The process of life relies on the flow of genetic information, moving from DNA to RNA and finally to protein. In eukaryotic cells, a gene is first copied into an initial RNA molecule called precursor messenger RNA (pre-mRNA) during transcription. This pre-mRNA is not yet ready to serve as a blueprint for protein synthesis. Before it can exit the nucleus and be translated by ribosomes, it must undergo significant modification. This necessary editing step removes non-coding sections, ensuring the final messenger RNA (mRNA) contains only the correct instructions for protein synthesis.
Defining the Immature Molecule: Pre-mRNA, Introns, and Exons
The newly created pre-mRNA molecule contains regions of genetic code classified as either exons or introns. Exons are the sequences that will be expressed and ultimately joined together to form the mature, protein-coding mRNA.
Introns, in contrast, are the intervening sequences that are transcribed but do not carry instructions for the final protein. They are the “extra information” that must be precisely excised from the pre-mRNA transcript. Introns can vary dramatically in size, ranging from a few dozen to tens of thousands of nucleotide base pairs.
For the genetic message to be accurately translated, the exons must be stitched together in the correct order. This requires the complete and accurate removal of every intron. This process of removing the introns and joining the exons is known as RNA splicing.
The Central Machinery: The Spliceosome Complex
The complex machinery responsible for this precise molecular surgery is called the spliceosome. This enormous, dynamic structure is one of the largest ribonucleoprotein (RNP) complexes found within the cell nucleus. The spliceosome is composed of five specialized small nuclear ribonucleoproteins (snRNPs), designated U1, U2, U4, U5, and U6.
These snRNPs contain both RNA and protein components. The small nuclear RNAs (snRNAs) within these particles recognize specific signal sequences on the pre-mRNA that mark the boundaries of the introns. These signal sequences include a conserved GU sequence at the 5′ end of the intron, an AG sequence at the 3′ end, and an internal branch point adenosine nucleotide.
The U1 snRNP first binds to the 5′ splice site, while the U2 snRNP recognizes and binds to the branch point adenosine. This initial binding establishes the framework for the spliceosome’s assembly. The entire complex, which can contain over 300 different proteins, then acts as a highly specialized molecular enzyme to catalyze the removal of the intron.
The Step-by-Step Process of RNA Splicing
The removal of the intron and the joining of the exons is accomplished through a highly coordinated series of biochemical reactions. The process involves two sequential transesterification reactions that break and form phosphodiester bonds without requiring an external energy source like ATP.
The first step involves the branch point adenosine. The hydroxyl group on its 2′ carbon acts as a nucleophile, attacking the phosphodiester bond at the 5′ splice site. This breaks the bond between the upstream exon and the intron, freeing the 5′ end of the intron.
The newly detached 5′ end of the intron then loops back and covalently attaches to the branch point adenosine, creating a unique structure known as the lariat intermediate. This lariat structure represents the excised intron, which is still temporarily bound to the spliceosome complex.
The second transesterification reaction occurs immediately after lariat formation. The newly freed 3′ hydroxyl group of the upstream exon becomes activated. This hydroxyl group then attacks the phosphodiester bond at the 3′ splice site.
This second attack simultaneously cleaves the intron from the downstream exon and joins the two flanking exons together, a process called ligation. The result is the mature mRNA transcript, now containing a continuous, protein-coding sequence. The intron, still in its lariat form, is then released and rapidly degraded within the nucleus.
Variation and Diversity: Alternative Splicing
The splicing mechanism is not always fixed; it offers a profound level of regulatory control through alternative splicing. This allows a single gene to encode multiple distinct messenger RNA molecules.
Alternative splicing is accomplished by selecting different combinations of exons from the same pre-mRNA transcript to be included in the final mature mRNA. For example, a pre-mRNA might have five exons, but the spliceosome might produce one mRNA containing exons 1, 2, 4, and 5, and a second mRNA containing exons 1, 3, 4, and 5. The excluded exon is treated as if it were an intron for that specific transcript.
This variation is common; current estimates suggest that nearly 95% of human genes with multiple exons undergo alternative splicing. This mechanism vastly increases the functional complexity of the human genome, allowing a relatively small number of genes to produce a much larger number of distinct protein variants, or isoforms. Alternative splicing is a fundamental mechanism for generating the biological diversity and specialization observed across different cells and tissues in a complex organism.