Targeting alternative splicing by RNAi: from the differential impact on splice variants to triggering artificial...

Area of Science:

• Molecular Biology
• Genetics
• Post-transcriptional Regulation

Background:

• Alternative splicing generates diverse transcript isoforms with varying functions.
• RNA interference (RNAi) is a key gene expression regulator, but its interplay with alternative splicing is unclear.
• Nonsense-mediated mRNA decay (NMD) and mRNA export influence transcript fate.

Purpose of the Study:

• To investigate the interactions between RNA interference (RNAi) and alternative splicing.
• To determine how splice variant characteristics affect RNAi efficacy.
• To explore the cross-kingdom conservation of these regulatory mechanisms.

Main Methods:

• Utilized artificial microRNAs (amiRNAs) for gene knockdown in Arabidopsis thaliana.
• Analyzed differential effects of amiRNAs on splice variants with varying protein-coding capacity, localization, and NMD sensitivity.
• Employed siRNAs in mammalian cells to assess cross-kingdom conservation.

Main Results:

• Splice variants differentially responded to amiRNA-mediated knockdown, even with shared target sites.
• Nuclear-localized and NMD-sensitive isoforms escaped degradation, masking RNAi activity.
• Similar regulatory patterns were observed in Arabidopsis SPL genes and mammalian cells, indicating conserved mechanisms.
• Artificial microRNAs demonstrated the ability to induce alternative splicing.

Conclusions:

• Nuclear localization and NMD sensitivity of splice variants can impede RNAi efficacy.
• Interactions between RNAi, alternative splicing, and NMD are crucial for post-transcriptional gene regulation.
• These findings reveal conserved cross-kingdom mechanisms governing transcript fate and gene expression.