RNA splicing

RNA splicing is a crucial post-transcriptional process in molecular biology in which a newly synthesised precursor messenger RNA (pre-mRNA) transcript is converted into a mature messenger RNA (mRNA) molecule. This transformation involves the precise removal of non-coding introns and the ligation of coding exons, permitting the production of a transcript competent for translation. Splicing forms a central component of gene expression in eukaryotes and contributes significantly to the diversity of proteins produced by a single genome.

Introns and Splicing Signals

Introns, from the terms intragenic region and intracistron, are non-coding segments of DNA and the corresponding regions within the pre-mRNA. They interrupt the coding sequence and must be excised for the production of functional mRNA. Introns occur in a wide range of genes, including those encoding proteins, ribosomal RNA, and transfer RNA.
Three principal intronic sequence elements are essential for correct splicing:

• 5′ splice (donor) site: Characterised by an almost invariant GU dinucleotide at the start of the intron, situated within a moderately conserved sequence.
• Branch site: Positioned upstream of the 3′ end of the intron and containing an essential adenine nucleotide that initiates lariat formation.
• 3′ splice (acceptor) site: Defined by a highly conserved AG dinucleotide at the end of the intron, preceded by a pyrimidine-rich tract composed largely of cytosine and uracil.

Consensus sequences for introns, expressed using IUPAC nucleic acid notation, include a GU-rich donor region and a Y-rich acceptor region. Variations in these motifs, along with differences in the spacing between the branchpoint and the acceptor site, strongly influence splice site selection. Point mutations within these regions can activate cryptic splice sites, leading to aberrant splicing and altered protein structure, often resulting in insertions, deletions, or truncations rather than single amino acid substitutions.

Formation and Activity of the Spliceosome

Splicing of most introns is catalysed by the spliceosome, a dynamic and complex ribonucleoprotein assembly composed of five small nuclear ribonucleoproteins (snRNPs) designated U1, U2, U4, U5, and U6. Assembly of the spliceosome occurs co-transcriptionally in the nucleus, with the RNA components of the snRNPs interacting directly with intronic elements to drive catalysis.
A sequence of ordered complexes forms during spliceosome maturation:

• Complex E: U1 binds to the 5′ splice site, while other factors interact with the branchpoint, polypyrimidine tract, and 3′ splice site.
• Complex A (prespliceosome): U2 displaces SF1 at the branch site in an ATP-dependent step.
• Complex B (precatalytic spliceosome): The U4/U5/U6 tri-snRNP binds, bringing the exons and intronic regions into proximity.
• Complex B*: Release of U1 permits further rearrangements, allowing U6 to pair with U2.
• Complex C (catalytic spliceosome): U4 dissociates and the U6–U2 complex catalyses the first transesterification reaction, in which the 5′ end of the intron is joined to the branchpoint adenine to form a lariat. A second transesterification ligates the two exons.
• Post-spliceosomal complex: The mature mRNA is released, the intron lariat is degraded, and snRNPs are recycled.

Canonical spliceosomal activity accounts for over 99 per cent of eukaryotic splicing. When intronic sequences deviate from the GU–AG pattern, non-canonical or minor splicing occurs.

The Minor Spliceosome

A distinct spliceosomal system, the minor spliceosome, removes a rare class of introns that possess different consensus sequences. While U5 is shared between the two systems, the minor spliceosome uses analogues of the major snRNPs: U11 (equivalent to U1), U12 (U2), U4atac (U4), and U6atac (U6). The existence of two spliceosomal systems illustrates the evolutionary complexity and diversity of intron processing mechanisms in eukaryotes.

Alternative Splicing Pathways

Splicing does not always proceed by simple excision of introns as discrete units. Several alternative pathways exist:

• Recursive splicing: Particularly associated with long introns, this process removes intronic segments in sequential steps. First described in the Ultrabithorax gene of Drosophila melanogaster, it has also been observed in humans.
• Trans-splicing: Exons from separate RNA molecules are joined together. This can occur between endogenous transcripts or between host and viral or artificial transcripts, expanding the regulatory and coding repertoire of the cell.
• Self-splicing: Some introns act as ribozymes and catalyse their own removal without protein assistance. Group I and group II introns utilise two sequential transesterification reactions. Group I introns typically require a guanine nucleoside as a cofactor, whereas group II introns form lariat intermediates akin to spliceosomal reactions. These introns are thought to be evolutionary precursors of spliceosomal machinery and may represent remnants of an ancient RNA-based biological world.

tRNA Splicing

tRNA splicing involves a pathway distinct from those of pre-mRNA. In Saccharomyces cerevisiae, the TSEN complex cleaves the intron from pre-tRNA at two positions, generating tRNA halves terminated with different chemical groups. A series of enzymes subsequently phosphorylate, process, and ligate the tRNA fragments into a mature molecule. Finally, a phosphotransferase removes a residual phosphate group. This pathway demonstrates the biochemical diversity of RNA processing mechanisms.

Splicing Pathways Across Evolution

Splicing occurs in all major domains of life, although its prevalence varies widely. Eukaryotes splice numerous protein-coding and non-coding transcripts, contributing to complex regulation and proteomic diversity. Prokaryotes, by contrast, engage in splicing far less frequently, and when it occurs, it typically involves self-splicing introns rather than spliceosomal processes. The evolutionary distribution suggests that spliceosomal machinery arose later in evolutionary history, refining gene expression and permitting the emergence of intricate regulatory networks.