RNA splicing is the processing of an RNA transcript by removing intervening sequences called introns and joining the remaining sequences, called exons. It is especially important in the maturation of precursor messenger RNA (pre-mRNA) in eukaryotes, although other RNA classes also undergo splicing. By determining which sequences remain in a transcript, splicing connects gene organization with RNA function and the production of proteins. Several distinct molecular mechanisms perform this process. (ncbi.nlm.nih.gov)
Biological context and discovery
During transcription, genetic information in DNA is copied into RNA. For an intron-containing gene, the initial transcript includes both introns and exons. Exons are not necessarily entirely protein-coding: they can also contain untranslated regions. Splicing acts on RNA rather than deleting introns from the DNA template. Together with addition of a 5′ cap and polyadenylation, it is a major component of eukaryotic pre-mRNA maturation. (ncbi.nlm.nih.gov)
The discontinuous organization of genes was established in 1977 through studies of adenovirus. Comparisons between viral DNA and mature RNA showed that sequences joined in RNA could be separated in DNA. Richard J. Roberts and Phillip A. Sharp received the 1993 Nobel Prize in Physiology or Medicine for discoveries of split genes. These findings changed the earlier view that a gene’s DNA sequence necessarily corresponded to one uninterrupted RNA sequence. (nobelprize.org)
Recognition and spliceosome assembly
Most nuclear pre-mRNA introns are removed by the spliceosome, a dynamic RNA–protein complex. Its major form contains five small nuclear RNAs—U1, U2, U4, U5, and U6—associated with proteins in small nuclear ribonucleoproteins, or snRNPs. Assembly brings distant regions of the transcript together and rearranges RNA–RNA and RNA–protein interactions to create a catalytic structure. (ncbi.nlm.nih.gov)
Recognition involves the 5′ splice site, an internal branch point, and the 3′ splice site. Most major-spliceosome introns begin with GU and end with AG when written in the RNA’s 5′-to-3′ direction. In mammals, a pyrimidine-rich tract near the 3′ end also contributes to recognition. These signals are variable rather than an infallible sequence code; additional regulatory elements help distinguish authentic boundaries from similar sequences elsewhere. (ncbi.nlm.nih.gov)
U1 initially recognizes the 5′ splice site, while U2 associates with the branch-point region. The U4/U6–U5 complex subsequently joins. Rearrangements release U1 and U4 and establish an active center involving U2 and U6, with U5 helping position the exons. A separate minor spliceosome processes a small class of introns using U11, U12, U4atac, U6atac, and the shared U5 RNA. (ncbi.nlm.nih.gov)
Reaction chemistry
Spliceosomal splicing normally proceeds through two transesterification reactions. First, the 2′ hydroxyl group of a branch-point adenosine attacks the phosphate at the 5′ splice site. This separates the upstream exon from the intron and creates an unusual 2′–5′ linkage, producing a looped intron intermediate called a lariat. (nature.com)
Second, the newly exposed 3′ hydroxyl group of the upstream exon attacks the phosphate at the 3′ splice site. The exons become joined through a conventional 3′–5′ linkage, and the intron lariat is released. The catalytic center uses magnesium ions coordinated within an RNA-rich structure; proteins stabilize and organize the machinery. Although these bond-exchange reactions do not directly consume ATP, ATP-dependent remodeling is required during the spliceosome’s assembly and functional cycle. (nature.com)
Alternative splicing and regulation
Alternative splicing produces different RNA isoforms from the same gene through different choices of splice sites. Common patterns include exon skipping, mutually exclusive exons, alternative 5′ or 3′ splice sites, and intron retention. This changes which transcript sequences are included, rather than simply rearranging exons into arbitrary orders. Different isoforms may encode proteins with distinct activities or alter untranslated regions that influence RNA behavior. (ncbi.nlm.nih.gov)
Splicing decisions depend on regulatory sequences and the proteins that bind them. Enhancers and silencers can occur within either exons or introns. RNA-binding proteins may promote or suppress particular splice choices, and their effects can depend on binding position. Differences among tissues and developmental states therefore contribute to regulated gene expression. Studies of neuronal RNA-binding proteins have directly mapped such position-dependent regulation. (nature.com)
Not every alternative transcript produces a stable protein. Some introduce premature termination signals or otherwise become substrates for nonsense-mediated decay. Splicing can therefore regulate transcript abundance as well as protein diversity. (nature.com)
Other mechanisms and experimental study
Some introns act as ribozymes and can catalyze their own removal under suitable conditions. Group I introns initiate splicing using a guanosine cofactor, whereas group II introns commonly use an internal adenosine and form a lariat. Such introns occur in several biological settings, including genes within mitochondria and chloroplasts. Self-splicing capability does not imply independence from protein assistance inside cells. Introns in some transfer RNAs instead undergo cleavage and ligation by specialized enzymes. (ncbi.nlm.nih.gov)
Splicing is investigated by comparing transcript sequences with genomic sequences and by measuring exon–exon junctions. RNA sequencing detects reads crossing these junctions and reveals tissue-dependent isoforms. Cryo-electron microscopy resolves spliceosomal intermediates, connecting molecular structure with individual reaction steps. Genetic and biochemical assays can establish how sequence changes affect splice-site recognition or catalytic activity. (nature.com)