originally derived from 7SL signal recognition particle RNA.

Crosslinking and immunoprecipitation

(CLIP). A method used to identify the RNA targets bound by an RNA-binding proteinof-interest that employs crosslinking, immunoprecipitation and stringent purification of protein–RNA complexes by SDS-PAGE.

NOVA RBPs

A joint name for RNA-binding proteins encoded by two partially redundant genes that are expressed in the brain, neuro-oncological ventral antigen 1 (NOVA1) and NOVA2.

NMD exons

(Nonsense-mediated decay exons). Exons that contain a premature termination codon and are therefore targeted for NMD.

Intrasplicing

An unconventional splicing mechanism in which splicing to a 3′ splice site reconstitutes a new 3′ splice to be used in a subsequent splicing step.

RS site exons

(Recursive splice site exons). Exons that follow an RS site and which are required for the exon definition mechanism that initiates splicing at the RS site.

Spliceosome

A macromolecular machine consisting of small nuclear ribonucleoproteins (snRNPs) and additional RNA-binding proteins (RBPs) that coordinate and catalyse the splicing reaction.

Chimeric transcripts

Transcripts that are formed when sections of two or more different genes are joined together in a new transcript either by splicing or as a result of chromosomal fusions.

Axonogenesis

The generation and outgrowth of axons during neuronal development.

Ataxia telangiectasia

An autosomal recessive disorder involving cerebellar degeneration, immunodeficiency, chromosomal instability, radiosensitivity and cancer predisposition. It is caused by mutations in ataxia telangiectasia mutated (ATM).

Laron syndrome

An autosomal recessive disorder characterized by short stature caused by mutations in growth hormone receptor (GHR).

Duchenne muscular dystrophy

A progressive proximal muscular dystrophy caused by mutations in dystrophin (DMD).

Hyperphenylalaninaemia

A neurologic disorder caused by autosomal recessive mutations in the genes encoding enzymes involved in the synthesis or regenerationof the BH4 (tetrahydrobiopterin) cofactor. The most common form is caused by mutations in PTS (6-pyruvoyltetrahydropterin synthase).

Autophagy

Intracellular pathway responsible for regulated disassembly of unnecessary or dysfunctional cellular components after their targeting to lysosomes.

β-thalassaemia

A genetic blood disorder characterized by a defective synthesis of the β-globin chains of haemoglobin, thus causing abnormal erythropoiesis and anaemia.

Aptamers

Oligonucleotide (or peptide) molecules that have secondary and tertiary structures that strongly bind to specific proteins or other cellular targets.

Co-transcriptional decay

RNA surveillance mechanism that acts in the nucleus while transcripts are still associated with the chromatin template.

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Sibley, C., Blazquez, L. & Ule, J. Lessons from non-canonical splicing. Nat Rev Genet 17, 407–421 (2016). https://doi.org/10.1038/nrg.2016.46

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