RNA structure is a key regulatory element in pathological ATM and CFTR pseudoexon inclusion events

Abstract

Genomic variations deep in the intronic regions of pre-mRNA molecules are increasingly reported to affect splicing events. However, there is no general explanation why apparently similar variations may have either no effect on splicing or cause significant splicing alterations. In this work we have examined the structural architecture of pseudoexons previously described in ATM and CFTR patients. The ATM case derives from the deletion of a repressor element and is characterized by an aberrant 5′ss selection despite the presence of better alternatives. The CFTR pseudoexon instead derives from the creation of a new 5′ss that is used while a nearby pre-existing donor-like sequence is never selected. Our results indicate that RNA structure is a major splicing regulatory factor in both cases. Furthermore, manipulation of the original RNA structures can lead to pseudoexon inclusion following the exposure of unused 5′ss already present in their wild-type intronic sequences and prevented to be recognized because of their location in RNA stem structures. Our data show that intrinsic structural features of introns must be taken into account to understand the mechanism of pseudoexon activation in genetic diseases. Our observations may help to improve diagnostics prediction programmes and eventual therapeutic targeting.

Novel roles of U1 snRNP in alternative splicing regulation

“…Very recently, the intronic "splicing code" that regulates inclusion of this pseudoexon has been further investigated and shown to involve several factors beside U1snRNP such as RNA secondary structure and trans-acting proteins as SF2/ASF. 68,69 In a second case, binding of hnRNP E1 and U1snRNP to a weak 5' splice site was observed to efficiently silence pseudoexon inclusion in the GHR gene, although the exact mechanism still remains unknown, 70 thus preventing the development of Laron syndrome (Fig. 3C).…”

An antisense microwalk reveals critical role of an intronic position linked to a unique long-distance interaction in pre-mRNA splicing

“…Methods to identify splicing motifs are continuing to evolve (Chasin 2007;Singh 2007a;Hertel 2008;Wang and Burge 2008;Yu et al 2008). An additional regulatory role is provided by RNA structures that enforce accessibility to splicing elements, as well as bring two distantly located cis-elements in close proximity (Graveley 2005;Buratti et al 2007;Singh et al 2007;Shepard and Hertel 2008;Warf et al 2009). Unraveling the mechanism by which splicing factors, RNA regulatory sequences, and structural motifs coordinate to regulate alternative splicing is an area of growing interest for evolving strategies to cure many human diseases associated with defective splicing (Garcia-Blanco 2006;Cooper et al 2009;Tazi et al 2009;Ward and Cooper 2010).…”

Pure intronic rearrangements leading to aberrant pseudoexon inclusion in dystrophinopathy: a new class of mutations?

“…Despite extensive efforts made to elucidate the splicing code [Barash et al, 2010], the factors that drive splicing decisions and allow differentiation of exons from long flanking introns are far from being understood. Pre-mRNA secondary structure is increasingly recognized as a general modifier of splicing events, and in particular, would play a role in helping the splicing machinery to distinguish between real exons and PE sequences [Buratti et al, 2007]. Conserved stem-loop regions within introns can regulate donor-site usage and splicing efficiency as reported for ATM and CFTR PEs [Buratti et al, 2007] or for tau exon 10 alternative splicing [Donahue et al, 2006].…”

“…Pre-mRNA secondary structure is increasingly recognized as a general modifier of splicing events, and in particular, would play a role in helping the splicing machinery to distinguish between real exons and PE sequences [Buratti et al, 2007]. Conserved stem-loop regions within introns can regulate donor-site usage and splicing efficiency as reported for ATM and CFTR PEs [Buratti et al, 2007] or for tau exon 10 alternative splicing [Donahue et al, 2006]. Stem-loop variants that destabilize this structure result in increased splicing of tau exon 10 and contribute to neurodegenerative disorders [Liu and Gong, 2008].…”