Spliceosomal Complexes

The spliceosome is a multi-protein/RNA complex that catalyzes excision of introns from pre-mRNA. Five snRNA molecules (U1, U2, U4, U5, and U6) entwined in conserved protein complexes play key roles in the pre-mRNA processing reaction. These snRNA/protein complexes are referred to as small nuclear ribonucleoproteins (snRNPs). The five snRNP subunits are thought to assemble on conserved elements within the intron in a regulated manner, undergo extensive structural rearrangements, and form the active spliceosome. Proteins not stably associated with snRNPs participate in the many rearrangements that occur during the splicing reaction and still additional accessory factors, operating primarily in higher eukaryotes, regulate splice site selections that can result in a diversity of gene products from a single gene.

One model of pre-mRNA splicing, derived from in vitro assays, posits that the spliceosome is assembled in a step-wise fashion. In this model spliceosome assembly begins with the recognition of the 5’ splice site and branch point sequences of the pre-mRNA by the U1 snRNP and the U2 snRNP, respectively (Complex A). After binding of the U4/U6.U5 tri-snRNP, the U4/U6 snRNA duplex is replaced by a U2/U6 snRNA duplex (Complex B). Furthermore, the U1 snRNA base pairing at the 5’ splice site is disrupted and exchanged for base pairing between the 5’ splice site and the U6 snRNA. The subsequent release of the U1 and U4 snRNPs marks the transition from an inactive to an active spliceosome composed of the U5 and U2/U6 snRNPs (Complex B* and C). 5’ splice site cleavage and lariat formation, followed by 3’ splice site cleavage and exon ligation, occur within the activated spliceosome. The dynamic nature of the pre-mRNA splicing reaction has hampered progress in analyzing the structure of the spliceosome.

Despite the complexity of the spliceosome, steady progress has been made in identifying its constituents. In the yeasts, Saccharomyces cerevisiae and Schizosaccharomyces pombe, splicing factors were first identified genetically and called prp for pre-mRNA processing factors. Conditionally lethal prp mutants were isolated based on the accumulation of pre-mRNA when cells were shifted to a restrictive temperature. Subsequent complementation cloning and DNA sequence analysis revealed the identities of these factors. Identification of a multitude of proteins co-purifying with snRNAs has been another fruitful approach to identify splicing factors. Recently, technical advances in epitope tagging and/or mass spectrometry have allowed more comprehensive identification of splicing factors and their regulators. The number of proteins now thought to be associated with the spliceosome is well over 50. The challenge for the future will be to learn how these many factors organize into a catalytic machine and to determine their role(s) in the splicing reaction.

Cdc5p-TAP Complex

Prp19p