Ence, followed by the subsequent selection of nearby “opportunistic” acceptor or donor sites. Alternatively, other frequent mechanisms leading to pseudoexon activation involve the creation of enhancer or loss of silencer splicing regulatory elements. Conversely, the trans-acting factors involved in pseudoexon inclusion are less known, although hnRNP proteins seem to have an important modifier role [16]. We previously described a deep-intronic homozygous mutation (IVS6-320A.T) that causes the inclusion of a 75-bp pseudoexon between exons 6 and 7 of the fibrinogen gamma-chain gene (FGG) transcript in a patient affected by congenital afibrinogenemia [17]. This mutation reinforces a pre-existing cryptic donor splice site by extending its complementarity to U1snRNA, eventually resulting in the activation of a 
pseudoexon. We also suggested that, apart from the cryptic splice-site activation, the modulation of normally silent regulatory elements could also play a role in this mutationinduced pseudoexon inclusion [17]. In the present work, we address this issue by functionally dissecting both the cis-acting elements and the trans-acting regulatory factors that contribute to the regulation of this pseudoexon insertion event.sequence similar to the cryptic one -which is totally neglected by the splicing machinery in the wild-type context- suggested the existence of splicing regulatory mechanisms modulating the inclusion of this pseudoexon (Figure S1A). This prompted us to investigate in more detail the in-cis and in-trans elements involved in this pseudoexon activation/suppression.hnRNP F Regulates Pseudoexon Inclusion in the FGG mRNAAs a first step in the study of regulatory elements controlling pseudoexon inclusion, we analyzed the 75-bp pseudoexon sequence and noticed the presence of three G-runs motifs (named G1, G2, and G3): two are located at the 59 of the pseudoexon (positions 21 to +4 and +13/15), the third towards the 39 end (position 60?2) (Figure 1). As hnRNP H and F are known to bind G-runs, acting either as splicing-enhancer or splicing-inhibitory factors depending on gene and cellular context [8,18,19], we explored their effect on FGG pseudoexon inclusion by performing siRNA-mediated silencing of the two proteins (Figure 2A). The pT-FGG-IVS6-320A.T minigene (containing the mutant IVS6320A.T FGG genomic region spanning 1,858 bp from intron 4 to intron 7, cloned into the pTargeT vector) [17] was thus cotransfected into HeLa cells (not expressing fibrinogen) with siRNAs against hnRNP F or hnRNP H. The efficacy of protein knockdowns was verified and quantitated by Western blotting (Figure 2A, left and central panels). Interestingly, real-time AKT inhibitor 2 biological activity RTPCRs showed that knockdown of hnRNP H results in a SPDB manufacturer nonsignificant increase of pseudoexon inclusion, 
whereas hnRNP F depletion significantly represses pseudoexon recognition (Figure 2A, right panel). A similar result was found after double knockdown of hnRNP F and H (data not shown), suggesting a prominent role of hnRNP F in the modulation of FGG pseudoexon splicing. The lack of response to 16574785 hnRNP H might raise the question whether a sufficient level of knockdown of this protein was obtained. However, silencing of hnRNP H was performed using exactly the same protocol and reaching the same level of silencing (85 ) that we previously showed to determine the activation of a cryptic acceptor splice site in the thrombopoietin gene [20]. In complementary experiments, the overexpression of hnRNP F resul.Ence, followed by the subsequent selection of nearby “opportunistic” acceptor or donor sites. Alternatively, other frequent mechanisms leading to pseudoexon activation involve the creation of enhancer or loss of silencer splicing regulatory elements. Conversely, the trans-acting factors involved in pseudoexon inclusion are less known, although hnRNP proteins seem to have an important modifier role [16]. We previously described a deep-intronic homozygous mutation (IVS6-320A.T) that causes the inclusion of a 75-bp pseudoexon between exons 6 and 7 of the fibrinogen gamma-chain gene (FGG) transcript in a patient affected by congenital afibrinogenemia [17]. This mutation reinforces a pre-existing cryptic donor splice site by extending its complementarity to U1snRNA, eventually resulting in the activation of a pseudoexon. We also suggested that, apart from the cryptic splice-site activation, the modulation of normally silent regulatory elements could also play a role in this mutationinduced pseudoexon inclusion [17]. In the present work, we address this issue by functionally dissecting both the cis-acting elements and the trans-acting regulatory factors that contribute to the regulation of this pseudoexon insertion event.sequence similar to the cryptic one -which is totally neglected by the splicing machinery in the wild-type context- suggested the existence of splicing regulatory mechanisms modulating the inclusion of this pseudoexon (Figure S1A). This prompted us to investigate in more detail the in-cis and in-trans elements involved in this pseudoexon activation/suppression.hnRNP F Regulates Pseudoexon Inclusion in the FGG mRNAAs a first step in the study of regulatory elements controlling pseudoexon inclusion, we analyzed the 75-bp pseudoexon sequence and noticed the presence of three G-runs motifs (named G1, G2, and G3): two are located at the 59 of the pseudoexon (positions 21 to +4 and +13/15), the third towards the 39 end (position 60?2) (Figure 1). As hnRNP H and F are known to bind G-runs, acting either as splicing-enhancer or splicing-inhibitory factors depending on gene and cellular context [8,18,19], we explored their effect on FGG pseudoexon inclusion by performing siRNA-mediated silencing of the two proteins (Figure 2A). The pT-FGG-IVS6-320A.T minigene (containing the mutant IVS6320A.T FGG genomic region spanning 1,858 bp from intron 4 to intron 7, cloned into the pTargeT vector) [17] was thus cotransfected into HeLa cells (not expressing fibrinogen) with siRNAs against hnRNP F or hnRNP H. The efficacy of protein knockdowns was verified and quantitated by Western blotting (Figure 2A, left and central panels). Interestingly, real-time RTPCRs showed that knockdown of hnRNP H results in a nonsignificant increase of pseudoexon inclusion, whereas hnRNP F depletion significantly represses pseudoexon recognition (Figure 2A, right panel). A similar result was found after double knockdown of hnRNP F and H (data not shown), suggesting a prominent role of hnRNP F in the modulation of FGG pseudoexon splicing. The lack of response to 16574785 hnRNP H might raise the question whether a sufficient level of knockdown of this protein was obtained. However, silencing of hnRNP H was performed using exactly the same protocol and reaching the same level of silencing (85 ) that we previously showed to determine the activation of a cryptic acceptor splice site in the thrombopoietin gene [20]. In complementary experiments, the overexpression of hnRNP F resul.
