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

作者：Singh Natalia N; Hollinger Katrin; Bhattacharya Dhruva; Singh Ravindra N^*

来源：RNA-A Publication of the RNA Society, 2010, 16(6): 1167-1181.

DOI：10.1261/rna.2154310

• SMN
• alternative splicing
• intron 7
• exon 7
• ISS-N1
• GC-rich
• antisense oligonucleotide
• ASO
• SMA

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.

• 出版日期2010-6