Mechanisms and Regulation of Alternative Pre-mRNA Splicing

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.

The landscape of human mutually exclusive splicing

“…The reconstruction resulted in a set of 6,541 MXE candidates in 1,542 protein‐coding genes, including 1,058 (68.6%) genes for which we predicted 1,722 completely novel exons in previously intronic regions (Fig 1B). Most introns in human genes are extremely long necessitating careful and strict validation of the MXE candidates to exclude false‐positive predictions (Lee & Rio, 2015). …”

“…Surprisingly, the majority of the annotated MXEs are of this type (91 of 122; 75%) as well as many exons previously annotated as other splice types (44 of 662), but only few of the novel MXEs predicted in intronic regions (25 of 615; Appendix Fig S3A and D). These numbers suggest that splicing of the remaining 484 MXE clusters is tightly regulated by other mechanisms (Fig 2B) such as RNA–protein interactions, interactions between small nuclear ribonucleoproteins and splicing factors (Lee & Rio, 2015), and competitive RNA secondary structural elements (Graveley, 2005; Yang et al , 2012; Lee & Rio, 2015). Competing RNA secondary structures are, however, usually not conserved across long evolutionary distances.…”

“…Alternative splicing of pre‐messenger RNAs is a mechanism common to almost all eukaryotes to generate a plethora of protein variants out of a limited number of genes (Matlin et al , 2005; Nilsen & Graveley, 2010; Lee & Rio, 2015). High‐throughput studies suggested that not only 95–100% of all multi‐exon genes in human are affected (Pan et al , 2008; Wang et al , 2008; Gerstein et al , 2014) but also that alternative splicing patterns strongly diverged between vertebrate lineages implying a pronounced role in the evolution of phenotypic complexity (Barbosa‐Morais et al , 2012; Merkin et al , 2012).…”

Low‐density lipoprotein receptor‐related protein 6 regulates alternative pre‐mRNA splicing

“…The exclusion or inclusion of alternative exons highly depends on the position and context of splicing cis ‐regulatory sequences within alternative exons or the flanking introns, including exonic splicing enhancers or silencers (ESEs or ESSs) and intronic splicing enhancers or silencers (ISEs or ISSs) that recruit splicing regulators 16, 17. Using the RIP approach, we observed that both mature Itgb1D mRNA and Itgb1 pre‐mRNA, which share the 81‐bp AED sequence, were detected in LRP6 protein‐immunoprecipitated transcripts (Figure 3A), implicating that the AED was the potential cis ‐regulatory element.…”

“…Regulation of this process is highly complicated, depending on loosely defined cis ‐acting regulatory sequence elements, trans‐acting protein factors and cellular responses to varying environmental conditions. Deciphering the splicing code requires understanding of splicing regulatory RNA‐binding proteins (RBPs) and their cis‐acting binding sites 16. There are virtually no data on the sequence preferences of RBPs in most organisms.…”