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

Singh, Natalia
Hollinger, Katrin
Singh, Ravindra
Bhattacharya, Dhruva
Singh, Ravindra
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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 (10C), which is neither conserved nor associated with any known splicing motif. Significance of 10C 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 10C and yet promoted SMN2 exon 7 skipping. Another 14-mer ASO (F14) that sequestered both, 10C and adjacent hnRNP A1 motifs, led to a strong stimulation of SMN2 exon 7 inclusion. The inhibitory role of 10C 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 10C 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.

<p>This is an article from <em>RNA</em> 16 (2010): 1167, doi:<a href="" target="_blank">10.1261/rna.2154310</a>. Posted with permission.</p>
SMN, alternative splicing, intron 7, exon 7, ISS-N1, GC-rich, antisense oligonucleotide, ASO, SMA