Introduction
RNA modification is a field of study that has evolved tremendously since the first discovery of pseudouridine (Ψ) in 1957. From the identification of basic RNA modifying enzymes, we have now progressed to understanding the complex and dynamic nature of RNA modifications- collectively termed the "epitranscriptome." This landscape encompasses a wide array of chemical modifications that impact RNA at multiple levels, affecting cellular processes from localization and stability to translation regulation. Researchers have developed various methods for mapping and understanding these modifications, leveraging advancements in sequencing technologies, mass spectrometry, and cryo-electron microscopy. This article delves into the major research methods for studying RNA modifications and discusses their significance.
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Types of RNA Modifications
RNA modifications can be broadly categorized based on the type of nucleotide they modify. Some of the most studied modifications include N6-methyladenosine (m6A), 5-methylcytidine (m5C), and pseudouridine (Ψ). While the majority of modifications were initially found on tRNA and rRNA, many have also been identified on mRNAs and other non-coding RNAs. These modifications are not mere molecular decorations but heavily influence RNA metabolism and functionality.
Fig. 1 The modifications on mRNA, tRNA, and rRNA (Lauman R., Garcia B. A. 2020).
mRNA Modifications
Modifications such as m6A, m5C, and 2′-O-methylribose (Nm) were first discovered on mRNA in the 1970s. Among these, m6A is the most prevalent and well-studied. Advances in sequencing technology have enabled the precise mapping of m6A sites on mRNA, uncovering that one-third of mammalian mRNAs contain multiple m6A modifications, predominantly located around the stop codon.
rRNA Modifications
Ribosomal RNAs (rRNAs) are extensively modified, with ribose 2′-O-methylation and pseudouridylation being the most common. Employing techniques such as cryo-electron microscopy (cryo-EM) has allowed the visualization of these modifications within the highly structured ribosome. Modifications on rRNA are believed to affect ribosome function, potentially contributing to differential protein synthesis under various cellular conditions.
tRNA Modifications
tRNAs are another heavily modified RNA species, with modifications found throughout the molecule, particularly in the anticodon loop. These modifications are vital for maintaining the accuracy of translation and ensuring the stability and proper folding of tRNA molecules. Dynamic changes in tRNA modifications have been observed under stress conditions, linking them to cellular adaptive responses.
RNA Modification Profiling Techniques
The study of RNA modifications has evolved from early chemical and chromatographic methods to sophisticated high-throughput sequencing and direct detection technologies. The major techniques employed for transcriptome-wide RNA modification profiling include mass spectrometry, cryo-EM, next-generation sequencing (NGS), and nanopore-based methods. Each of these methods has distinct advantages and challenges, offering complementary insights into the RNA modification landscape.
Mass Spectrometry and Cryo-Electron Microscopy-Based Methods
Mass spectrometry, particularly LC-MS/MS, has been instrumental in directly identifying and quantifying RNA modifications based on their mass and fragmentation patterns. LC-MS/MS analyzes RNA samples treated with specific ribonucleases to produce oligonucleotides. These oligonucleotides are then identified and characterized based on their molecular weights and fragmentation spectra. This method provides precise information about the type and location of modifications, with improvements allowing simultaneous detection of multiple modification types.
Cryo-EM has become a powerful tool for visualizing RNA modifications within the intact ribosome complex. This technique involves flash-freezing ribosomes and imaging them at high resolution, providing structural details of modified nucleotides. Cryo-EM has revealed over 130 rRNA modifications in the human ribosome, offering insights into how these modifications might influence ribosome function and protein synthesis. However, not all modifications are readily resolved by cryo-EM, and some discrepancies with sequencing data highlight the need for complementary approaches.
Next-Generation Sequencing-Based Methods
Next-generation sequencing (NGS) technologies have revolutionized our understanding of RNA modifications by providing high-throughput and detailed mapping capabilities. NGS-based methods for RNA modification profiling can be broadly categorized into three strategies: antibody-based, chemical-based, and enzyme-based techniques.
The first category uses antibodies that specifically recognize modified nucleotides to immunoprecipitate RNA fragments. Techniques such as m6A-seq and MeRIP-seq rely on this immunoprecipitation approach, followed by sequencing the enriched RNA fragments. While early versions of these methods had limited resolution (100–200 nt), advancements like photo-crosslinking-assisted protocols (miCLIP, m6ACE-Seq) have achieved single-base resolution.
Chemical-based methods utilize selective chemical treatments to convert modified nucleotides into other bases, which are then detected by sequencing. For example, bisulfite treatment converts unmethylated cytosine to uridine, allowing for the identification of methylated cytosines. Similar methods include Pseudo-Seq and AlkAniline-Seq for different modifications. These methods are efficient and cost-effective but can be limited by incomplete conversion and overlapping modification types.
Enzyme-based methods use enzymes to selectively modify specific nucleotides, aiding in their detection by sequencing. Examples include Nm-REP-seq for 2′-O-methylation and various methods for m6A detection (Mazter-Seq, m6A-REF-Seq, DART-Seq). Combining enzyme treatments with immunoprecipitation further enhances resolution, as seen in protocols such as Aza-IP for m5C detection.
Nanopore-Based Methods
Nanopore sequencing represents a leap in RNA modification detection by allowing the direct sequencing of native RNA molecules. This technology measures changes in electrical conductivity as RNA passes through a nanopore, detecting modifications based on unique disruption patterns in the current signal. Techniques like EpiNano, MINES, and nanom6A use machine learning to identify modifications from these patterns. Neural network-based approaches like xPore and m6Anet compare signals from modified and unmodified samples to pinpoint modification sites.
Nanopore sequencing offers the advantage of identifying modifications directly without the need for conversion or specific antibodies. However, challenges include the requirement for substantial RNA input and higher base-calling error rates compared to NGS. Continued improvements in chemistry and software are expected to enhance accuracy and reduce input amounts, making nanopore sequencing a promising tool for epitranscriptome studies.
Conclusion
RNA modifications are integral to understanding cellular processes and disease mechanisms. The continued development and integration of advanced sequencing technologies, mass spectrometry, and cryo-electron microscopy will further illuminate the complexity of the epitranscriptome. As these methodologies continue to evolve, they hold the promise of uncovering new modifications and elucidating their roles in health and disease, paving the way for novel therapeutic strategies.
References
- Goh W. S. S., Kuang Y. Heterogeneity of chemical modifications on RNA. Biophysical Reviews. 2024, 16 (1): 79-87.
- Meyer K. D., et al. Comprehensive analysis of mRNA methylation reveals enrichment in 3' UTRs and near stop codons. Cell. 2012, 149(7): 1635-1646.
- Chen B., et al. Analytical methods for deciphering RNA modifications. Analytical Chemistry. 2018, 91 (1): 743-756.
- Lauman R., Garcia B. A. Unraveling the RNA modification code with mass spectrometry. Molecular Omics. 2020, 16 (4): 305-315.
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