Skip to main content

Molecular Biology

Exons, Introns, & Splicing

Eukaryotic gene structure and processing, Exons, Introns, and Splicing, is a crucial concept because, in eukaryotes (like humans!), the coding information within a gene is often interrupted, like a sentence with irrelevant phrases inserted in the middle. Splicing is the editing process that removes the interruptions to create a coherent message

The Structure: Exons and Introns

Imagine a gene on a chromosome as a segment of DNA that holds the instructions for building a protein (or a functional RNA molecule). In eukaryotes, this segment isn’t usually one continuous block of code. Instead, it’s composed of two types of sequences:

  • Exons (Expressed Sequences): These are the segments of the gene that contain the actual coding information – the sequences that will ultimately be translated into amino acids or become part of a functional RNA (like rRNA or tRNA). Think of these as the essential words in the instruction manual
  • Introns (Intervening Sequences): These are non-coding segments that lie between the exons within the gene sequence. They interrupt the flow of coding information. Think of these as interrupting, non-essential phrases or commentary scattered within the instructions. Introns can vary greatly in number and size, sometimes being much longer than the exons they separate

Important Note Prokaryotes (bacteria) generally lack introns. Their coding sequences are typically continuous

The Initial Product: Pre-mRNA

When a eukaryotic gene containing introns is transcribed by RNA Polymerase II, the initial RNA molecule produced is called pre-messenger RNA (pre-mRNA) or heterogeneous nuclear RNA (hnRNA). This pre-mRNA molecule is a direct copy of the entire gene sequence, including both exons and introns

This pre-mRNA is not yet ready to be translated into a protein. It needs to be processed, and the most critical processing step is splicing

The Process: Splicing

Splicing is the molecular editing process that removes the introns from the pre-mRNA and joins the exons together in the correct order. This creates a mature, continuous coding sequence in the messenger RNA (mRNA) molecule

The Machinery: The Spliceosome

Splicing is carried out by a large and dynamic molecular machine called the spliceosome. The spliceosome is composed of:

  • Small Nuclear RNAs (snRNAs): Several types (U1, U2, U4, U5, U6). These RNAs are the key players; they recognize the sequences signaling the intron boundaries and likely catalyze the splicing reactions
  • Proteins: Numerous proteins combine with the snRNAs to form small nuclear ribonucleoprotein particles (snRNPs – pronounced “snurps”). These snRNPs, along with other protein factors, assemble on the pre-mRNA to form the active spliceosome

The Signals: Splice Sites

How does the spliceosome know exactly where to cut? There are short, conserved consensus sequences at the boundaries of introns that act as signals:

  • 5’ Splice Site (Donor Site): Located at the 5’ end of the intron (the junction between the upstream exon and the intron). Typically contains a GU sequence in the RNA (GT in the DNA coding strand)
  • 3’ Splice Site (Acceptor Site): Located at the 3’ end of the intron (the junction between the intron and the downstream exon). Typically contains an AG sequence in the RNA (AG in the DNA coding strand)
  • Branch Point: An Adenine (A) nucleotide located within the intron, usually 15-45 bases upstream of the 3’ splice site. This A plays a crucial role in the splicing chemistry

The Mechanism (Simplified)

  1. Recognition snRNPs (like U1 and U2) bind to the 5’ splice site and the branch point, respectively
  2. Assembly Other snRNPs (U4/U6 and U5) join, bringing the splice sites and branch point close together, forming the active spliceosome
  3. First Cut The RNA is cleaved at the 5’ splice site
  4. Lariat Formation The freed 5’ end of the intron loops around and forms a unique 2’-5’ phosphodiester bond with the branch point Adenine, creating a lariat (lasso-shaped) structure
  5. Second Cut The RNA is cleaved at the 3’ splice site, releasing the intron lariat
  6. Ligation The two adjacent exons are joined (ligated) together by a standard 5’-3’ phosphodiester bond
  7. Release The mature mRNA (now containing only exons) is released, and the spliceosome disassembles. The intron lariat is degraded

Alternative Splicing: Generating Diversity

One of the most significant consequences of the exon-intron structure is alternative splicing. This means that the exons of a single pre-mRNA transcript can be spliced together in different combinations. For example:

  • An exon might be skipped entirely
  • Mutually exclusive exons might be chosen (either exon A or exon B is included, but not both)
  • Alternative 5’ or 3’ splice sites might be used, making an exon shorter or longer

Why is this important? Alternative splicing allows a single gene to produce multiple different mRNA transcripts, which in turn can be translated into different protein isoforms (variants). This vastly increases the coding potential of the genome, allowing complex organisms to produce a wide array of proteins from a relatively limited number of genes. Different protein isoforms can have different functions, localizations, or regulatory properties

Clinical Laboratory Relevance

Understanding exons, introns, and splicing is critical in the clinical molecular lab:

  • Genetic Disease Diagnosis: A significant proportion of disease-causing mutations affect splicing. These mutations can occur:
    • Directly within the splice site consensus sequences (GU/AG), destroying the signal
    • Near splice sites, interfering with snRNP binding
    • Within exons or introns, creating cryptic splice sites (sequences that resemble real splice sites and are mistakenly used by the spliceosome)
    • In splicing regulatory elements (enhancers/silencers) within exons or introns
  • Consequences of Splicing Mutations: These mutations can lead to:
    • Exon skipping: An entire exon is left out of the mature mRNA
    • Intron retention: An intron (or part of it) is included in the mature mRNA
    • Use of cryptic splice sites: Leads to abnormally short or long exons, often causing frameshifts or incorporating incorrect amino acids
    • Result: Often produces truncated, unstable, or non-functional proteins. Examples include some forms of cystic fibrosis, beta-thalassemia, spinal muscular atrophy (SMA), Duchenne muscular dystrophy, and many cancers
  • Diagnostic Testing: Molecular tests (like PCR amplification followed by sequencing) are used to identify mutations in splice sites or surrounding regions. RNA analysis (RT-PCR followed by sequencing or fragment analysis) can directly visualize the effects of a mutation on the splicing pattern (e.g., showing exon skipping)
  • Therapeutics: Splicing modulation is an exciting area of drug development. Antisense oligonucleotides (ASOs) can be designed to bind to specific pre-mRNA sequences and alter splicing outcomes – for example, forcing the inclusion of a skipped exon (e.g., treatments for SMA like Spinraza and Duchenne muscular dystrophy like Exondys 51)
  • Cancer Biology: Aberrant splicing patterns are increasingly recognized as hallmarks of cancer, contributing to tumor growth, metastasis, and drug resistance. Mutations in splicing factor genes themselves can also drive cancer

Key Terms

  • Exon: A segment of a gene’s DNA or RNA molecule containing information coding for a protein or peptide sequence (or functional RNA); retained after splicing
  • Intron: A non-coding segment of a gene’s DNA or RNA molecule that lies between exons; removed by splicing
  • Splicing: The process of removing introns from pre-mRNA and joining exons together
  • Pre-mRNA (Primary Transcript): The initial RNA molecule transcribed from a eukaryotic gene, containing both exons and introns
  • Mature mRNA: The fully processed mRNA molecule, containing only exons, ready for translation
  • Spliceosome: A large RNA-protein complex that catalyzes the removal of introns from pre-mRNA
  • snRNP (Small Nuclear Ribonucleoprotein Particle): Complexes of small nuclear RNAs (snRNAs) and proteins that are components of the spliceosome
  • 5’ Splice Site (Donor Site): Consensus sequence (usually GU) marking the beginning of an intron
  • 3’ Splice Site (Acceptor Site): Consensus sequence (usually AG) marking the end of an intron
  • Branch Point: An Adenine residue within the intron crucial for lariat formation
  • Lariat: The looped structure formed by the intron during splicing after cleavage at the 5’ splice site and bonding to the branch point
  • Alternative Splicing: A regulated process where different combinations of exons from the same gene are joined, producing multiple mRNA and protein isoforms from a single gene
  • Cryptic Splice Site: A sequence within an exon or intron that resembles a consensus splice site and may be inappropriately used if the normal site is mutated