Determining small RNA-interacting proteomes using endogenously modified tRNA-derived RNAs.

Vera Oberbauer; Aleksej Drino; Matthias R Schaefer

doi:10.1016/bs.mie.2024.11.005

Home/Archive/PubMed

PubMed · 39952715

Determining small RNA-interacting proteomes using endogenously modified tRNA-derived RNAs.

Vera Oberbauer·Aleksej Drino·Matthias R Schaefer

View original source ↗Export metadata ↓

Abstract

tRNA-derived RNAs (tDRs), resulting from enzyme-mediated hydrolysis of tRNAs, have been implicated as active small RNAs in various molecular processes. While the molecular modes of action for these small RNAs remain unclear, attempts to decipher the mechanistic details of tDR functionality have mostly used synthetic tDR sequences. Since parental tRNAs are extensively post-transcriptionally modified, tDR functionality is likely affected by chemical modifications. To help approach the biological function of endogenously modified tDRs, this contribution details a protocol that allows purifying specific tDRs carrying post-transcriptional modifications from both in vivo and in vitro sources. Purified tDRs can be used for various downstream applications including differential affinity capture of tDR-binding proteins, the details of which are also described in this contribution.

Explore related subjects

RNA, Transfer Proteome RNA Processing, Post-Transcriptional Humans RNA-Binding Proteins RNA, Small Untranslated Proteomics RNA modifications Stress response TRNA TRNA fragments Journal Article Research Support, Non-U.S. Gov't

Keep this discovery

Explore connections, maps & timelines

BibTeX RIS

Vera Oberbauer, Aleksej Drino, Matthias R Schaefer. 2024-12-03. Determining small RNA-interacting proteomes using endogenously modified tRNA-derived RNAs.. https://doi.org/10.1016/bs.mie.2024.11.005

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related citations

PubMed2026

Genes near tRNAs are enriched in translational machinery.

Transfer RNAs (tRNAs) are known for delivering amino acids to the growing polypeptide chain during translation. They can also influence gene expression, especially in times of nutrient starvation, through differential tRNA expression and modification. Transfer RNAs have a highly consistent cloverleaf structure, but relatively few known regulatory elements govern this conserved structure despite the 20 different standard isotypes. This study examines gene enrichment patterns near tRNA genes across 1149 fungal genomes. Genes enriched in proteasome regulation, ion transport, and rRNA were found to be significantly closer to tRNAs than other pathways. These results were consistent across KEGG overrepresentation analysis (ORA), KEGG gene set enrichment analysis (GSEA), and gene ontology (GO) analysis. Proteasome, ion transport, and RNA are all important aspects of protein production and regulation, suggesting that genes required for the synthesis and quality control of proteins, including tRNAs, are located near each other. Protein regulation is an energetically expensive process, and local co-regulation could increase efficiency and stress impacts on proteins.

RNA, Transfer↗

PubMed2026

Quantitative RNA modification mapping by mass spectrometry with isobaric tags and nucleobase fragment analysis.

RNA modifications regulate RNA stability, translation, stress responses, and disease processes, yet their function remains poorly understood due to technical limitations in sequence analysis. Here, we present an RNA-specific isobaric tandem mass tagging (RMT) platform for omic-scale quantitative mapping of RNA modifications. The platform combines RNA-specific tags adapted from proteomics with an end-to-end workflow spanning sample preparation through data processing. Validation using synthetic oligonucleotides and total tRNA from Pseudomonas aeruginosa yielded reproducible quantification, with coefficients of variation below 5%. Together with nucleobase fragment analysis, we identified and quantified 24 RNA modifications in PA14 tRNAs, including previously undescribed m2A38 and Gm/Cm39, and assigned their corresponding writer enzymes. Further analyses of tRNAs from writer knockout strains and stressed cells revealed dynamic modification patterns, modification interdependencies, and their potential roles in stress adaptation. This method provides a robust, cost-effective platform for quantitative RNA modification mapping, enabling deeper biological insights.

RNA, Transfer↗

PubMed2026

tRNA methylation: functional insights and epitranscriptomic regulation.

tRNAs, one of the most conserved and abundant RNAs, are central components of protein synthesis, transferring genetic information from DNA to proteins through a precise base-pairing mechanism. Post-transcriptional modifications of tRNAs by tRNA modifying enzymes are essential for maintaining their normal physiological functions, including methylation, isomerization and glycosylation. tRNA methylation, particularly 1-methyladenosine (m1A), 5-methylcytidine (m5C), and 7-methylguanosine (m7G), are among the most abundant and diverse types of post-transcriptional modifications of tRNA, which promote the stability of tRNA secondary and tertiary structures and allow for proper translation. In addition, tRNA methylation affects the production and function of tsRNA (tRNA-derived small RNA), small fragments of RNA that further regulate gene expression and protein synthesis. In our review, we discuss the relevant biological functions of tRNA methylation, including tRNA stability, protein translation, and tsRNA biogenesis.

RNA, Transfer↗