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A spliceosome is a large ribonucleoprotein (RNP) complex found primarily within the cell nucleus of eukaryotic cells. The spliceosome is assembled from snRNA (snRNA) and numerous proteins. Small nuclear RNA (snRNA) molecules bind to specific proteins to form a small nuclear ribonucleoprotein complex (snRNP, pronounced "snurps"), which in turn combines with other snRNPs to form a large ribonucleoprotein complex called a spliceosome. The spliceosome removes from a transcribed pre-mRNA, a type of primary transcript. This process is generally referred to as RNA splicing. An analogy is a film editor, who selectively cuts out irrelevant or incorrect material (equivalent to the introns) from the initial film and sends the cleaned-up version to the director for the final cut.
However, sometimes the RNA within the intron acts as a ribozyme, splicing itself without the use of a spliceosome or protein enzymes.
The assembly of the spliceosome occurs on each pre-mRNA (also known as heterogeneous nuclear RNA, hn-RNA) at each exon:intron junction. The pre-mRNA introns contains specific sequence elements that are recognized and utilized during spliceosome assembly. These include the 5' end splice site, the branch point sequence, the polypyrimidine tract, and the 3' end splice site. The spliceosome catalyzes the removal of introns, and the ligation of the flanking exons.
Introns typically have a GU nucleotide sequence at the 5' end splice site, and an AG at the 3' end splice site. The 3' splice site can be further defined by a variable length of polypyrimidines, called the polypyrimidine tract (PPT), which serves the dual function of recruiting factors to the 3' splice site and possibly recruiting factors to the branch point sequence (BPS). The BPS contains the conserved adenosine required for the first step of splicing.
Many proteins exhibit a zinc-binding motif, which underscores the importance of zinc in the splicing mechanism. The first molecular-resolution reconstruction of U4/U6.U5 triple small nuclear ribonucleoprotein (tri-snRNP) complex was reported in 2016.
Cryo-EM has been applied extensively by Shi et al. to elucidate the near-/atomic structure of spliceosome in both yeast and humans. The molecular framework of spliceosome at near-atomic-resolution demonstrates Spp42 component of U5 snRNP forms a central scaffold and anchors the catalytic center in yeast. The atomic structure of the human spliceosome illustrates the step II component Slu7 adopts an extended structure, poised for selection of the 3'-splice site. All five metals (assigned as Mg2+) in the yeast complex are preserved in the human complex.
The presence of a pseudouridine residue in U2 snRNA, nearly opposite of the branch site, results in an altered conformation of the RNA-RNA duplex upon the U2 snRNP binding. Specifically, the altered structure of the duplex induced by the pseudouridine places the 2' OH of the bulged adenosine in a favorable position for the first step of splicing. The U4/U5/U6 tri-snRNP (see Figure 1) is recruited to the assembling spliceosome to form complex B, and following several rearrangements, complex C is activated for catalysis. (1999). 9780879693800, Cold Spring Harbor Lab. Press. ISBN 9780879693800 It is unclear how the tri-snRNP is recruited to complex A, but this process may be mediated through protein-protein interactions and/or base pairing interactions between U2 snRNA and U6 snRNA.
The U5 snRNP interacts with sequences at the 5' and 3' splice sites via the invariant loop of U5 snRNA and U5 protein components interact with the 3' splice site region.
Upon recruitment of the tri-snRNP, several RNA-RNA rearrangements precede the first catalytic step and further rearrangements occur in the catalytically active spliceosome. Several of the RNA-RNA interactions are mutually exclusive; however, it is not known what triggers these interactions, nor the order of these rearrangements. The first rearrangement is probably the displacement of U1 snRNP from the 5' splice site and formation of a U6 snRNA interaction. It is known that U1 snRNP is only weakly associated with fully formed spliceosomes, and U1 snRNP is inhibitory to the formation of a U6-5' splice site interaction on a model of substrate oligonucleotide containing a short 5' exon and 5' splice site. Binding of U2 snRNP to the branch point sequence (BPS) is one example of an RNA-RNA interaction displacing a protein-RNA interaction. Upon recruitment of U2 snRNP, the branch binding protein SF1 in the commitment complex is displaced since the binding site of U2 snRNA and SF1 are mutually exclusive events.
Within the U2 snRNA, there are other mutually exclusive rearrangements that occur between competing conformations. For example, in the active form, stem loop IIa is favored; in the inactive form a mutually exclusive interaction between the loop and a downstream sequence predominates. It is unclear how U4 is displaced from U6 snRNA, although RNA has been implicated in spliceosome assembly, and may function to unwind U4/U6 and promote the formation of a U2/U6 snRNA interaction. The interactions of U4/U6 stem loops I and II dissociate and the freed stem loop II region of U6 folds on itself to form an intramolecular stem loop and U4 is no longer required in further spliceosome assembly. The freed stem loop I region of U6 base pairs with U2 snRNA forming the U2/U6 helix I. However, the helix I structure is mutually exclusive with the 3' half of an internal 5' stem loop region of U2 snRNA.
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