Using In Vitro and In-cell SHAPE to Investigate Small Molecule Induced Pre-mRNA Structural Changes

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• Elucidating RNA structural changes is crucial for developing RNA-targeting small molecules.
• Spinal muscular atrophy (SMA) is a genetic disorder caused by mutations in the survival motor neuron (SMN) gene.
• The SMN gene produces the SMN protein, essential for motor neuron survival.

Purpose of the Study:

• To detail and compare in vitro and in-cell selective 2 -hydroxyl acylation analyzed by primer extension (SHAPE) protocols.
• To investigate RNA structural changes in the presence of the experimental SMA drug, SMN-C2.
• To analyze the structure of exon 7 of the SMN2 gene pre-mRNA.

Main Methods:

• In vitro SHAPE: RNA transcription, folding with SMN-C2, acylation with NAI, primer extension, and PAGE analysis.
• In-cell SHAPE: In situ acylation in living cells, PCR amplification, and next-generation sequencing.
• Comparative analysis of the two SHAPE methodologies regarding cost, accuracy, and scalability.

Main Results:

• In vitro SHAPE is cost-effective but may not fully represent cellular RNA structures due to missing protein interactions.
• In-cell SHAPE provides a more accurate cellular RNA secondary structure without radioactive materials and is scalable to ~1,000 nucleotides.
• Both in vitro and in-cell SHAPE yielded similar RNA models for SMN2 pre-mRNA exon 7.

Conclusions:

• In-cell SHAPE offers advantages in accuracy and scalability for studying RNA structures in a cellular context.
• In vitro SHAPE remains a viable, cost-effective option for initial RNA structure analysis.
• The study provides valuable insights into RNA structural dynamics relevant to SMA drug development.