Optimal antisense target reducing INS intron 1 retention is adjacent to a parallel G quadruplex

Area of Science:

• Molecular Biology
• RNA Biology
• Gene Expression Regulation

Background:

• Splice-switching oligonucleotides (SSOs) are typically used to inhibit exon usage.
• Antisense strategies to promote intron removal and enhance gene expression have not been developed.
• A specific human haplotype exhibits low proinsulin expression due to inefficient intron 1 splicing.

Purpose of the Study:

• To develop and characterize antisense strategies using SSOs to promote intron removal and increase gene expression.
• To identify and validate a specific target motif for SSO binding that reduces intron 1 retention.
• To investigate the structural and mechanistic basis of SSO-mediated intron removal.

Main Methods:

• Utilized splice-switching oligonucleotides (SSOs) targeting specific motifs in human INS intron 1.
• Employed reporter constructs and antisense microwalks to map optimal SSO target sites.
• Applied circular dichroism spectroscopy and nuclear magnetic resonance to analyze RNA structures (G-quadruplexes, hairpin-loops).
• Investigated interactions with heterogeneous nuclear ribonucleoproteins (hnRNPs) F and H.

Main Results:

• Demonstrated SSO-mediated reduction of INS intron 1 retention in a low proinsulin-producing human haplotype.
• Identified a specific splicing silencer motif, targeted by SSOs, that significantly reduces intron 1-containing mRNAs.
• Characterized the formation of stable G-quadruplex structures and hairpin-loops at the target site, influencing SSO binding.
• Revealed potential interference by hnRNPs F and H with conformational transitions at the target site.

Conclusions:

• Developed a novel antisense strategy using SSOs to promote weak intron removal from the 5' UTR, enhancing gene expression.
• The findings suggest that targeting competing RNA structures with SSOs can facilitate intron removal.
• This approach offers a new avenue for developing strategies to enhance gene expression, particularly for conditions associated with inefficient splicing.