Models for normal and defective BRCA1 exon 18 splicing

Our results indicate that the primary determinant of the T6 BRCA1 exon 18 exclusion is not the disruption of an ASF/SF2 dependent enhancer but the creation of a new silencer element recognized specifically by the splicing inhibitory factors hnRNP A1/A2 and DAZAP1.

Based on our analysis we propose a model for WT and T6 BRCA1 exon 18 splicing regulation (figure 4.1). BRCA1 exon 18 WT may contain two weak ESE regions localized at position +4-9 and +23-32 and two downstream silencers at position at position 35-44 and 64-74 and its definition is largely dependent on the 3’ and 5’ ss (Fig. 4.1 A). The two enhancer regulatory elements are dispensable for the definition of the BRCA1 WT exon, as their single deletions did not induce exon skipping (Fig.

3.5.IB). Only when deleted in combination can they induce exon skipping (Fig.

3.5.2B, lane 8) and the weak central enhancer element probably mediates its effect through its binding to ASF/SF2.

The G to T mutation in position +6 of exon 18 induces exon skipping through the creation of a strong silencer regulatory element located at the 5 ’ end of the exon that binds to the inhibitory splicing factors hnRNP A l, hnRNP A2, and DAZAP1.

Several mechanisms have been proposed to explain hnRNP A l mediated splicing repression; our data extended these finding by proposing an involvement of DAZAP1 in these models. In the simplest scenario, silencer-bound hnRNP A1/A2 and DAZ API factors can directly antagonize another key splicing factor involved in exon definition. The hnRNP A l, hnRNP A2, and DAZAP1 binding is very close to the 3’ss and this could interfere with the early spliceosome assembly by blocking the access to general splicing factor such as U2AF65, U2AF35 and/or U2 snRNP to the acceptor site (Fig 4. lb) (Tange, Damgaard et al. 2001). However, it is also possible that the initial binding of hnRNP A l, hnRNP A2 and DAZAP1 at the T6 sequence

could nucleate the formation of a chain of inhibitory proteins along the other two silencer elements located in the middle and at the end of the exon. These two silencers, as the T6 mutant sequence, contain TAG, which might represent the “core”

sequences involved in this process. This model for cooperative repression involves the creation of a “zone of silencing” due to the propagation of the repressor proteins that will cover the entire exon 18, leading to its exclusion, as previously suggested for a hnRNP A l silencer element described in HIV-1 tat exon 3 systems (Zhu, Mayeda et al. 2001).

Recently, a concomitant disruption of an exonic splicing enhancer not dependent on ASF/SF2 has been suggested to be involved in the exon skipping induced by the T6 mutant (Kashima, Rao et al. 2007). The presence of this putative enhancer was suggested by the fact that the siRNA-mediated silencing of hnRNP A1/A2 did not have a significant effect on T6 aberrant skipping (Kashima, Rao et al. 2007). We have carefully evaluated this point by performing co-transfection experiments with the SMN2 and T6 BRCA1 minigenes and in both cases we observed that silencing of hnRNP A1/A2 rescued both defective exons (Fig. 3.4.4). We do not have a clear explanation for these differences in the results. An inhibitory effect on the BRCA1 recognition may differ depending on the variable concentration of splicing factors, like DAZAP1, due to the use of different cell types. These factors might efficiently replace hnRNP A1/A2 at the T6 mutant silencer or can act on other inhibitory splicing regulatory elements, thus modulating the siRNA rescue splicing efficiency.

However, even if our results clearly indicate that the T6 mutant creates a strong silencer, we cannot exclude the simultaneous disruption of a weak enhancer region present in the 5’ end of the BRCA1, as suggested by our double delition analysis (Fig. 3.5.2B lane 8), in agreement with Kashima et al. (Kashima, Rao et al. 2007).

A

E x o n 1 7 E x o n 1 8 E x o n 1 9

? ASF/SF2

WT ^{j 3'ss i l l} H i 1 5'ss ^{Exon 18}

inclusion

• ' / 4. 4.

CT GAG TT

B

F/SF2

Mut T6 ^15'ss

CT TAG TT

Zone of silencing

Exon 18 exclusion

Exon 18 exclusion

Figure 4.1: Splicing regulatory elements in BRCA1 exon 18 and creation of an ESS by the T6 mutant. (A) WT exon 18 contains two weak ESEs (light grey boxes) and two ESSs (black boxes) and its inclusion is largely dependent on the 3’ and 5’ss definition. (B-C) The G to T mutation in position +6 creates a strong silencer element at the 5’ end of the exon binding to hnRNP A1/A2 and DAZAP1 and may also disrupt a weak splicing enhancer. The ASF/SF2 ESE located in the middle of exon 18 stimulates BRCA1 exon 18 splicing only in the context of the T6 mutant. The mechanism of inhibition could be mediated by blocking 3’ss recognition (B) or by forming a zone of silencing that covers the entire exon (C).

In fact, the partial exon 18 skipping obtained by the double deletion of the 4-9 and 23-32 regions (Fig. 3.5.2B, lane 8) might be due to the presence of two nearby weak enhancer sequences, which may contribute in a synergic manner to the definition of the WT exon. Thus, with the limitation provided by the gross deletion analysis, which can interfere with RNA secondary structure and acceptor site accessibility or reduce the exon definition below a critical threshold, the 5’ end on BRCA1 exon 18 might contain a weak enhancer, which becomes relevant only when the downstream enhancer is deleted.

However, the extent of splicing inhibition mediated by the A23-32 deletion in the context of the single T6 substitution was more severe than the splicing inhibition mediated by the presence of the A4-9 deletion (Fig. 3.5.2B, lane 4 vs. 8). This result indicated that the inhibitory effect of the T6 mutation is more repressive than the removal of the weak enhancer between position +4-9, further indicating the creation of a silencer element as the main cause of pathological exon 18 skipping.

FUTURE DIRECTIONS

In this thesis the role of exonic regulatory elements in the aberrant splicing of the human BRCA1 exon 18 have been investigated. This study brings some insights into the rather complex regulation of BRCA1 exon 18, whose T6 mutation provoke the substantial exon 18 skipping.

The data collected in this thesis clearly suggest that the T6 BRCA1 exon 18 splicing is modulated by several other cis-acting elements distributed along the exon. These elements were identified mainly by large deletion analysis and accordingly, it will be necessary to better characterize their specific contribution in the regulation of BRCA1 exon 18 splicing. Further experiments will characterize in more detail the nucleotide composition and the binding properties of these sequences. In addition, it would be important to explore the potential role of intronic regions, which, as described for the SMN case (Miyajima, Miyaso et al. 2002; Miyaso, Okumura et al.

2003; Singh, Singh et al. 2006; Kashima, Rao et al. 2007) might contribute to the splicing regulation.

In this thesis a novel role for DAZAP1 in the pre-mRNA splicing regulation has been described. An obvious question that needs to be addressed is the involvement of this splicing factor in the regulation of the similar SMN1/2 model. The behavior of BRCA1 T6 exon 18 mutation in fact, is analogous to that which promotes exon skipping in SMN2 exon 7. It will be interesting to compare the two models, in particular a possible role of DAZAP1 in the SMN2 exon 7 splicing regulation.

DAZAP1 might be also involved in the control of other alternative splicing events.

Using an exon array in DAZAP1 depleted cells it will be possible to study the role of this protein in general and/or in specific splicing events.

Co-transfection experiments suggested that the 23-32 region of the BRCA1 exon 18 is an enhancer element connected to the ASF/SF2 factor. This hypothesis will be further explored by performing binding experiment in order to confirm the presence of ASF/SF2 factor in the 23-32 exonic region. Moreover, the result obtained by the double deleted construct (A4-9/A23-32) supports the notion of a weak ESE in the vicinity of position +6, even if not related to ASF/SF2. Additional trans-acting factors could be involved in the recognition of this weak enhancer and their presence will be investigated by binding experiments.

A more general mechanistic problem will also be considered in order to understand the role of hnRNP A l, -A2 and DAZ API complex in the splicing inhibition. This binding may inhibit the recognition of the 3’ss by U2AF35 or in interfering with subsequent steps of spliceosome assembly. A set up of an in vitro system will allow the study of the different splicing steps. It is also possible, as indicated in the model (Fig. 4.1), that a cooperative binding of hnRNP A1/A2 and DAZAP1 covers the entire exon 18 and produces exon skipping. To this aim, the potential contribution of other silencers containing the TAG motifs, localized within the last part of exon 18, is going to be checked. The binding of hnRNP A l, -A2 and DAZ API to the first inhibitory element, surrounding the position +6, might promote a subsequent cooperative binding to the other putative ESSs localized at the end of the exon 18.