Tetracyclines Modify Translation by Targeting Key Human rRNA Substructures

Area of Science:

• Molecular Biology
• Pharmacology
• Cancer Research

Background:

• Tetracyclines possess known antimicrobial effects.
• Clinically validated anti-inflammatory and anti-cancer properties of tetracyclines are established.
• The precise molecular targets and mechanisms underlying these non-antimicrobial effects remain largely unknown.

Purpose of the Study:

• To identify the molecular targets of tetracyclines responsible for their anti-cancer effects.
• To elucidate the mechanisms by which tetracyclines exert anti-proliferative activity in human cancer cells.
• To investigate the interaction of tetracyclines with human ribosomes and their impact on translation and cellular stress responses.

Main Methods:

• Quantitative mass spectrometry-based proteomics to identify tetracycline targets.
• In-cell click selective crosslinking with RNA sequence profiling (icCL-seq) to map tetracycline binding sites on ribosomal RNA (rRNA).
• Analysis of tetracycline-induced changes in ribosomal translation and the integrated stress response (ISR).

Main Results:

• Human 80S ribosomes were identified as direct targets of tetracyclines (Col-3 and doxycycline).
• Specific binding sites for tetracyclines on key human rRNA substructures were mapped at nucleotide resolution using icCL-seq.
• Tetracycline analogs demonstrated chemical discrimination of these rRNA binding sites.
• Tetracyclines were found to subtly modify human ribosomal translation and selectively activate the integrated stress response (ISR).

Conclusions:

• Tetracyclines target specific rRNA substructures within human 80S ribosomes.
• Activation of the integrated stress response (ISR) and modulation of translation are key mechanisms.
• These ribosome-targeting effects, ISR activation, and translation inhibition correlate with the anti-proliferative properties of tetracyclines in human cancer cells.