Spliceosome

The spliceosome is a complex of RNA and protein subunits that removes non-coding intervening sequences (introns) from precursor mRNA, a process generally referred to as splicing. The spliceosome is composed of five small nuclear ribonucleoproteins (snRNPs) (pronounced "snurps") and a range of non-snRNP associated protein factors.

The snRNPs that make up the spliceosome are named U1, U2, U4, U5, and U6, and participate in several RNA-RNA and RNA-protein interactions. The RNA component of the snRNP is rich in uridine (the U nucleotides).

The canonical assembly of the spliceosome occurs anew on each pre-mRNA. The pre-mRNA contains specific sequence elements that are recognized and utilized during spliceosome assembly. These include the 5' splice, the branch point sequence, the polypyrimidine tract, and the 3' splice site. The spliceosome catalyzes the removal of the intervening sequences and the ligation of the flanking exons. Introns are typically defined as having a GU at the 5' splice site and an AG at the 3' 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.

A group of less abundant snRNPs, U11, U12, U4atac, and U6atac, together with U5, are subunits of the so-called minor spliceosome that splices a rare class of pre-mRNA introns, denoted U12-type.

Alternative splicing
Alternative splicing (the re-combination of different exons) is a major source of genetic diversity in eukaryotes. Splice variants have been used to account for the relatively small number of genes in the human genome. For years the estimate widely varied with top estimates reaching 100,000 genes, but now, thanks to the Human Genome Project we know the figure is closer to 30,000 genes. However, almost every human gene is thought to have at least two isoforms.

RNA Splicing
In 1977, work by the Sharp and Roberts labs revealed that genes of higher organisms are “split” or present in several distinct segments along the DNA molecule. The coding regions of the gene are separated by non-coding DNA that is not involved in protein expression. The split gene structure was found when adenoviral mRNAs were hybridized to endonuclease cleavage fragments of single stranded viral DNA. It was observed that the mRNAs of the mRNA-DNA hybrids contained 5' and 3' tails of non-hydrogen bonded regions. When larger fragments of viral DNAs were used, forked structures of looped out DNA were observed when hybridized to the viral mRNAs. It was realized that the looped out regions or intervening sequences are excised from the precursor mRNAs in a process Sharp coined “splicing”. The split gene structure was subsequently found to be common to most eukaryotic genes.

Spliceosome assembly
The canonical model for formation of the spliceosome active site involves an ordered, stepwise assembly of discrete snRNP particles on the pre-mRNA substrate. The first recognition of pre-mRNAs involves U1 snRNP binding to the 5' splice site of the pre-mRNA and other non-snRNP associated factors to form the commitment complex or early (E) complex in mammals. The commitment complex is an ATP independent complex that commits the pre-mRNA to the splicing pathway. U2 snRNP is recruited to the branch region through interactions with the E complex component U2AF (U2 snRNP auxiliary factor) and possibly U1 snRNP. In an ATP dependent reaction, U2 snRNP becomes tightly associated with the branch point sequence (BPS) to form complex A. A duplex formed between U2 snRNA and the pre-mRNA branch region bulges out the branch adenosine specifying it as the nucleophile for the first transesterification. The presence of a pseudouridine residue in U2 snRNA nearly opposite the branch site results in an altered conformation of the RNA-RNA duplex upon 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 is recruited to the assembling spliceosome to form complex B, and following several rearrangements, complex C (the spliceosome) is activated for catalysis. It is unclear how the triple 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 triple 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 or 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 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; a number of RNA helicases have 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.