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Updated: Jul 4, 2025
Characterizing RNA Modifications in Single Neurons Using Mass Spectrometry
Published on: April 21, 2022
Mass Spectrometry-Based Pipeline for Identifying RNA Modifications Involved in a Functional Process: Application to
Amandine Amalric1,2, Aurore Attina2, Amandine Bastide1
1IGF, INSERM, Université de Montpellier, CNRS, 34090 Montpellier, France.
Abstract:
Cancer onset and progression are known to be regulated by genetic and epigenetic events, including RNA modifications (a.k.a. epitranscriptomics). So far, more than 150 chemical modifications have been described in all RNA subtypes, including messenger, ribosomal, and transfer RNAs. RNA modifications and their regulators are known to be implicated in all steps of post-transcriptional regulation. The dysregulation of this complex yet delicate balance can contribute to disease evolution, particularly in the context of carcinogenesis, where cells are subjected to various stresses. We sought to discover RNA modifications involved in cancer cell adaptation to inhospitable environments, a peculiar feature of cancer stem cells (CSCs). We were particularly interested in the RNA marks that help the adaptation of cancer cells to suspension culture, which is often used as a surrogate to evaluate the tumorigenic potential. For this purpose, we designed an experimental pipeline consisting of four steps: (1) cell culture in different growth conditions to favor CSC survival; (2) simultaneous RNA subtype (mRNA, rRNA, tRNA) enrichment and RNA hydrolysis; (3) the multiplex analysis of nucleosides by LC-MS/MS followed by statistical/bioinformatic analysis; and (4) the functional validation of identified RNA marks. This study demonstrates that the RNA modification landscape evolves along with the cancer cell phenotype under growth constraints. Remarkably, we discovered a short epitranscriptomic signature, conserved across colorectal cancer cell lines and associated with enrichment in CSCs. Functional tests confirmed the importance of selected marks in the process of adaptation to suspension culture, confirming the validity of our approach and opening up interesting prospects in the field.
Insights
Researchers identified a specific set of RNA modifications (epitranscriptomics) crucial for cancer stem cell (CSC) survival and adaptation in challenging environments, like suspension cultures.
Area of Science:
- Molecular Biology
- Cancer Research
- Epigenetics
Background:
- Cancer development involves genetic and epigenetic changes, including RNA modifications (epitranscriptomics).
- Over 150 RNA modifications exist across mRNA, rRNA, and tRNA, impacting post-transcriptional regulation.
- Dysregulated RNA modifications contribute to carcinogenesis and cancer cell adaptation to stress.
Purpose of the Study:
- To identify RNA modifications enabling cancer cells to adapt to stressful, inhospitable environments.
- To investigate RNA marks facilitating cancer stem cell (CSC) survival in suspension culture, a model for tumorigenic potential.
Main Methods:
- Cultured cells under conditions favoring CSC survival.
- Enriched RNA subtypes (mRNA, rRNA, tRNA) and hydrolyzed RNA.
- Analyzed nucleosides using LC-MS/MS and bioinformatic analysis.
- Performed functional validation of identified RNA modifications.
Main Results:
- The RNA modification landscape changes with cancer cell phenotype under growth constraints.
- Discovered a conserved epitranscriptomic signature in colorectal cancer cell lines linked to CSC enrichment.
- Validated the role of specific RNA marks in adaptation to suspension culture.
Conclusions:
- RNA modifications play a significant role in cancer cell adaptation and survival.
- The identified epitranscriptomic signature offers insights into CSC biology.
- This approach is promising for understanding and potentially targeting cancer evolution.
