Targeted m⁶A Reader Proteins To Study Epitranscriptomic Regulation of Single RNAs

Area of Science:

• Molecular Biology
• Epigenetics
• RNA Biology

Background:

• Post-transcriptional gene regulation is crucial for mammalian protein production.
• Chemical modifications of RNA, like N6-methyladenosine (m6A), are key regulatory mechanisms.
• The interplay of m6A regulators (writers, erasers, readers) at the single-transcript level remains unclear.

Purpose of the Study:

• To develop a tool for investigating the regulatory effects of specific m6A readers on single RNA transcripts in live cells.
• To elucidate the competitive interplay of m6A regulators in controlling RNA fate.

Main Methods:

• Development of programmable dPspCas13b-m6A reader proteins by fusing catalytically inactive PspCas13b to m6A reader proteins (YTHDF1, YTHDF2).
• Utilizing guide RNA (gRNA) for targeted delivery of reader proteins to specific RNA of interest.
• Demonstration of reader protein function on reporter constructs and endogenous mRNA targets.

Main Results:

• Fused reader proteins (YTHDF2, YTHDF1) retained their known functions: YTHDF2 induced RNA degradation, and YTHDF1 enhanced translation.
• The system successfully targeted endogenous mRNA transcripts.
• Tethering YTHDF2 to endogenous mRNA led to its decay.

Conclusions:

• dPspCas13b-based tools enable transcript-specific studies of RNA regulation in live cells.
• These tools enhance understanding of RNA regulation at the single-transcript level.
• The developed tools have potential applications in synthetic biology for controlling gene expression via RNA manipulation.