Secondary Structure Analysis by SHAPE‐MaP of the EGFR pre‐mRNA Transcript to Identify Target Regions for RNA Anti‐sense Therapies

Abstract

Glioblastoma Multiforme (GBM) is the most common primary brain malignancy with a median survival of 10–14 months. A common aberration in GBM is the overexpression of epidermal growth factor receptor (EGFR), dysregulated in 57% of all GBM. Standard care increases survival to only 14–15 months indicating a necessity to generate novel therapeutics. Our strategy is to deliver a gene directly to the CNS using an adeno‐associated virus gene transfer vector encoding antisense RNA therapeutics. This therapy targets critical splicing elements surrounding Intron 10 of the EGFR pre‐mRNA transcript. Cryptic poly‐a signals within intron 10 have the potential to activate the expression of a therapeutic isoform of EGFR. Alternative splicing and polyadenylation is regulated by secondary structure of the pre‐mRNA transcript. To improve our therapeutic strategy, we have analyzed the EGFR secondary structure using selective 2′ hydroxyl acylation and primer extension followed by mutational profiling (SHAPE‐MaP). The SHAPE reagent 1M7 reacts with the 2′ hydroxyl of RNA molecules when the RNA molecule is in a conformationally flexible position creating a 2′ O‐adduct. The modified RNA is reverse transcribed, incorporating mismatches at the acylated positions; a comparison of unmodified to modified RNA allows us to determine RNA nucleotides that are involved in secondary structure, part of RNA‐binding‐protein complexes, or single stranded. The EGFR DNA sequence of Exon 10, Intron 10, Exon 11 was cloned into a plasmid. The plasmid was PCR amplified with a forward T7 promoter primer and reverse primer corresponding to exon 11, this generated the template for T7 RNA transcription. RNA was T7 transcribed and purified by RNA gel electrophoresis. RNA was treated with 1M7 (+) or DMSO (−), reverse transcribed under SHAPE conditions with Superscript II then converted to cDNA. Reverse transcription conditions for SHAPE experiments incorporate Mn++ as the divalent cation, which allows the enzyme to incorporate nucleotide mismatches. 3.5 million reads generated using Nanopore MinION sequencing corresponded to (+) and (−) conditions with a mean read length of 936 nucleotides and a quality score of 9.7. Sequencing output was run through the ShapeMapper2 pipeline. The median read depth at each nucleotide was at least 92K for all conditions after elimination of reads under quality control score of 15. SHAPE‐MaP reactivity profiles were generated using RNAfold. Splicing elements within the targeted region were identified and compared to reactivity profiles. SRp40 binding motifs show high accessibility upstream of the 5′ splice site. The exon‐intron junction of the 5′ splice site is structurally available while the 3′ intron‐exon junction shows moderate accessibility. We identified a putative poly‐a signal exists within intron 10 and the literature shows expression of this shortened EGFR isoform in various cell types. Importantly, this cryptic poly‐a signal shows no reactivity indicating high secondary structures blocking recognition by the poly‐a machinery. Based on these structural profiles, we generated RNA antisense molecules to unravel the hidden poly‐a signal to activate expression of the short isoform. In conjunction, we used eCLIP to identify target specific RNA‐binding proteins.