CAR-SPLASH identifies nascent pre-mRNA structures implicated in kinetic coupling and alternative splicing

Area of Science:

• Molecular Biology
• RNA Biology
• Gene Regulation

Background:

• Pre-messenger RNA (pre-mRNA) splicing is kinetically coupled to transcription, with transcription speed influencing alternative splicing (AS) outcomes.
• The precise molecular mechanisms underlying this kinetic coupling are not fully understood.
• A proposed mechanism involves elongation rate-dependent alternative folding of nascent pre-mRNA.

Purpose of the Study:

• To investigate RNA structures within nascent pre-mRNA that may mediate kinetic coupling to splicing.
• To identify and characterize novel regulatory elements controlling alternative splicing.
• To explore the potential of targeting these structures with antisense oligonucleotides (ASOs) for splice modulation.

Main Methods:

• Modification of Sequencing of Psoralen Crosslinked, Ligated And Selected Hybrids (SPLASH) to Chromatin Associated RNA (CAR)-SPLASH.
• Application of CAR-SPLASH to cells with wild-type and slow mutant RNA polymerase II.
• Disruption of identified RNA duplexes using antisense oligonucleotides (ASOs).

Main Results:

• Identification of over 3,000 intramolecular RNA duplexes in nascent pre-mRNA, with over 400 located near splice sites.
• ASO-mediated disruption of specific duplexes, termed 'RNA kinetic switches,' significantly impacted alternative splicing.
• These effects on AS of NISCH, GAK, and MEGF8 exons were dependent on transcription elongation rates.

Conclusions:

• RNA kinetic switches mediate kinetic coupling between transcription and splicing by modulating nascent RNA structure folding.
• Transcription speed influences RNA folding, which in turn affects alternative splicing outcomes.
• Nascent RNA structures represent potential targets for splice-modifying ASOs.