Sequence-dependent structural dynamics of primate adenosine-to-inosine editing substrates

Area of Science:

• Molecular Biology
• Genetics
• Biophysics

Background:

• Humans exhibit higher adenosine-to-inosine (A-to-I) RNA editing levels compared to other primates.
• The underlying molecular mechanisms driving this difference are not fully understood.
• Alu Sg elements are key substrates for A-to-I RNA editing.

Purpose of the Study:

• To investigate the role of sequence variations in Nup50 Alu Sg elements in differential A-to-I RNA editing across primates.
• To characterize the structural and dynamic properties of human, chimp, and rhesus Nup50 Alu Sg RNA substrates.
• To correlate RNA structural dynamics with the efficiency of adenosine deaminase acting on RNA (ADAR) binding and catalysis.

Main Methods:

• Sequence analysis of Nup50 Alu Sg elements in human, chimp, and rhesus.
• Construction and characterization of synthetic RNA substrates (HuAp5, ChAp5, RhAp5) using 2-aminopurine (2-Ap) substitution for fluorescence monitoring.
• Thermal melting (T(M)) analysis using UV and steady-state fluorescence (SSF) to assess RNA stability.
• Time-resolved fluorescence (TRF) spectroscopy to resolve RNA folding dynamics and identify conformational states.

Main Results:

• Subtle sequence variations exist in Nup50 Alu Sg elements among humans, chimps, and rhesus monkeys.
• Human RNA constructs exhibit higher thermal stability (T(M) ~70°C) compared to chimp (T(M) ~60°C) and rhesus (T(M) ~54°C).
• TRF analysis revealed three distinct fluorescence lifetimes, supporting a two-intermediate model for RNA folding and unfolding dynamics.
• At physiological temperature (37°C), the human construct favors a more populated folded state and intermediate J, conducive to ADAR enzyme activity.

Conclusions:

• Sequence variations in Nup50 Alu Sg elements significantly impact RNA structure and dynamics.
• The enhanced stability and specific conformational states of human RNA substrates facilitate efficient A-to-I editing by ADAR.
• These findings suggest that localized RNA sequence differences are a key factor contributing to the elevated A-to-I editing levels observed in humans.