Downloaded from genesdev.cshlp.org on October 1, 2021 - Published by Cold Spring Harbor Laboratory Press REVIEW Pre-mRNA splicing and human disease Nuno Andre´Faustino1,3 and Thomas A.Cooper 1,2,4 Departments of 1Pathology and 2Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030, USA; 3Graduate Program in Basic and Applied Biology, ICBAS, University of Oporto, Portugal The precision and complexity of intron removal during snRNP binds the branch site via RNA:RNA interactions pre-mRNA splicing still amazes even 26 years after the between the snRNA and the pre-mRNA (Fig. 1B). Spli- discovery that the coding information of metazoan genes ceosome assembly is highly dynamic in that complex is interrupted by introns (Berget et al. 1977; Chow et al. rearrangements of RNA:RNA, RNA:protein, and pro- 1977). Adding to this amazement is the recent realiza- tein:protein interactions take place within the spliceo- tion that most human genes express more than one some. Coinciding with these internal rearrangements, mRNA by alternative splicing, a process by which func- both splice sites are recognized multiple times by inter- tionally diverse protein isoforms can be expressed ac- actions with different components during the course of cording to different regulatory programs. Given that the spliceosome assembly (for example, see Burge et al. 1999; vast majority of human genes contain introns and that Du and Rosbash 2002; Lallena et al. 2002; Liu 2002). The most pre-mRNAs undergo alternative splicing, it is not catalytic component is likely to be U6 snRNP, which surprising that disruption of normal splicing patterns joins the spliceosome as a U4/U6 · U5 tri-snRNP (Villa can cause or modify human disease. The purpose of this et al. 2002). review is to highlight the different mechanisms by A splicing error that adds or removes even 1 nt will which disruption of pre-mRNA splicing play a role in disrupt the open reading frame of an mRNA; yet exons human disease. Several excellent reviews provide de- are correctly spliced from within tens of thousands of tailed information on splicing and the regulation of splic- intronic nucleotides. This remarkable precision is, in ing (Burge et al. 1999; Hastings and Krainer 2001; Black part, built into the mechanism of intron removal be- 2003). The potential role of splicing as a modifier of hu- cause once the spliceosome is assembled, the base-paired man disease has also recently been reviewed (Nissim- snRNAs target specific phosphate bonds for cleavage. Rafinia and Kerem 2002). The challenge for the spliceosome comes in recognizing the correct splice sites prior to the cut-and-paste reac- tions. The short and degenerate splice sites contain only Constitutive splicing and the basal splicing machinery half of the information necessary for splice-site recogni- The typical human gene contains an average of 8 exons. tion (Lim and Burge 2001) because bona fide splice sites Internal exons average 145 nucleotides (nt) in length, and must be distinguished from pseudo splice-site sequences introns average more than 10 times this size and can that resemble classical splice sites but are never used. be much larger (Lander et al. 2001). Exons are defined Pseudo splice sites can outnumber bona fide splice sites by rather short and degenerate classical splice-site se- within a pre-mRNA by an order of magnitude (Sun and quences at the intron/exon borders (5Ј splice site, 3Ј Chasin 2000). Auxiliary cis-elements, known as exonic splice site, and branch site; Fig. 1A). Components of the and intronic splicing enhancers (ESEs and ISEs) and basal splicing machinery bind to the classical splice-site exonic and intronic splicing silencers (ESSs and ISSs; Fig. sequences and promote assembly of the multicompo- 1B), aid in the recognition of exons (see below). nent splicing complex known as the spliceosome. The It is now clear that exon recognition is accomplished spliceosome performs the two primary functions of by the accumulated recognition of multiple weak sig- splicing: recognition of the intron/exon boundaries and nals, resulting in a network of interactions across exons catalysis of the cut-and-paste reactions that remove in- as well as across introns (Fig. 1B; Berget 1995; Reed trons and join exons. The spliceosome is made up of five 1996). It is also clear that different constitutive exons are small nuclear ribonucleoproteins (snRNPs) and >100 recognized by different mechanisms and require differ- proteins. Each snRNP is composed of a single uridine- ent sets of auxiliary elements in addition to the classical rich small nuclear RNA (snRNA) and multiple proteins. splice-site sequences. The significance of these observa- The U1 snRNP binds the 5Ј splice site, and the U2 tions is threefold. First, there are a considerable number of disease-causing mutations in exons or introns that disrupt previously unrecognized auxiliary cis-elements 4Corresponding author. as well as the well-known classical splice sites (Fig. 1C). E-MAIL [email protected]; FAX (713) 798-5838. Article and publication are at http://www.genesdev.org/cgi/doi/10.1101/ Second, because exons differ in their requirements for gad.1048803. recognition, mutations that disrupt the function of the GENES & DEVELOPMENT 17:419–437 © 2003 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/03 $5.00; www.genesdev.org 419 Downloaded from genesdev.cshlp.org on October 1, 2021 - Published by Cold Spring Harbor Laboratory Press Faustino and Cooper Figure 1. Classical and auxiliary splicing signals (n=G,A,U,orC;y=pyrimidine;r=purine).(A) Clas- sical splice sites: The classical splicing signals found in the major class (>99%) of human introns are re- quired for recognition of all exons. There is also a minor class of introns using different classical se- quences and different spliceosome components (Tarn and Steitz 1997). (B) Classical and auxiliary splicing elements and binding factors: Factors that bind clas- sical and auxiliary splicing elements. Auxiliary ele- ments within exons (ESEs and ESSs) and introns (ISEs and ISSs) are commonly required for efficient splicing of constitutive and alternative exons. Intronic ele- ments also serve to modulate cell-specific use of alter- native exons by binding multicomponent regulatory complexes. (C) Cis-acting splicing mutations. Muta- tions that disrupt cis-acting elements required for pre- mRNA splicing can result in defective splicing that causes disease. splicing machinery will have different effects on dif- plexes on the cis-acting elements surrounding the regu- ferent subsets of exons. Third, variability in the basal lated splice sites (Grabowski 1998; Smith and Valcarcel splicing machinery among different cell types could 2000). The straightforward model is that these com- cause cell-specific sensitivities to individual splicing plexes serve to enhance or inhibit recognition of the mutations. classical splice sites by the basal splicing machinery. Ac- tivating and repressing activities coexist within cells (Charlet et al. 2002a), and it remains unclear why acti- Alternative splicing vation dominates in one cell type whereas repression dominates in another. Importantly, mutations that per- Alternative splicing is the joining of different 5Ј and 3Ј turb this balance can result in aberrant regulation of al- splice sites, allowing individual genes to express mul- ternative splicing, causing the expression of protein iso- tiple mRNAs that encode proteins with diverse and even forms that are inappropriate for a cell type or develop- antagonistic functions. Up to 59% of human genes gen- mental stage. erate multiple mRNAs by alternative splicing (Lander et al. 2001), and ∼80% of alternative splicing results in changes in the encoded protein (Modrek and Lee 2002), Human disease caused by disruption revealing what is likely to be the primary source of hu- of pre-mRNA splicing man proteomic diversity. Alternative splicing generates segments of mRNA variability that can insert or remove To define the diverse mechanisms by which defects in amino acids, shift the reading frame, or introduce a ter- pre-mRNA splicing result in a primary cause of disease, mination codon (Fig. 2). Alternative splicing also affects we have classified splicing mutations into four catego- gene expression by removing or inserting regulatory ries (Fig. 3). These categories are based on two criteria. elements controlling translation, mRNA stability, or First, does the mutation affect expression of a single gene localization. by disrupting a splicing cis-element, or does the muta- A large fraction of alternative splicing undergoes cell- tion have an effect in trans on multiple genes by disrupt- specific regulation in which splicing pathways are modu- ing a component of the splicing machinery or of a splic- lated according to cell type, developmental stage, gender, ing regulatory complex? Second, does the mutation or in response to external stimuli. In the best character- cause aberrant splicing (expression of unnatural mRNAs) ized models of vertebrate cell-specific alternative splic- by creating unnatural splicing patterns or aberrant regu- ing, regulation is mediated by intronic repressor and ac- lation of splicing (the inappropriate expression of natural tivator elements distinct from the classical splicing se- mRNAs) by disrupting use of alternatively used splice quences. Cell specificity emerges primarily from two sites? features: First, the repression of splicing in the inappro- Cis-acting mutations can affect the use of constitutive priate cell type is combined with activation of splicing in splice sites (Fig. 3A) or alternative splice sites (Fig. 3B). the appropriate cell type; and, second, combinatorial Disrupted constitutive splicing most often results in loss control is exerted by multiple components involving co- of gene expression due to aberrant splicing (see below). operative assembly of activation and/or repression com- On the other hand, a cis-acting mutation that inactivates 420 GENES & DEVELOPMENT Downloaded from genesdev.cshlp.org on October 1, 2021 - Published by Cold Spring Harbor Laboratory Press Pre-mRNA splicing and human disease Figure 2.