Structural imprints in vivo decode RNA regulatory mechanisms

Area of Science:

• Molecular Biology
• Genomics
• Biochemistry

Background:

• Visualizing molecular behavior within living cells is a significant biological challenge.
• RNA structure is critical for gene expression regulation, but current in vivo methods offer incomplete profiles.
• Understanding physiological RNA structures is limited, with existing techniques covering only two of the four RNA bases.

Purpose of the Study:

• To present a novel biochemical approach for global RNA secondary structure profiling in living cells.
• To enable the first comprehensive view of all four RNA bases' structures within a cellular environment.
• To investigate the differences between in vivo and in vitro RNA structural dynamics.

Main Methods:

• Development and application of in vivo click selective 2 -hydroxyl acylation and profiling experiment (icSHAPE).
• Global transcriptome-wide RNA structure profiling in mouse embryonic stem cells.
• Comparison of RNA structures obtained in vivo versus in vitro.

Main Results:

• icSHAPE provides the first global view of RNA secondary structures for all four bases in living cells.
• In vivo RNA structural dynamics differ from in vitro conditions, distinguishing RNA classes and regulatory elements.
• Structural signatures at translational and ribosome pause sites are conserved, indicating sequence programming.
• Dynamic structural footprints reveal RNA-protein interaction interfaces and RNA modification sites, consistent with atomic data.
• Accurate genome-wide prediction of RNA-protein interactions and N(6)-methyladenosine (m(6)A) modification is enabled.

Conclusions:

• icSHAPE technology opens new avenues for RNA structural genomics in living cells.
• Physiological RNA structures play key roles in controlling gene expression.
• The study reveals precise RNA structural dynamics that mediate interactions with proteins and modifications.