Targeted blocking of gene splicing can dysregulate intron-embedded primary microRNAs

Addressing whether antisense oligonucleotides (ASOs)-based targeting of genes embedded with intronic noncoding RNAs (ncRNAs) affects the expression and function of intronic ncRNAs is crucial to the success of ASOs in clinical trials. While studying zebrafish posterior pituitary development-an important neuroendocrine interface-we found that an ASO targeting the slit3 splice site, but not the translation start site, disrupts pituitary axonal morphogenesis. In addition to altered slit3 splicing, we observed increased expression of slit3, its intron-embedded microRNA mir-218a-1, its longest intron, and the paralogous slit2 gene. The ASO-induced phenotype does not occur when mature mir-218a-1 is blocked by an ASO or in mir-218a-1^-/- mutants, or when the splicing of the exon immediately upstream of the mir-218a-1 embedded intron is blocked by an ASO. The axonal phenotype also phenocopies in samples injected with mir-218a mimic in a dose-dependent manner. Our results indicate that despite the ASO-induced genetic compensation response, intron-retained transcripts can escape the nonsense-mediated decay (NMD) machinery, become stabilized, and lead to increased intronic primary microRNA expression and function. As prematurely terminated intron-retained transcripts can translocate to axons and affect neuronal function, our study warrants further validation for other classes of ncRNAs. Moreover, the idiosyncratic phenotypes observed with translation- versus splice-blocking ASOs can serve as markers to identify the role of intronic microRNAs.