Splicing Moclulation Of Spinal,nuscular Atrophy-associated Gene SMN2 By Cis-acting Regulatory Elements

Background and objective:RNA splicing is a key step in the post transcriptional process of eukaryotic gene expression.It has been found that more than 95%human genes undergo alternative splicing,giving rise to different coding transcripts.The key step in RNA splicing is to accurately identify splice sites,which relies on complex interplays between various cis-acting elements and their cognate trans-acting factors.Therefore,study of cis-acting elements and their binding proteins is important in the understanding regulation of RNA splicing.However,studies on SREs are highly limited,particularly considering that there are over 1000 RNA binding proteins(RBPs)and their specific binding motifs and potential effects on alternative splicing are largely unknown.More importantly,many splicing-assocated RBPs are linked to human diseases including motor neuron degeneration diseases and more recently various types of cancer.Therefore,our project will not only help find valuable cis-acting elements and their mechansims in regulating alternative splicing but also provide a theoretical foundation for future therapeutical intervention.Methods:Using the spinal muscular atrophy(SMA)-related gene SMN2 as an alternative splicing model,we first built a pentameric motif library at the site downstream the 5' splice site of intron 7 using mutagenesis in a previously described SMN2 minigene.All 2014 minigene plasmids were individually transfected into HEK293 cells and their splicing patterns were analyzed by fluorescence-labelled RT-PCR.Data was assessed with computational tool GESA in collaboration with Cold Spring Harbor Laboratory to identify top splicing stimulatory or inhibitory 3-bp and-4 bp motifs in both position-dependend and independent manners.The leading motifs along their flanking sequences were validated and characterized in different positons in SMN2 intron 7 and in the context of other alternatively spliced genes.Results:A complete pertameric RNA splicing library was established in the context of the SMN2 minigene.The splicing patterns of all minigenes with all 1024 different motifs were defined,providing a valuable source for future studies on alternative splicing regulation.Top 20 inhibitory and stimulatory motifs were identified using GESA.Validation of these top motifs along with flanking sequences uncovered several key common splicing regulatory motifs.We characterized a few of them including two inhibitory SREs(TGCACC and GCAAACCTT)as well as two stimulatory SREs(poly-C and poly-T).Our data also verified a long-distance RNA-RNA interation that contributes SMN2 exon 7 skipping.Meanwhile,our data helped optimize previously-established SREs.For example,we found that UGCAUGC is a stronger version of UGCAUG,the core motif for RNA binding protein Rbfoxl.Further characterization of the SREs identified in our study are still underway and will be our future directions in studying regulation of alternative splicing.Conclusion:We have for the first time established a complete library of a pentameric SRE library.The splicing patterns of all 1024 motifs have been defined.Top 20 inhibitory and stimulatory motifs have been identified.Several key SREs have been characherized including two strong inhibitory SREs TGCACC and GCAAACCTT and stimulatory SREs Poly-C and Poly-T.Our data also confirms an inhibitory nature of long distance RNA-RNA interaction and optimized a previously-defined stimulatory SRE recognized by RNA-binding protein Rbfoxl.