Spliceosomal Splicing Cycle
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Spliceosomal Splicing Cycle
During the final step in formation of a mature, functional mRNA, the introns are removed and exons are spliced together. The discovery that introns are removed during splicing came from electron microscopy of RNA-DNA hybrids between adenovirus DNA and the mRNA encoding hexon, a major virion capsid protein. For short transcription units, RNA splicing usually follows cleavage and polyadenylation of the 3’ end of the primary transcript. But for long transcription units containing multiple exons, splicing of exons in the nascent RNA usually begins before transcription of the gene is complete (Ref.1 & 2).

The splicing snRNPs (Ribonucleoproteins) associate with the pre-mRNA and with each other in an ordered sequence to form the spliceosome. This large ribonucleoprotein complex then catalyzes the two transesterification reactions that result in splicing of the exons and excision of the intron as a lariat structure (Ref.3). Although ATP hydrolysis is not required for the transesterification reactions, it is thought to provide the energy necessary for rearrangements of the spliceosome structure that occur during the cycle. The snRNP proteins in the spliceosome are distinct from the hnRNP proteins. In higher eukaryotes, the association of U2 snRNP with pre-mRNA is assisted by an hnRNP protein called U2AF, which binds to the pyrimidine-rich region near the 3’ splice site. The snRNPs involved in splicing are U1, U2, U5, U4, and U6 (Ref.4,5 & 6). They are named according to the snRNAs that are present. Each snRNP contains a single snRNA and several (<20) proteins. The U4 and U6 snRNPs are usually found as a single (U4/U6) particle. The snRNPs together contain -40 individual proteins. A common structural core for each snRNP consists of a group of 8 proteins. The other proteins in each snRNP are unique to it. The Sm proteins bind to the conserved sequence PuAU3_6Gpu, which is present in all snRNAs except U6. The U6 snRNP contains instead a set of Sm-like (Lsm) proteins. The Sm proteins maybe involved in the autoimmune reaction, although their relationship to the phenotype of the autoimmune disease is not clear (Ref.7). Some of the proteins in the snRNPs are involved directly in splicing; others may be required in structural roles or just for assembly or interactions between the snRNP particles. Most of the proteins that are involved in splicing are components of the snRNPs; some additional proteins are included in the spliceosome and are generally described as "splicing factors."

Splicing can be broadly divided into two stages: first the consensus sequences are recognized and the complex assembles; then the cleavage and ligation reactions change the structure of the substrate RNA. Recognition of the consensus sequences involves both RNAs and proteins. Certain snRNAs have sequences that are complementary to the consensus sequences or to one another, and base pairing between snRNA and pre-mRNA, or between snRNAs, plays an important role in splicing. The human U1 snRNP contains 8 proteins as well as the RNA. It contains several domains. The Sm-binding site is required for interaction with the common snRNP proteins. Domains identified by the individual stem-loop structures provide binding sites for proteins that are unique to U1 snRNP. U1 snRNA base pairs directly with the 5 splice site (Ref.7 & 8). The 5-terminal 11 nucleotides are single-stranded and include a stretch complementary to the consensus se¬quence at the 5 site of the intron. The extent of complementarity between U1 snRNA and actual sites is usually 4-6bp (because actual sites rarely conform perfectly to the consensus) necessary for U1 snRNA to pair with the 5 splice site.

The first stage in splicing is formation of the E Complex (Early Presplicing), which contains U1 snRNP, the splicing factor U2AF, and some other proteins. The E Complex is sometimes called the “Commitment Complex”, because its formation identifies a pre-mRNA as a substrate for formation of the splicing complex. Binding of U1 snRNP by base pairing with the 5 splice site is the first step. The recruitment of U1 snRNP involves an interaction between one of its proteins (U1-70k) and the protein ASF/SF2 (a general splicing factor in the SR class). The U2AF splicing factor binds to a pyrimidine tract downstream of the branch site. Both U1 snRNP and U2AF are needed for U2 snRNP to bind to the branch site. The U2 snRNA includes sequences complementary to the branch site. A sequence near the 5 end of the snRNA base pairs with the branch sequence in the intron. In yeast this typically involves formation of a 7bp duplex with the entire UACUAAC Box. Several proteins of the U2 snRNP are bound to the substrate RNA just upstream of the branch site. The addition of U2 snRNP to the E Complex generates the “A Presplicing Complex”. The binding of U2 snRNP requires ATP hydrolysis, and commits a pre-mRNA to the splicing pathway. There may be more than one way to form the E Complex. The most direct reaction is for both splice sites to be recognized across the intron. The presence of U1 snRNP at the 5 splice site is necessary for U2AF to bind near the branch site, making it possible that the 5 and 3 ends of the intron are brought together in this complex. The E Complex is converted to the A Complex when U2 snRNP binds at the branch site. The basic feature of this route for splicing is that the two splice sites are recognized without requiring any sequences outside of the intron (Ref.7).

An alternative route to form the spliceosome may be followed when the introns are long and the splice sites are weak. In such a case the 5 splice site is recognized by U1 snRNA in the usual way. However, the 3 splice site is recognized as part of a complex that forms across the next exon, in which the next 5 splice site is also bound by U1 snRNA. This U1 snRNA is connected by SR proteins to the U2AF at the branch site. When U2 snRNP joins to generate the A Complex, there is a rearrangement, in which the correct 5 splice site displaces the downstream 5 splice site in the complex. The important feature of this route for splicing is that sequences downstream of the intron itself are required. The stage at which the two ends of the intron are brought together may vary with the type of splicing reaction. It could happen in the E Complex when recognition occurs across the intron, but may not happen until the later B Complex stage when recognition requires downstream sequences. The B1 Complex is formed when a trimer containing the U5 and U4/U6 snRNPs binds. This complex is regarded as a spliceosome, since it contains the components needed for the splicing reaction. It is converted to the B2 Complex when U1 is released. The dissociation of U1 is necessary to allow other components to come into juxtaposition with the 5 splice site, most notably U6 snRNA. At this point U5 snRNA changes its position; initially it is close to exon sequences at the 5 splice site, but it shifts to the vicinity of the intron sequences. The catalytic reaction is triggered by the release of U4; this requires hydrolysis of ATP. The role of U4 snRNA may be to sequester U6 snRNA until it is needed. In the U6/U4 snRNP, a continuous length of 26 bases of U6 is paired with two separated regions of U4. When U4 dissociates, the region in U6 that is released becomes free to take up another structure. The first part of it pairs with U2; the second part forms an intramolecular hairpin. The interaction between U4 and U6 is mutually incompatible with the interaction between U2 and U6, and thus the release of U4 controls the ability of the spliceosome to proceed (Ref.6). The 5 splice site is actually close to the U6 sequence immediately on the 5 side of the stretch bound to U2. This sequence in U6 snRNA contacts the conserved GU at the 5 splice site, and in some cases the pairing may extend to sequences down¬stream of the GU. The base pairing between U2 and U6 creates a structure that resembles the active center of the Group-II introns. The formation of the lariat at the branch site is responsible for determining the use of the 3 splice site, since the 3 consensus sequence nearest to the 3 side of the branch becomes the target for the second transesterification. The second splicing reaction follows rapidly. Binding of U5 snRNP to the 3 splice site is needed for this reaction (Ref.9). There is no region of complementarity available in single-stranded form.

The snRNA components of the splicing apparatus interact both among themselves and with the substrate RNA by means of base pairing interactions, and these interactions allow for changes in structure that may bring reacting groups into apposition and may even create catalytic centers. Furthermore, the conformational changes in the snRNAs are reversible; for example, U6 snRNA is not used up in a splicing reaction and at completion must be released from U2, so that it can reform the duplex structure with U4 to undertake another cycle of splicing. Any particular snRNP may play more than one role in splicing. So the ability of U1 snRNP to promote binding of U2 snRNP to the branch site is independent of its ability to bind to the 5 splice site (Ref.6,9 & 10).