Spliceosome

A spliceosome is a large and complex molecular machine found primarily within the splicing speckles of the cell nucleus of eukaryotic cells. The spliceosome is assembled from snRNAs and protein complexes. The spliceosome removes introns from a transcribed pre-mRNA, a kind of primary transcript. This process is generally referred to as splicing. Only eukaryotes have spliceosomes and metazoans have a second spliceosome, the minor spliceosome.

Spliceosomal splicing cycle

Composition

Each spliceosome is composed of five small nuclear RNAs (snRNA), and a range of associated protein factors. When these small RNA are combined with the protein factors, they make an RNA-protein complex called snRNP.

The snRNAs that make up the major spliceosome are named U1, U2, U4, U5, and U6, and participate in several RNA-RNA and RNA-protein interactions. The RNA component of the small nuclear ribonucleic protein or snRNP (pronounced "snurp") is rich in uridine (the nucleoside analog of the uracil nucleotide).

The canonical assembly of the spliceosome occurs anew on each hnRNA (pre-mRNA). The hnRNA contains specific sequence elements that are recognized and utilized during spliceosome assembly. These include the 5' end splice, 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.

A group of less abundant snRNAs, 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. The minor spliceosome is located in the nucleus like its major counterpart, though there are exceptions in some specialised cells including anucleate platelets and the dendroplasm of neuronal cells.

New evidence derived from the first crystal structure of a group II intron suggests that the spliceosome is actually a ribozyme, and that it uses a two–metal ion mechanism for catalysis. In addition, many proteins exhibit a zinc-binding motif, which underscores the importance of zinc metal in the splicing mechanism.

Figure 1. Above are electron microscopy fields of negatively stained yeast (Saccharomyces cerevisiae) tri-snRNPs. Below left is a schematic illustration of the interaction of tri-snRNP proteins with the U4/U6 snRNA duplex. Below right is a cartoon model of the yeast tri-snRNP with shaded areas corresponding to U5 (gray), U4/U6 (orange) and the linker region (yellow).

Alternative splicing

Main article: 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, due to the Human Genome Project, the figure is believed to be closer to 20,000 genes. One particular Drosophila gene (Dscam, the Drosophila homolog of the human Down syndrome cell adhesion molecule DSCAM) can be alternatively spliced into 38,000 different mRNA.

RNA splicing

See also: Splicing (genetics)

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, the introns, are excised from the precursor mRNAs in a process Sharp named "splicing". The split gene structure was subsequently found to be common to most eukaryotic genes. Phillip Sharp and Richard J. Roberts were awarded the 1993 Nobel Prize in Physiology or Medicine for their discovery of introns and the splicing process.

Spliceosome assembly

The model for formation of the spliceosome active site involves an ordered, stepwise assembly of discrete snRNP particles on the hnRNA substrate. The first recognition of hnRNAs involves U1 snRNP binding to the 5' end splice site of the hnRNA 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 hnRNA 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 snRNP and the hnRNA 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 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 (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, 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 snRNAm, 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.

External links

• Spliceosomes at the US National Library of Medicine Medical Subject Headings (MeSH)
• 3D macromolecular structures of Spliceosomes from the EM Data Bank(EMDB)