Messenger RNA Splicing Signals

Abstract

Splicing is a post‐transcriptional processing step in which intervening sequences (introns) are excised and coding sequences (exons) are ligated together to create the mature mRNA (messenger ribonucleic acid) molecule. It is a sequential process that is facilitated by the information in the RNA (ribonucleic acid) sequence (splicing regulatory elements/signals) and numerous RNA‐binding proteins (trans factors). Splicing occurs through two biochemical steps, and is catalysed by a large ribonucleoprotein known as the spliceosome. The spliceosomal machinery recognises the core splicing signals and assembles in a stepwise fashion on the premRNA molecule. These signals include the obligate 5′ splice site, 3′ splice site and branch site sequence, as well as enhancer and silencer sequences which functionally interact with RNA binding proteins. A current research application in the field uses computational approaches to study splicing signals and predict the effect of single nucleotide variations on message processing.

Key Concepts

  • Splicing is a post‐transcriptional process in which certain sections of RNA (known as introns) are removed and other sections (known as exons) are ligated together, producing the mature mRNA molecule.
  • Splicing is catalysed by a large macromolecular machine known as the spliceosome.
  • The major elements required for splicing include the 5′ splice site (5′ss), the 3′ splice site (3′ss), the polypyrimidine tract and the branch site.
  • Most introns begin with a ‘GT’ sequence and end with an ‘AG’ sequence; however, there exists a class of intron with different boundary sequences that are recognised by a parallel spliceosome.
  • Outside of these major elements, there are additional enhancer and suppressor signals that interact with RNA‐binding proteins (RBPs) and affect splicing outcome.
  • Different combinations and positions of binding of RBPs can result in different splicing outcome.
  • Scientists do not yet have a complete understanding of all of the different splicing signals, and this topic is an area of active research.
  • Researchers are using computational machine learning approaches to predict the effects of sequence variation on splicing outcome.
  • Researchers are also using new methods of sequencing to determine which RNA sequences the RBPs are recognising.

Keywords: splice site selection; consensus sequences; splicing enhancers; exon definition; small nuclear ribonucleoprotein particles (snRNPs); exonic splicing enhancers; RNA‐binding proteins; intronic splicing silencers; identification of causal variants

Figure 1. The chemistry of pre‐mRNA splicing. (a) The two steps of splicing are indicated in cartoon form, (b) with an expanded view of the branch structure, including the unusual 2′–5′ phosphodiester bond.
Figure 2. Consensus sequences and base pairing between splicing consensus sequences and small nuclear RNAs (snRNAs). (a) Frequency matrices presenting the 5′ splice site, 3′ splice site, and branchpoint consensus sequences. (b) Base pairing between consensus sequences and U‐RNAs (U1: 5′ splice site; U2: branch site). The single unpaired nucleotide in the base pairing between U2 snRNA and the branch site is the A at which branch formation occurs. The U snRNA sequences shown are phylogenetically invariant, and the consensus sequences shown are similar in all eukaryotes. However, the sequence of any individual splice site is likely to differ from the sequence shown. As a result, the extent of base pairing is generally less than depicted here. Additional base pairing interactions between snRNAs and the pre‐mRNA, or among snRNAs, that occur late in the splicing process are not shown.
Figure 3. Examples of interactions among factors that recognise splicing signals. Appropriate spacing of splice sites across either (a) an intron or (b) an exon facilitates spliceosome assembly. (c) Recognition of an exonic splicing enhancer by an SR protein. (d) Repression of splicing mediated by heterogeneous nuclear RNA proteins (hnRNPs) bound at sites within introns.
Figure 4. Different outcomes of the failure to recognise a specific splicing signal. When a splicing signal (such as the 5′ splice site at the 3′ end of exon 2) is defective, one might expect the affected intron to be retained (alternative A). However, the most commonly observed result is exon skipping (alternative B). Another result often observed is splicing at a cryptic 5′ splice site (alternative C). These outcomes demonstrate the role of splicing signals other than the splice sites themselves.
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References

Amit M, Donyo M, Hollander D, et al. (2012) Differential GC content between exons and introns establishes distinct strategies of splice‐site recognition. Cell Reports 1 (5): 543–556.

Cho S, Hoang A, Sinha R, et al. (2011) Interaction between the RNA binding domains of Ser‐Arg splicing factor 1 and U1‐70K snRNP protein determines early spliceosome assembly. Proceedings of the National Academy of Sciences of the United States of America 108 (20): 8233–8238.

Mazza C, Segref A, Mattaj IW and Cusack S (2002) Large‐scale induced fit recognition of an m(7)GpppG cap analogue by the human nuclear cap‐binding complex. EMBO Journal 21 (20): 5548–5557.

Zhang Y, Madl T, Bagdiul I, et al. (2013) Structure, phosphorylation and U2AF65 binding of the N‐terminal domain of splicing factor 1 during 3′‐splice site recognition. Nucleic Acids Research 41 (2): 1343–1354.

Further Reading

Berget SM (1995) Exon recognition in vertebrate splicing. Journal of Biological Chemistry 270: 2411–2414.

Black DL (1995) Finding splice sites within a wilderness of RNA. RNA 1: 763–771.

Burge C and Karlin S (1997) Prediction of complete gene structures in human genomic DNA. Journal of Molecular Biology 268: 78–94.

Gesteland RF, Cech TR and Atkins JF (eds) (1999) The RNA World, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.

Lee YD and Rio C (2015) Mechanisms and regulation of alternative pre‐mRNA splicing. Annual Review of Biochemistry 84: 291–323.

Matera AG and Wang Z (2014) A day in the life of the spliceosome. Nature Reviews Molecular Cell Biology 15 (2): 108–121.

Moore MJ, Query CC and Sharp PA (1993) Splicing of precursors to mRNA by the spliceosome. In: Gesteland RF and Atkins JF (eds) The RNA World, pp. 303–357. Cold Spring Harbor, NY: Cold Spring Harbor Press.

Staley JP and Guthrie C (1998) Mechanical devices of the spliceosome: motors, clocks, springs and things. Cell 92: 315–326.

Taggart AJ, DeSimone AM, Shih JS, Filloux ME and Fairbrother WG (2012) Large‐scale mapping of branchpoints in human pre‐mRNA transcripts in vivo. Nature Structural and Molecular Biology 19 (7): 719–721.

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How to Cite close
Cygan, Kamil J, Taggart, Allison J, Fairbrother, William G, and Mount, Stephen M(Feb 2017) Messenger RNA Splicing Signals. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0000888.pub2]