siRNA Mediate RNA Interference Concordant with Early On-Target Transient Transcriptional Interference

Area of Science:

• Molecular Biology
• Gene Regulation
• Gene Therapy

Background:

• Exogenous siRNAs are used for gene regulation but can target both cytosolic mRNA and nuclear nascent RNA, complicating specificity.
• Multiple synostosis syndrome type 4 (SYSN4) is associated with a disrupted non-coding gene TOSPEAK/C8orf37AS1 and downregulated GDF6.
• A conserved enhancer for GDF6 is located within TOSPEAK, which overlaps with the SMALLTALK/C8orf37 gene.

Purpose of the Study:

• To investigate the challenges in achieving siRNA target specificity.
• To elucidate the molecular mechanisms underlying gene dysregulation in SYSN4.
• To explore the potential of siRNA for gene therapy in SYSN4.

Main Methods:

• siRNA targeting of SMALLTALK in fibroblast cell lines.
• Analysis of gene expression levels of SMALLTALK, TOSPEAK, and GDF6 over time.
• Assessment of post-transcriptional gene silencing (PTGS/RNAi) and transcriptional interference.

Main Results:

• siRNA targeting SMALLTALK induced PTGS/RNAi of SMALLTALK and transiently increased TOSPEAK and GDF6 levels.
• The siRNA caused early transcriptional interference of SMALLTALK in the nucleus, leading to increased TOSPEAK and subsequently GDF6 transcription.
• siRNA targeting SMALLTALK successfully restored GDF6 levels in SYSN4 family fibroblasts in culture.

Conclusions:

• siRNA targeting can cause unintended transcriptional interference, affecting the expression of overlapping genes.
• Understanding transcriptional interference is crucial for designing effective and specific RNA and DNA targeting technologies like siRNA and CRISPR.
• The findings provide a basis for developing gene therapy strategies for SYSN4 by modulating GDF6 levels.