Exploiting Co-Transcriptional Folding and Processing of Nascent Messenger RNA for Modulating Specific Exon Splicing

Abstract

RNA splicing, which involves intron removal and exon ligation, is a key step that transforms eukaryotic nascent messenger RNA (pre-mRNA) into mature mRNA. The ability to modulate inclusion or exclusion of specific exons to elicit switching between alternatively spliced variants, restoration or disruption codon-reading frame, and correcting aberrant splicing has applications for both biomedical research and therapy. However, the identification of potent pre-mRNA sites, that when targeted or masked via therapeutics efficiently induce specific exon splicing, remains a challenge. As the transcription elongation of a pre-mRNA, exon recognition and exon splicing occur simultaneously, we hypothesized that the dynamics of co-transcriptional pre-mRNA folding could be a key factor in determining the accessibility of target sites. By analyzing all possible optimal and sub-optimal local secondary structures of the pre-mRNA at each step of transcriptional analysis, we predicted the co-transcriptional accessibility profile and identified possible target sites that have the highest probability of being accessible throughout transcription process. To test the hypothesis, we used glycine decarboxylase (GLDC), an ‘oncogenic protein’ overexpressed in metastatic lung cancer cells, as a test case. Ten target sites each to induce specific exclusion of one of the two targeted exons of the GLDC gene, were identified to disrupt the codon reading frame to induce nonsense-mediated decay. In our wet experiments on NSCLC (non-small cell lung carcinoma) cell lines, specific exon exclusion were observed when each of the ten sites was masked; of these, three sites induced exon exclusion in more than 80% of total pre-mRNA. Notably, two sites predicted to be ineffective were validated to be so. Our results suggest that co-transcriptional binding accessibility can be used to aid the rational identification of potent target sites for modulating specific exon splicing.