El efecto de irradiación del derecho de propiedad

In most of the cases, vertebrate genes contain multiple short exons separated by intronic sequences that can be significantly longer. A typical human pre-mRNA molecule can be up to 30 kb long and contains about ten exons separated by much longer and variable intronic sequences (Hawkins 1988). The average length of an internal exon is around 137 nt (Hawkins 1988) and the exons that are more than 300 nt long or are shorter than 50 nt seem not to be favoured by the splicing machinery (Black 1991; Sterner and Berget 1993).

The discrepancy between the lengths of human exons compared to the introns led to the proposal of the “exon definition” model (Fig. 1.8A). In this model the exon is the unit recognized by the splicing machinery and the splice sites are first paired across exons, with subsequent spliceosome assembly proceeding through pairing of neighbour exon units (Robberson, Cote et al. 1990; Berget 1995).

Evidence for the exon definition model arises from the observation that the first and the last exon require a special mechanism for their recognition. It has been shown that 5’ capping and the 5 ’ss are necessary to define the first exon (Izaurralde, Lewis

et al. 1994). On the other hand, last exon is defined by 3’ss and polyadenylation signal (Niwa and Berget 1991). Moreover, it was shown that the exon length might affect splicing. A minimal separation of the sites seems to be required to prevent steric hindrance of the factors that recognize the splice sites bordering an exon (Black 1991). This indication is supported by the observation that a constitutively recognized internal exon was skipped by in vivo splicing machinery if its size was smaller that 50 nt (Dominski and Kole 1991). Complementary studies showed that the extension of the 18-nt N1 exon of the mouse c-src gene up to 109 nt leads to its constitutive inclusion. This finding suggests that the exon is normally skipped because it is too short to allow spliceosome assembly at both ends simultaneously (Black 1991). Further in vitro analysis on the expansion of internal exons to length above 300 nt causes the activation of cryptic splice site located within the exon or lead to exon skipping indicating that splicing efficiency is affected by length (Robberson, Cote et al. 1990). In fact, less than 1% of the known internal exons in vertebrate are longer than 400 nt (Hawkins 1988). Conversely the experiments obtained by expanding the central exon in the dihydrofolate reductase minigene with random DNA fragments from E.coli, demonstrated that its expansion up to 1200 nt does not compromise the correct inclusion into mRNA (Chen and Chasin 1994). To explain these data a compensatory relationship between exon and intron size has been proposed. In fact, Sterner et al. observed that long internal exons are problematic for recognition if they are flanked by long introns, suggesting that these exons might be flanked by short introns (Sterner, Carlo et al. 1996). In this case, the splice sites can be paired across introns rather than exons as proposed by the "intron definition" model (Fig.l.8B). This model in fact suggests that introns are the units recognized by the splicing machinery proposing a sort of scanning mechanism where the 5' and 3' splice signals are initially recognized and paired across the intron

(Guthrie 1991; Berget 1995). Intron definition is thought to be the predominant way of splicing in transcripts containing short introns and long exons (Sterner, Carlo et al.

1996). Evidence supporting this mechanism arises from observations made in yeast, where RNA messengers often have a unique intron and its length is usually below 100 nt (Goguel and Rosbash 1993).

In Drosophila there are two different classes of introns: long, vertebrate-like introns that possess a 3’ pyrimidine tract and short, yeast-like introns that lack this consensus sequence (Talerico and Berget 1994). 50% of small introns are less than 100 nt and are often flanked by large exons (Hawkins 1988). Mutants of 5’ss and expansion of the size of these small introns showed the induction of intron retention and cryptic splice site activation, respectively supporting the intron definition model as way of recognition (Talerico and Berget 1994).

All these observations suggest mechanistic differences in the process of splice site selection in pre-mRNAs containing small exons or small introns. It has been indicated that splice sites are initially paired across the shortest distance so both exon definition and intron definition might occur in different parts of the pre-mRNA of the same gene (Sterner, Carlo et al. 1996).

(a) Exon definition model

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Figure 1.8: Exon definition versus intron definition models.

(A) Exon definition model: a vertebrate gene consists o f multiple short exons separated by considerably longer introns. In this model the exon is recognized as a unit during early spliceosome assembly. Multiple factors interact with exonic sequences in order to defining the 5’ and the 3’ borders of the exon. SR proteins can help this “cross exon” recognition through the binding exonic splicing enhancers (ESEs) and recruiting U1 snRNP to the 5’ss, U2AF65 and U2AF35 subunits to the PPT and to the 3’ss, respectively.

(B) Intron definition model: it has been proposed for the systems in which pre- mRNA has small introns. In this case the intron, rather than exon, is the recognized unit. Multiple factors favour the 5’ and the 3’ ss pairing at the intron ends. SR proteins function in a “cross intron” recognition complex by bridging together the U 1 snRNP bound to the upstream 5’ss and the U2AF65 and U2AF35 subunits bound to the PPT and to the AG of the downstream 3’ss, respectively. Adapted from Ram and Ast (Ram and Ast 2007).