RNase 4 improves bottom-up modification mapping of E. coli total tRNAs using HILIC-MS/MS

Area of Science:

• Biochemistry
• Molecular Biology
• Genomics

Background:

• RNA modifications are crucial for tRNA function in protein synthesis.
• Mass spectrometry is a standard but underutilized tool for mapping RNA modifications due to technical challenges.
• Current methods face limitations with RNase diversity, RNA mixture complexity, and ion-pairing reagents.

Purpose of the Study:

• To develop and validate a novel bottom-up liquid chromatography-tandem mass spectrometry (LC-MS/MS) workflow for global tRNA modification mapping.
• To overcome limitations of existing mass spectrometry approaches for RNA analysis.
• To achieve high sequence coverage for E. coli tRNA isoacceptors.

Main Methods:

• Utilized a bottom-up LC-MS/MS workflow with hydrophilic interaction liquid chromatography (HILIC) and no ion-pairing reagents.
• Employed orthogonal digestions with RNase 4 and a folded digestion scheme with RNase T1.
• Achieved 75-100% sequence coverage for most E. coli tRNA isoacceptors.

Main Results:

• The HILIC-MS/MS workflow demonstrated performance comparable to traditional methods.
• Discovered a new methylation site (Gm17) in tRNA Gly and confirmed an s4U8 modification in tRNA Arg.
• Validated the dependence of acp3U47 insertion on m5U54 in tRNA Phe in a trmA mutant.

Conclusions:

• RNase 4 enhances bottom-up tRNA sequencing, enabling high-quality E. coli tRNA analysis.
• The developed HILIC-MS/MS method provides a robust alternative for global tRNA modification mapping.
• This approach facilitates the discovery and characterization of RNA modifications and their regulatory pathways.