El encargo de protección y fomento del derecho de propiedad

The transcriptional process includes the formation of a pre-initiation complex, the transcription initiation, elongation, termination and finally the dissociation of RNA polymerase II (Pol II) from the DNA template. Then the pre-mRNA transcript undergoes several processing steps including capping, splicing, polyadenylation, surveillance and export (Fig. 1.9). In vitro each of these modifications can occur independently from the others, as found in reconstituted systems on purified pre- mRNA substrates. However, in vivo these reactions influence one another’s efficiency revealing a functional relationship among them and indicating that most mRNA processing reactions occur co-transcriptionally (Bentley 2002; Bentley 2005).

These observations led to the proposal that the RNA modifications occur in a “gene expression factory” composed of machines linked together for the purposes of efficiency and regulation (Bentley 2002; Bentley 2005).

To date, two models have been proposed to explain the connection between the different steps of RNA transcription and processing. The “recruitment model”

derived from the observation that several trans-acting factors can interact directly with the RNA Pol II and thus increasing their own concentration in the proximity of the nascent transcript (Bentley 2005). The carboxy-terminal domain (CTD) of the RNA Pol II in fact has been shown to play a central role in coupling transcription to pre-mRNA processing acting as assembly platform for proteins involved both in transcription and pre-mRNA processes regulation (Bentley 2005). In particular, recent studies reported an important connection between SRp20 and the CTD tail for the EDA alternative exon regulation. Mature mRNAs transcribed by a RNA Pol II, lacking the CTD tail, showed a much higher percentage of EDA inclusion compared to the ones transcribed by the entire RNA Pol II. The CTD-dependent silencing

seems to be mediated by its ability to recruit the SRp20 splicing factor (de la Mata and Komblihtt 2006).

An alternative to the recruitment model is the “kinetic model”, mainly linked to the RNA Pol II transcriptional elongation rate (Komblihtt, de la Mata et al. 2004). This model was originally proposed by experiments in which RNA Pol II pausing sites were artificially introduced into a gene, delaying the transcription of a splicing inhibitory element and therefore resulting in higher inclusion levels of an alternative exon (Roberts, Gooding et al. 1998). Further evidence supported this model indicating that transcription can affect splicing acting at different cis- and trans­

acting levels (Komblihtt, de la Mata et al. 2004). For instance differences in promoter architecture have been shown to affect the subsequent selection of the fibronectin EDA alternative exon and the CFTR exon 9 (Cramer, Pesce et al. 1997;

Cramer, Caceres et al. 1999; Pagani, Stuani et al. 2003). However, in vivo most of the genes have a single promoter and the regulation of splicing has been reported to occur through the binding of different transcription factors. In line with this view it has been described that transcriptional activators can affect alternative splicing (Nogues, Kadener et al. 2002). Additionally, the presence of enhancer sequences, like SV40 next to the promoter can stimulate RNA Pol II elongation while the deletion of this enhancer causes a reduction in exon skipping (Kadener, Fededa et al.

2002).

A less explored factor involved in regulation of splicing via RNA Pol II elongation is the chromatin packaging. Changes in its stmcture due to acetylation can alter splicing. It was reported in fact that cell treatment with a potent inhibitor of histone deacetylation inhibits EDA inclusion (Nogues, Kadener et al. 2002). Recently, a role of SWI/SNF chromatin remodelling complex was also described for alternative splicing. This factor was shown to stimulate the inclusion of alternative exons in the

CD45 gene by decreasing the RNA pol II elongation rate and facilitating the recruitment of the splicing machinery (Batsche, Yaniv et al. 2006).

A more direct proof for the kinetic model derived from studies on RNA Pol II elongation rate (Roberts, Gooding et al. 1998; de la Mata, Alonso et al. 2003). A slow Pol II and/or the presence of internal transcriptional stalling sites, results in an increased inclusion of alternative exon harbouring a weak 3’ss. By contrast, when a highly processive RNA Pol II transcribes the same pre-mRNA, the weak alternative splice site is unable to compete with the stronger downstream 3’ss, which results in skipping of the alternative exon (de la Mata, Alonso et al. 2003). Recently a reciprocal coupling between splicing and transcriptional elongation has also been reported. Splicing proteins have been involved in transcriptional elongation in vitro (Fong and Zhou 2001) and specific depletion of SC35 showed RNA Pol II accumulation and attenuated elongation in vivo (Lin, Coutinho-Mansfield et al.

2008). Furthermore, it was also described that an efficient RNA Pol II transcription is strictly connected with the presence of intronic sequences (Furger, O'Sullivan et al.

2002). Taking these evidences together a complex view has emerged from the studies focused on the coupling between transcription and pre-mRNA processing suggesting that both recruitment of factors to the CTD and RNA Pol II kinetic are involved in this connection (Komblihtt, de la Mata et al. 2004).

Transcription

initiation Transcription elongation

5’<*pping packaging splicing

: Transcription

; term ination I 3 ’-processfftg

mRNA surveillance

mRNA export

Figure 1.9: Coupling mRNA transcription and processing.

The production o f a correct mRNA is a coordinated and multiple-step process that occurs in the nucleus of eukaryotic cells. RNA Pol II, and specifically the C-terminal domain (CTD) of its largest subunit directs these processes in a way that links CTD phosphorylation changes to different factors binding. These proteins in turn affect the subsequent processing steps and/or help to recruit processing/packaging factors to the nascent transcript. The maturation of pre-mRNA includes the capping at its 5’

end, the introns removal during splicing, and the cleavage and the polyadenylation of the 3’ end. After going through the mRNA surveillance system, the matured mRNA is exported to the cytoplasm for translation. Each stage of RNA Pol II transcription and the steps o f co-transcriptional processing are indicated. Figure adapted from Li and Manley (Li and Manley 2006).