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Mechanisms that increase protein diversity include the usage of multiple transcription start sites, alternative pre-mRNA processing, polyadenylation, pre- mRNA editing and post-translational modifications. Among these mechanisms, alternative pre-mRNA splicing is considered the most important source of protein diversity. This process was described in nearly all metazoan organisms as a mode of producing functionally diverse polypeptides from a single gene (Maniatis and Tasic 2002). Alternative splicing in fact generates a large number of mRNAs from the surprisingly low number of human genes encoding proteins with slight or opposing functional differences, with profound biological consequences (Lopez 1998).

The process of alternative splicing is highly regulated in developmental stages and in different tissues (Black 2003). Moreover the selection of the correct splicing variants in a given cell type and/or in a particular cell condition is considered to be regulated by multiple (sometimes overlapping) exonic and/or intronic splicing enhancers and silencers (Cartegni, Chew et al. 2002; Ladd and Cooper 2002). Because a single primary transcript can have several regions that each undergo alternative splicing, the resulting combinatorial effects of selecting different splice sites can be very pronounced, and genes that code for tens to hundreds of different isoforms are common (Graveley 2001). One of the most striking examples of alternative splicing complexity has been described in Drosophila melanogaster axon guidance receptor gene, Down syndrome cell-adhesion molecule (Dscam). The pre-mRNA of this gene can potentially produce 38,016 different mature transcripts by alternative splicing (Schmucker, Clemens et al. 2000). Even if only a subset of these mRNAs is ever produced in vivo, this combinatorial use of alternative exons still represents an incredible source of diversity, especially given that the entire Drosophila genome consists of only -14,000 genes (Adams, Celniker et al. 2000).

At first, in humans, splicing was thought to be only a minor processing pathway affecting about 5% of all genes (Sharp 1994), but over time, it became clear that it is very abundant. Bioinformatic studies showed that 59% of the 245 genes present on chromosome 22 are alternatively spliced, and DNA microarray experiments indicate that 74% of all human genes are alternatively spliced (Johnson, Castle et al. 2003), suggesting that alternative splicing of human genes is the rule and not the exception.

Every conceivable pattern of alternative splicing is present in nature (Fig. 1.7). Exons can have different 5’ss or 3’ss that can be alternatively used. Single cassette exons can reside between two constitutive exons such that the alternative exon is either included or skipped. Alternatively, multiple cassette exons can reside between two constitutive exons such that the splicing machinery must choose between them. In these systems, special mechanisms must enforce the exclusive choice (Smith and Nadal-Ginard 1989). Introns can be retained in the mRNA and become translated.

The 5-terminal exons of a mRNA can be switched through the use of alternative promoters and alternative splicing. Similarly, the 3-terminal exons can be switched by combining alternative splicing with alternative polyadenylation sites. In addition, these individual patterns can be combined in a single transcription unit to produce a complex array of splice isoforms. Moreover, changes in alternative splicing can modulate transcript expression levels subjecting mRNAs to nonsense-mediated decay (NMD) by creating a stop codon within the coding sequence or by altering the structure of the gene product by inserting, or deleting, novel protein parts (Faustino and Cooper 2003).

The mechanisms that determine which splice site has to be utilized and/or which exon has to be chosen in different cell types or developmental stages have still not been precisely defined.

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Figure 1.7: Patterns of alternative splicing.

Alternative splicing generates different segments within mRNAs.

Alternative promoters: selection of one of multiple first exons results in variability at the 5’ end of the mRNA. Red indicates variable regions within the mRNA and encoded protein (7). Alternative splicing of internal exons: the alternative splicing patterns for internal exons include the cassette exon (2), alternative 5’ splice sites (5), alternative 3’ splice sites (4), intron retention (5), and mutually exclusive exons (6).

Alternative terminal exons: selection of one of multiple terminal exons results from a competition between cleavage at the upstream poly(A) site or splicing to the downstream 3’ splice site (7). There are also examples of competition between a 5’

splice site and a poly (A) site within an upstream terminal exon (8). Variability at the 3’ end of the mRNA produces either proteins with different C termini or mRNAs with different 3,-UTRs. Figure adapted from Faustino and Cooper (Faustino and Cooper 2003).

^ Much progress has been made in identifying the “combinatorial code” composed by cis-acting elements and trans-acting factors involved in the regulation of alternative splicing. High-throughput technologies like large-scale sequencing and microarrays analysis are providing opportunities to address these key questions (Ben-Dov, Hartmann et al. 2008).

1.5.1 Combinatorial control o f trans-acting factors.

The information that determines an alternative splicing event is very difficult to characterize. Along with the main determinants of exon-intron definition, such as splice sites or enhancer/silencer elements, many more splicing factors have been involved in splicing regulation. Alternative splicing in mammals in fact is largely controlled by combinatorial binding of basal splicing factors to the pre-mRNA.

Although several tissue-specific factors associated to a particular alternative splice site event have been identified (Markovtsov, Nikolic et al. 2000; Ladd, Charlet et al.

2001), the model for splice site choice consists of a selective usage of an exon due to the binding of a distinct set of generic splicing factors (Smith and Valcarcel 2000;

Mabon and Misteli 2005). This combinatorial model is supported by the observation that in vivo and in vitro the counteracting activities of multiple antagonistic factors can regulate alternative splicing, suggesting that the physiological concentration of competing splicing factors is important for regulation of splice site selection (Caceres, Stamm et al. 1994; Hanamura, Caceres et al. 1998).

For example, hnRNP A l can modulate splice site selection by antagonizing the activity of ASF/SF2 protein (Caceres, Stamm et al. 1994). It was observed that high relative concentrations of A l favour the choice of distal 5 ’ss, an excess of ASF/SF2 result in the proximal 5’ss choice both in vitro and upon overexpression in vivo

(Mayeda and Krainer 1992; Caceres, Stamm et al. 1994). The same competitive effect between hnRNP A l and ASF/SF2 has been reported also in the case of alternative 3’ss selection. In the same manner hnRNP A l promotes the use of the distal 3’ss, while ASF/SF2 promotes the use of the proximal 3’ss (Caceres, Stamm et al. 1994; Bai, Lee et al. 1999).

In addition to these indirect data, a recent work based on a quantitative single-cell imaging provided new evidence for the combinatorial model for alternative splicing reporting the first in vivo evidence for a distinct association of splicing factors with alternative spliced pre-mRNAs (Mabon and Misteli 2005).