Molecular basis of the human ApoAII exon 3 splicing

Among human genes, CFTR intron Vlll/exon 9 and apolipoprotein All intron Il/exon 3 boundaries share the characteristic feature given by the presence of a peculiar tract of alternating pyrimidines and purines close to the 3’ splice site. In the case of CFTR gene, the pyrimidine rich tract is composed by a stretch of Ts in a row and a stretch of alternating pyrimidines and purines (microsatellite TG dinucleotide repeats) and both tracts are polymorphic for their length. On the other hand, in the case of ApoAII gene the pyrimidine rich tract is made exclusively of alternating pyrimidines and purines (microsatellite TG dinucleotide repeats) and it is also polymorphic for its length in both genes. This apparent sequence similarity, concerning the TG tract, is contrasted by the different splicing pattern exhibited by the two genes. In fact, CFTR exon 9 undergoes alternative splicing to a variable extent, depending on the variations in length of the pyrimidine rich tract, whereas ApoAII exon 3 is constitutively included in mRNA. Previous studies of our group have shown in the CFTR intron Vlll/exon 9 context, the stretch of pyrimidines alternated with purines (within the UG tract) alone is not equivalent to a functional continuous polypyrimidine tract, contrarily to what has been observed for the apolipoprotein All gene. Moreover, the comparison of splice sites strength of human ApoAII exon 3 and human CFTR exon 9 has outlined an apparent contradiction between the splicing behavior of the two exons and the strength of the splice sites. In fact, both the 3’ and the 5’ splice sites of CFTR exon 9 display a good match with the consensus whereas the match of the ApoAII exon 3 splice sites is not good. Altogether these observations prompted us to investigate the mechanisms underlying the constitutive splicing of ApoAII exon 3 and, in particular, to characterize the cis-acting elements and the trans-acting factors involved in ApoAII exon 3 definition to assure its constitutive splicing. In order to study in vivo the splicing mechanism of ApoAII exon 3, we set up an eukaryotic expression system by cloning the whole ApoAII gene, from its promoter to the poly-A signal. Then, the effects of point mutations, deletions or substitutions on splicing of exon 3 were analyzed by RT-PCR after transient transfection in Hep3B cell line. Deletion or replacement of the UG repeats at the 3’ splice site of intron 2 resulted in a significant increase in exon 3 skipping, indicating the importance of this alternated arrangement of U and G as a functional polypyrmidine tract or at least as an important sequence able to lead the exon 3 definition. Furthermore, UV-crosslinking assays showed that the (UG)I6 repeats of ApoAII intron 2 are recognized by TDP-43, a protein that binds specifically the UG tract within the context of the 3' end of CFTR intron VIII and that affects negatively CFTR exon 9 splicing. Next, we characterized the exonic cis-acting elements able to affect the splicing efficiency of ApoAII exon 3. Transient transfections of different constructs of the ApoAII gene system carrying deletions or point mutations showed that the region spanning from nucleotide 87 to 113 of human ApoAII exon 3 is important for its close to the 5’ splice site. In order to identify trans-acting factor/s able to bind the 9nt-ESE within ApoAII exon 3, both Electro Mobility Shift Assay (EMSA) and UV-crosslinking coupled to immunoprécipitation assays were carried out. EMSA showed a broad band of shifted material with the ESEwt RNA, whereas no significant shift was seen with mutated ESE RNA. This suggested that one or more proteins interact specifically with the wild type ApoAII exon 3 across the 9nt-sequence. Then, UV-crosslinking followed by immunoprécipitation with monoclonal antibodies anti-SR proteins ASF/SF2 and anti-SC-35 showed that ESEwt but not mutated ESE RNA was able to immunoprecipitate a band whose molecular weight corresponds to that of ASF/SF2 and, even if to a lower extent, also a band whose molecular weight corresponds to that of SC35. Thus, these results provided an evidence that at least two SR proteins are able to interact with the sequence across the ESE sequence. Subsequently, the relevance of ESE position within an internal intron (and therefore with other flanking cis-acting elements) was also tested. Both in vitro and in vivo experiments showed that the ApoAII exon 3 ESE works only if it is localized within an internal exon and not if it is placed within the first or the last exon. These results also suggested the presence of other splicing regulatory elements within the flanking intronic regions of ApoAII exon 3. Thus, to explore intron 2 and 3 for the presence of cis-acting elements able to affect the splicing of exon 3, a series of deletions within both introns were carried out. Thus, we found at least one regulatory element placed within intron 3 that regulates positively exon 3 inclusion. In conclusion, the constitutive splicing of ApoAII exon 3 seems to be the result of the balance between positive and negative action of the regulatory elements found in the exon 3 and its flanking introns. Future studies will be aimed at identifying the factors interacting with the intronic regulatory elements and at defining a possible model to explain the mechanism of ApoAII exon 3 constitutive splicing by integrating the network of interactions among the identified cis-acting elements and trans-acting factors

An exonic splicing enhancer offsets the atypical GU-rich 3' splice site of human apolipoprotein A-II exon 3.

Human apolipoprotein A-II (apoA-II) intron 2/exon 3 junction shows a peculiar tract of alternating pyrimidines and purines (GU tract) that makes the acceptor site deviate significantly from the consensus. However, apoA-II exon 3 is constitutively included in mRNA. We have studied this unusual exon definition by creating a construct with the genomic fragment encompassing the whole gene from apoA-II and its regulatory regions. Transient transfections in Hep3B cells have shown that deletion or replacement of the GU repeats at the 3′ splice site resulted in a decrease of apoA-II exon 3 inclusion, indicating a possible role of the GU tract in splicing. However, a 3′ splice site composed of the GU tract in heterologous context, such as the extra domain A of human fibronectin or cystic fibrosis transmembrane conductance regulator exon 9, resulted in total skipping of the exons. Next, we identified the exonic cis-acting elements that may affect the splicing efficiency of apoA-II exon 3 and found that the region spanning from nucleotide 87 to 113 of human apoA-II exon 3 is essential for its inclusion in the mRNA. Overlapping deletions and point mutations (between nucleotides 91 and 102) precisely defined an exonic splicing enhancer (ESEwt). UV cross-linking assays followed by immunoprecipitation with anti-SR protein monoclonal antibodies showed that ESEwt, but not mutated ESE RNA, was able to bind both alternative splicing factor/splicing factor 2 and SC35. Furthermore, overexpression of both splicing factors enhanced exon 3 inclusion. These results show that this protein-ESE interaction is able to promote the incorporation of exon 3 in mRNA and suggest that they can rescue the splicing despite the noncanonical 3′ splice site

Depletion of TDP 43 overrides the need for exonic and intronic splicing enhancers in the human apoA-II gene

Exon 3 of the human apolipoprotein A-II (apoA-II) gene is efficiently included in the mRNA although its acceptor site is significantly weak because of a peculiar (GU)(16) tract instead of a canonical polypyrimidine tract within the intron 2/exon 3 junction. Our previous studies demonstrated that the SR proteins ASF/SF2 and SC35 bind specifically an exonic splicing enhancer (ESE) within exon 3 and promote exon 3 splicing. In the present study, we show that the ESE is necessary only in the proper context. In addition, we have characterized two novel sequences in the flanking introns that modulate apoA-II exon 3 splicing. There is a G-rich element in intron 2 that interacts with hnRNPH1 and inhibits exon 3 splicing. The second is a purine rich region in intron 3 that binds SRp40 and SRp55 and promotes exon 3 inclusion in mRNA. We have also found that the (GU) repeats in the apoA-II context bind the splicing factor TDP-43 and interfere with exon 3 definition. Significantly, blocking of TDP-43 expression by small interfering RNA overrides the need for all the other cis-acting elements making exon 3 inclusion constitutive even in the presence of disrupted exonic and intronic enhancers. Altogether, our results suggest that exonic and intronic enhancers have evolved to balance the negative effects of the two silencers located in intron 2 and hence rescue the constitutive exon 3 inclusion in apoA-II mRNA