Alternative Splicing Simulator

About RNA Splicing

RNA splicing is the process by which introns (non-coding sequences) are removed from pre-mRNA and exons (coding sequences) are joined together to form mature mRNA. This process is carried out by the spliceosome, a large molecular machine composed of five small nuclear RNAs (snRNAs) and hundreds of proteins.

The Splicing Mechanism

Splicing occurs in two sequential transesterification reactions:

• Step 1: The 2'-OH of an adenosine in the branch point attacks the 5' splice site (donor), creating a lariat intermediate
• Step 2: The free 3'-OH of the upstream exon attacks the 3' splice site (acceptor), joining the exons and releasing the lariat intron

Splice Site Recognition

Splice sites are recognized by conserved consensus sequences:

• 5' splice site (donor): GT dinucleotide with upstream exonic and downstream intronic sequences
• Branch point: Adenosine residue typically 20-50 nucleotides upstream of 3' splice site
• Polypyrimidine tract: Pyrimidine-rich region between branch point and 3' splice site
• 3' splice site (acceptor): AG dinucleotide at the intron-exon boundary

Exon Definition vs Intron Definition

In vertebrates, where exons are typically shorter than introns, the spliceosome preferentially recognizes exons rather than introns. SR proteins bound to exonic splicing enhancers (ESEs) recruit splicing factors to the flanking splice sites, defining the exon boundaries. This "exon definition" model explains how the splicing machinery locates small exons within vast intronic sequences.

Alternative Splicing Mechanisms

Alternative splicing generates multiple mRNA isoforms from a single gene:

• Exon skipping (cassette exons): Optional exons that can be included or excluded
• Alternative 5' splice sites: Multiple donor sites leading to different exon boundaries
• Alternative 3' splice sites: Multiple acceptor sites creating different exon ends
• Intron retention: Introns retained in the mature mRNA
• Mutually exclusive exons: Only one of several exons is included

SR Proteins and Splicing Regulation

SR proteins (serine/arginine-rich proteins) are key splicing regulators that:

• Bind to exonic splicing enhancers (ESEs) within exons
• Recruit U1 snRNP to the 5' splice site and U2AF to the 3' splice site
• Promote exon inclusion by strengthening splice site recognition
• Enable exon definition by bridging across short exons

Biological Significance

Alternative splicing dramatically increases proteomic diversity:

• Over 95% of human multi-exon genes undergo alternative splicing
• Enables tissue-specific and developmentally regulated gene expression
• Mutations affecting splicing cause many human diseases (e.g., spinal muscular atrophy, beta-thalassemia)
• Splicing regulation is a major target for therapeutic intervention

Historical Context

RNA splicing was discovered in 1977 independently by Richard Roberts and Phillip Sharp, who shared the 1993 Nobel Prize in Physiology or Medicine. The discovery that genes are "split" (containing introns) revolutionized molecular biology and revealed unexpected complexity in gene expression. The exon definition model was proposed in the 1990s to explain how short exons are recognized in vertebrate genes with long introns.