Spliceosome

Abstract: Pre-mRNA splicing is catalyzed by the spliceosome, a multimegadalton ribonucleoprotein (RNP) complex comprised of five snRNPs and numerous proteins. Intricate RNA-RNA and RNP networks, which serve to align the reactive groups of the pre-mRNA for catalysis, are formed and repeatedly rearranged during spliceosome assembly and catalysis. Both the conformation and composition of the spliceosome are highly dynamic, affording the splicing machinery its accuracy and flexibility, and these remarkable dynamics are largely conserved between yeast and metazoans. Because of its dynamic and complex nature, obtaining structural information about the spliceosome represents a major challenge. Electron microscopy has revealed the general morphology of several spliceosomal complexes and their snRNP subunits, and also the spatial arrangement of some of their components. X-ray and NMR studies have provided high resolution structure information about spliceosomal proteins alone or complexed with one or more binding partners. The extensive interplay of RNA and proteins in aligning the pre-mRNA's reactive groups, and the presence of both RNA and protein at the core of the splicing machinery, suggest that the spliceosome is an RNP enzyme. However, elucidation of the precise nature of the spliceosome's active site, awaits the generation of a high-resolution structure of its RNP core.

Abstract: Precursor messenger RNA (pre-mRNA) splicing is a critical step in the posttranscriptional regulation of gene expression, providing significant expansion of the functional proteome of eukaryotic organisms with limited gene numbers. Split eukaryotic genes contain intervening sequences or introns disrupting protein-coding exons, and intron removal occurs by repeated assembly of a large and highly dynamic ribonucleoprotein complex termed the spliceosome, which is composed of five small nuclear ribonucleoprotein particles, U1, U2, U4/U6, and U5. Biochemical studies over the past 10 years have allowed the isolation as well as compositional, functional, and structural analysis of splicing complexes at distinct stages along the spliceosome cycle. The average human gene contains eight exons and seven introns, producing an average of three or more alternatively spliced mRNA isoforms. Recent high-throughput sequencing studies indicate that 100% of human genes produce at least two alternative mRNA isoforms. Mechanisms of alternative splicing include RNA-protein interactions of splicing factors with regulatory sites termed silencers or enhancers, RNA-RNA base-pairing interactions, or chromatin-based effects that can change or determine splicing patterns. Disease-causing mutations can often occur in splice sites near intron borders or in exonic or intronic RNA regulatory silencer or enhancer elements, as well as in genes that encode splicing factors. Together, these studies provide mechanistic insights into how spliceosome assembly, dynamics, and catalysis occur; how alternative splicing is regulated and evolves; and how splicing can be disrupted by cis- and trans-acting mutations leading to disease states. These findings make the spliceosome an attractive new target for small-molecule, antisense, and genome-editing therapeutic interventions.

Abstract: A mechanism is proposed for the RNA-catalyzed reactions involved in RNA splicing and RNase P hydrolysis of precursor tRNA. The mechanism postulates that chemical catalysis is facilitated by two divalent metal ions 3.9 A apart, as in phosphoryl transfer reactions catalyzed by protein enzymes, such as the 3',5'-exonuclease of Escherichia coli DNA polymerase I. One metal ion activates the attacking water or sugar hydroxyl, while the other coordinates and stabilizes the oxyanion leaving group. Both ions act as Lewis acids and stabilize the expected pentacovalent transition state. The symmetry of a two-metal-ion catalytic site fits well with the known reaction pathway of group I self-splicing introns and can also be reconciled with emerging data on group II self-splicing introns, the spliceosome, and RNase P. The role of the RNA is to position the two catalytic metal ions and properly orient the substrates via three specific binding sites.

Abstract: Complex and intricate RNA splicing mechanisms are crucial for gene regulation and for maximizing proteomic diversity. This Review discusses how alterations to splicing mechanisms — such as mutations in pre-mRNAs, or mutations and dysregulation of core spliceosome proteins and other RNA-binding proteins — results in diverse molecular consequences and various diseases. Opportunities for therapeutic correction of these defects are also explored.