Splicing of Pre‐mRNA

Abstract

The majority of eukaryotic messenger RNAs (mRNAs) are initially transcribed as intron‐containing precursor mRNAs (pre‐mRNAs). Removal of introns from each pre‐mRNA by splicing is necessary to generate a mature mRNA. Pre‐mRNA splicing is catalysed by the spliceosome, a large ribonucleoprotein complex that contains five small nuclear RNAs and numerous protein factors and forms anew on each intron of nascent transcripts. In higher eukaryotes, alternative splicing of pre‐mRNA generates multiple mRNA isoforms from a single gene and is regulated by cellular RNA‐binding proteins. Alternative splicing not only increases proteome diversity but also has the potential to regulate gene expression levels post‐transcriptionally. Thus, alterative splicing may have a great impact on a wide range of physiological and developmental processes. Defects in pre‐mRNA splicing underlie a considerable number of genetic diseases and cancers and may fine‐tune disease severity.

Key Concepts

  • In most eukaryotic pre‐mRNAs, the coding sequences (exons) are interrupted by stretches of non‐coding sequences (introns).
  • The spliceosome uses a two‐step transesterification reaction to remove introns and join exons of pre‐mRNAs.
  • The snRNAs and several conserved spliceosomal protein factors are implicated in the key catalytic steps of splicing.
  • Alternative splicing extends proteome diversity and regulates gene expression levels.
  • Alternative splicing is determined by interplay between trans‐acting regulatory factors and cis‐elements of pre‐mRNAs.
  • Alteration of cis‐elements and aberrant control of trans‐acting factors can lead to diseases.

Keywords: pre‐mRNA splicing; spliceosome; alternative splicing; snRNA; RNA‐binding protein

Figure 1. The mechanism of splicing. (a) The consensus sequences of a typical metazoan intron (R, purine; Y, pyrimidine). (b) Schematic of the two consecutive steps of transesterification. The two red arrows indicate the two nucleophilic attacks of the splicing reaction. In the first step, the 2′‐hydroxyl of the branch‐point adenosine attacks the 5′ splice site, and in the second step, the 3′‐hydroxyl of exon I attacks the 3′ splice site, resulting the ligated exons and a lariat intron.
Figure 2. The stepwise assembly of the spliceosome. The U1, U2, U4/U6 and U5 snRNPs and spliceosomal protein factors sequentially assemble onto and dissociate from the pre‐mRNA. The representative intermediate splicing complexes are E, A, B, B* and C; complex B* represents the catalytically active spliceosome. After two sequential transesterification reactions, ligated exons and excised introns are generated. DEAD box RNA helicases (red) facilitate conformational changes of the splicing complexes and may control the fidelity of the splicing reaction. The Prp19 protein complex is crucial for multiple steps in splicing including the catalytic activation of the spliceosome.
Figure 3. Alternative splicing and regulation. (a) Five basic modes of alternative splicing. The coloured boxes are exons in differentially alternative splicing modes. (b) Trans‐acting splicing regulators (activators or suppressors; not shown) bind to responsive cis‐elements, that is, exonic splicing enhancers (ESEs), exonic splicing silencers (ESSs), intronic splicing enhancers (ISEs) and intronic splicing silencers (ISSs), to determine the splice site or exon usage. (c) Cellular signalling can modulate CD44 v5 exon inclusion. Upon extracellular stimuli, Sam68 is phosphorylated by ERK via the Ras signalling pathway and displaces or inhibits hnRNP A1. Sam68 acts cooperatively with SRm160 to promote v5 inclusion. Brm is a component of the chromatin remodelling SWI/SNF complex and can induce accumulation of RNA polymerase II on the v5 exon and thus promotes v5 inclusion.
Figure 4. Splicing‐related genetic diseases. (a) An intronic mutation near the 5′ splice site of IKBKAP intron 20 induces skipping of exon 20 in familiar dysautonomia. (b) The C‐to‐T transition at position 6 of SMN2 exon 7 essentially causes exon 7 skipping. In addition, two intronic splicing silencers (ISSs) are located in intron 7. Antisense oligonucleotides that block these ISSs can promote exon 7 inclusion and thus may be in SMA therapeutics.
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Further Reading

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Tu, Chi‐Chiang, and Tarn, Woan‐Yuh(Feb 2015) Splicing of Pre‐mRNA. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0005037.pub3]