Spliceosome

The spliceosome is a large, dynamic complex of RNA and proteins that performs RNA splicing in eukaryotic cells. It removes introns from precursor messenger RNA (pre-mRNA) and joins the surrounding exons. Its principal components are small nuclear ribonucleoprotein particles and numerous associated proteins, which assemble and rearrange around the RNA substrate. Splicing proceeds through two chemical reactions, branching and exon ligation, rather than through simple cutting followed by an independent repair process. (nature.com)

Composition and organization

The major spliceosome assembles from five small nuclear RNAs (snRNAs), named U1, U2, U4, U5, and U6, together with their associated proteins. Each RNA–protein particle is a small nuclear ribonucleoprotein (snRNP, often pronounced “snurp”). U4 and U6 are initially paired, and their particle associates with U5 to form the U4/U6.U5 tri-snRNP. Recruitment of this preassembled unit is an important step in constructing the spliceosome. (nature.com)

The snRNAs have distinct, overlapping functions:

These functions depend on extensive RNA–RNA and RNA–protein interactions. The mature catalytic apparatus is therefore not simply the sum of five independently acting particles. (nature.com)

The spliceosome’s composition changes during its cycle. Proteins involved in initial recognition may be displaced before catalysis, whereas other factors enter to stabilize reaction-ready configurations. Human structures show that the U2-associated SF3B complex initially holds the branch-point region away from the catalytic center; subsequent remodeling releases and repositions it. (nature.com)

Recognition of the RNA substrate

A spliceosome must identify and pair the appropriate boundaries of an intron. Early recognition involves the 5′ splice site and an internal branch-point sequence, selected through interactions involving U1 and U2, respectively. The branch-point adenosine later participates directly in the first chemical reaction. Recognition and catalysis are separate stages: an RNA region can be bound by the assembling machinery without yet being positioned to react. (nature.com)

Early assembly can follow two organizational routes:

• Intron definition: U1 and U2 associate with recognition sites across the same intron.
• Exon definition: U2 binds upstream of an exon, while U1 recognizes the 5′ splice site downstream of that exon.

An exon-defined assembly must subsequently become organized across the intron that will be removed. Structural studies of human complexes have revealed intermediates in this cross-exon to cross-intron transition, connecting initial recognition to the eventual pairing of splice sites. (nature.com)

Assembly and activation

Spliceosome assembly is commonly described using named complexes. These labels identify experimentally distinguishable configurations rather than permanently separate molecular machines.

1. A complex, or prespliceosome. U1 and U2 establish an early assembly that brings the selected 5′ splice site and branch-point region into a common complex.
2. B complex, or precatalytic spliceosome. Recruitment and rearrangement of the U4/U6.U5 tri-snRNP produce a more complete assembly. It contains the components needed for splicing but is not yet catalytically ready. (nature.com)
3. Activated and branching-competent complexes. RNA and protein rearrangements establish the catalytic architecture and then position the branch-point adenosine for reaction. These stages are commonly called Bact and B*. In human spliceosomes, the RNA helicases PRP2 and Aquarius drive successive steps in catalytic activation. (nature.com)
4. C and C complexes.* The C complex contains the products of branching. Further remodeling produces a configuration competent for exon ligation, termed C*. (nature.com)

Many transitions consume ATP. This expenditure principally supports molecular remodeling, including changes in RNA contacts and displacement of bound proteins. It should be distinguished from the two bond-exchange reactions that actually remove the intron and join the exons. (nature.com)

Chemical mechanism

Spliceosomal splicing consists of two transesterification reactions: one phosphodiester bond is exchanged for another in each step.

Branching. The 2′ hydroxyl group of the branch-point adenosine attacks the phosphate at the 5′ splice site. This separates the upstream exon from the intron and creates a 2′–5′ linkage between the branch-point nucleotide and the intron’s 5′ end. The resulting intermediate consists of a free upstream exon and a branched intron still attached to the downstream exon. The branched RNA has a loop-and-tail configuration called a lariat.

Exon ligation. The newly exposed 3′ hydroxyl group of the upstream exon attacks the phosphate at the 3′ splice site. The two exons become joined by a conventional 3′–5′ phosphodiester linkage, and the intron is excised as a lariat. Both reactions use the same catalytic center, with substrate rearrangement allowing it to perform the second step. (nature.com)

The spliceosome is an RNA-based catalyst, or ribozyme, whose activity depends on proteins and metal ions. Biochemical experiments demonstrated that U6 RNA directly positions catalytic metals for both reactions. In particular, replacing selected RNA oxygen atoms with sulfur and testing rescue by sulfur-compatible metals provided evidence that the RNA participates directly in catalysis, rather than serving only as a scaffold. (nature.com)

Product release and recycling

Completion of exon ligation does not immediately dismantle the spliceosome. The spliced RNA must first be released. Experiments in yeast established that the ATP-dependent factor PRP22 acts late in the pathway and is required for release of the spliced messenger RNA. (nature.com)

The remaining intron-containing complex is subsequently disassembled. The RNA-dependent ATPase Prp43 is required for release of the lariat intron in yeast, separating product formation from the later breakdown of the machinery. These release and disassembly stages make spliceosomal components available for further rounds of assembly. (pubmed.ncbi.nlm.nih.gov)

Major and minor spliceosomes

Eukaryotic pre-mRNA splicing includes two related systems. The major spliceosome processes U2-type introns. The minor spliceosome processes the much rarer U12-type introns and uses U11, U12, U4atac, and U6atac in place of U1, U2, U4, and U6; U5 is shared between the systems. (pubmed.ncbi.nlm.nih.gov)

The minor spliceosome follows a related catalytic strategy but is not merely an interchangeable version of the major machinery. An activated human minor-spliceosome structure showed recognition of the branch-point sequence by U12 and of the 5′ splice site by U6atac. It also identified proteins that stabilize its distinctive catalytic organization. (pubmed.ncbi.nlm.nih.gov)

Alternative splicing and biological significance

The spliceosome participates in alternative splicing, in which different splice-site choices produce different RNA products from the same precursor. Regulation can act at early recognition and assembly stages, including stabilization of the 5′ splice site and the organization of complexes across exons. Thus, the machinery both executes RNA processing and provides stages at which splice-site selection can be controlled. (nature.com)

Changes in spliceosomal components can alter this selection. For example, studies of cancer-associated mutations in SF3B1, a U2-associated protein, demonstrated altered branch-point use and activation of alternative 3′ splice sites. This finding connects a specific molecular change in the recognition machinery to reproducible changes in RNA processing; it does not imply that every spliceosomal mutation has the same effect. (nature.com)

Discovery and structural investigation

In 1985, biochemical studies identified large RNA–protein assemblies associated with pre-mRNA splicing. Edward Brody and John Abelson described a yeast complex and proposed the name spliceosome. That year, Paula Grabowski, Sharon Seiler, and Phillip Sharp reported a multicomponent complex containing precursor RNA and characteristic splicing intermediates in a mammalian in-vitro system. (pubmed.ncbi.nlm.nih.gov)

Subsequent biochemical and structural work resolved how its components recognize RNA, exchange interactions, and carry out catalysis. Cryo-electron microscopy has been especially important in visualizing prespliceosomal, precatalytic, catalytically activated, and exon-ligation configurations. These structures connect molecular architecture with experimentally defined stages of the reaction. (nature.com)

Structural models nevertheless represent particular captured states. Human and yeast machinery share central features but differ in some remodeling requirements—for example, the additional role of Aquarius in human catalytic activation. Mechanistic interpretation therefore requires distinguishing conserved catalytic principles from organism-specific factors and experimentally stabilized intermediates. (nature.com)

References

1. Prespliceosome structure provides insights into spliceosome assembly and regulationnature.com
2. Structure of a pre-catalytic spliceosomenature.com
3. Structural insights into the cross-exon to cross-intron spliceosome switchnature.com
4. Structural basis of catalytic activation in human splicingnature.com
5. RNA catalyses nuclear pre-mRNA splicingnature.com
6. Structure of a spliceosome remodelled for exon ligationnature.com
7. Requirement of the RNA helicase-like protein PRP22 for release of messenger RNA from spliceosomesnature.com
8. Prp43 is an essential RNA-dependent ATPase required for release of lariat-intron from the spliceosomepubmed.ncbi.nlm.nih.gov
9. Structure of the activated human minor spliceosomepubmed.ncbi.nlm.nih.gov
10. Cancer-associated SF3B1 mutations affect alternative splicing by promoting alternative branchpoint usagenature.com
11. The "spliceosome": yeast pre-messenger RNA associates with a 40S complex in a splicing-dependent reactionpubmed.ncbi.nlm.nih.gov
12. A multicomponent complex is involved in the splicing of messenger RNA precursorspubmed.ncbi.nlm.nih.gov