Translational regulation by oxidative desulfuration of tRNA modifications

Area of Science:

• Molecular Biology
• Biochemistry
• Structural Biology

Background:

• Transfer RNA (tRNA) modifications are crucial for accurate protein synthesis.
• 5-Methyl-2-thiouridine (xm5s2U) is a key tRNA modification at the anticodon wobble position, enhancing decoding efficiency.
• The thiocarbonyl group in xm5s2U is prone to oxidative desulfuration, forming 4-pyrimidinone derivatives (xm5h2U).

Purpose of the Study:

• To identify and confirm the cellular formation of xm5h2U derivatives in human and mouse tissues.
• To investigate the functional consequences of xm5h2U modification on tRNA decoding and aminoacylation.
• To elucidate the structural basis for altered codon recognition by xm5h2U-modified tRNAs.

Main Methods:

• Spike-in experiments to confirm cellular formation of xm5h2U.
• In vitro translation system using reconstituted human systems to assess codon recognition.
• Cryogenic electron microscopy (cryo-EM) to determine structural changes at the ribosomal A-site.

Main Results:

• xm5h2U derivatives were identified and confirmed in human cells and mouse tissues.
• Desulfurized tRNAs with mcm5h2U modification exhibited impaired codon recognition and reduced aminoacylation for specific tRNAs (lysine, glutamate, glutamine).
• Cryo-EM revealed the structural basis for altered AAA/AAG codon decoding by mcm5h2U at the ribosomal A-site.

Conclusions:

• Oxidative desulfuration of tRNA modifications dynamically regulates codon recognition and protein synthesis.
• This mechanism plays a role in cellular responses to oxidative stress in human and mammalian cells.
• The findings provide insights into how tRNA modification status impacts protein synthesis fidelity.