title: An antisense microwalk reveals critical role of an intronic position linked to a unique long-distance interaction in pre-mRNA splicing.
abstract: Here we report a novel finding of an antisense oligonucleotide (ASO) microwalk in which we examined the position-specific role of intronic residues downstream from the 5' splice site (5' ss) of SMN2 exon 7, skipping of which is associated with spinal muscular atrophy (SMA), a leading genetic cause of infant mortality. Our results revealed the inhibitory role of a cytosine residue at the 10th intronic position ((10)C), which is neither conserved nor associated with any known splicing motif. Significance of (10)C emerged from the splicing pattern of SMN2 exon 7 in presence of a 14-mer ASO (L14) that sequestered two adjacent hnRNP A1 motifs downstream from (10)C and yet promoted SMN2 exon 7 skipping. Another 14-mer ASO (F14) that sequestered both, (10)C and adjacent hnRNP A1 motifs, led to a strong stimulation of SMN2 exon 7 inclusion. The inhibitory role of (10)C was found to be tightly linked to its unpaired status and specific positioning immediately upstream of a RNA:RNA helix formed between the targeting ASO and its intronic target. Employing a heterologous context as well as changed contexts of SMN2 intron 7, we show that the inhibitory effect of unpaired (10)C is dependent upon a long-distance interaction involving downstream intronic sequences. Our report furnishes one of the rare examples in which an ASO-based approach could be applied to unravel the critical role of an intronic position that may not belong to a linear motif and yet play significant role through long-distance interactions.
authors.affiliation: Department of Biomedical Sciences, College of Veterinary Medicine, Iowa State University, Ames, Iowa 50011, USA.
authors.forename: Natalia N
authors.initials: NN
authors.lastname: Singh
authors.forename: Katrin
authors.initials: K
authors.lastname: Hollinger
authors.forename: Dhruva
authors.initials: D
authors.lastname: Bhattacharya
authors.forename: Ravindra N
authors.initials: RN
authors.lastname: Singh
mesh_terms: D020029:Base Pairing; D001483:Base Sequence; D005075:Biological Evolution; D005091:Exons / Q000235:genetics; D015894:Genome, Human; D034441:Heterogeneous-Nuclear Ribonucleoproteins / Q000235:genetics*; D006801:Humans; D007438:Introns / Q000235:genetics; D009154:Mutation; D016376:Oligonucleotides, Antisense; D012322:RNA Precursors / Q000235:genetics*; D012326:RNA Splicing; D016372:RNA, Antisense / Q000235:genetics*; D017384:Sequence Deletion; D055540:Survival of Motor Neuron 2 Protein / Q000235:genetics
chemical_list: D034441:Heterogeneous-Nuclear Ribonucleoproteins; D016376:Oligonucleotides, Antisense; D012322:RNA Precursors; D016372:RNA, Antisense; C526477:SMN2 protein, human; D055540:Survival of Motor Neuron 2 Protein