mRNA epitranscriptomics

The success of COVID-19 mRNA vaccines brought the importance and utility of RNA modifications to the forefront of research and health care. There are over 170 chemical modifications identified in the biological universe and counting. About 2% of all protein-coding genes can be considered to be involved in the installation and maintenance of the homeostasis of these modifications. In eukaryotes, tRNAs are the most modified, followed by rRNAs, spliceosomal RNAs, other noncoding RNAs, and finally mRNAs, which have been the primary driver of recent sequencing technology developments and other efforts to expand our understanding of the importance, diversity, and application potential of RNA modification biology.

The term “epitranscriptomics” (beyond transcriptomics) was coined in 2012 to emphasize the ubiquitous and dynamic aspects of RNA modification biology. Since then, RNA modifications have captured the attention not only of the RNA biology community, but of the broader research community as well. Although epitranscriptomics was initially phrased for mRNA modifications, today, all RNA modifications are under this umbrella and the field is still on the trajectory of unprecedented growth. Beyond the RNA field, the National Academies of Sciences, Engineering, and Medicine (NASEM) commissioned a special report, Charting a Future for Sequencing RNA and Its Modifications: A New Era for Biology and Medicine, that was published in March 2024. The NASEM report describes the current state of RNA modification detection technologies, as well as challenges, future directions, and funding recommendations. Both of us were members of this committee and were active participants in discussions of these crucial issues that, once resolved, will propel RNA modification studies to a whole new level. Understanding the prevalence, regulation, and function of RNA modifications in all their forms will usher in a new era of applying and manipulating RNA modifications to improve human health and mitigate human disease.

This special issue presents the perspectives of experts who work on mRNA modifications. The focus on mRNA modifications in particular is deliberate, as knowledge of modifications in mRNA and technologies used to study them still face significant challenges, and substantial questions remain to be answered. The authors who contributed to this issue span diverse areas of expertise, not only in terms of the modifications they study, but also in their biological focus, including plants, viruses, mammals, and diverse disease models. The issue follows the outline of describing each modification type separately, beginning with N⁶-methyladenosine (m⁶A), then inosine (I), pseudouridine (Ψ), N¹-methyladenosine (m¹A), 5-methylcytosine (m⁵C), 2′-O-methylation (Nm), and N⁴-acetylcytosine (ac⁴C).

m⁶A is the most abundant mRNA modification in multicellular eukaryotes. In their Perspective, Zaccara and Jaffrey discuss m⁶A regulation by cytosolic YTH domain-containing proteins (YTHDF1, 2, and 3). They focus on divergent models that have been proposed for the function of these three proteins, providing a detailed review of discrepant data sets and opposing viewpoints that have emerged in recent years. The authors conclude by providing their opinions on future experiments and the importance of mechanistic studies to further understand the function of these proteins in different cell types and contexts. Horner and Thompson describe the challenges to map and define m⁶A function in viral RNAs. Although many studies have identified the location of m⁶A in viral RNAs and its global regulatory roles in viral infection, major challenges remain to assign these modification sites and events during different stages of the viral life cycle, which would enable more in-depth understanding of these modifications. They also present a framework for future studies to advance understanding of m⁶A regulation in viral infection. Song, Cai, and Jia provide a detailed review of current knowledge in the area of m⁶A regulation in plants. They overview research that has been done exploring how m⁶A writer, reader, and eraser proteins contribute to plant development and disease/stress resistance, and they identify critical areas of future research that will be needed to better understand m⁶A biology and to harness the power of m⁶A regulation for improving crop yield and combating plant disease.

A-to-I editing is also a frequently occurring feature of mRNAs and noncoding RNAs. Catalyzed by ADAR enzymes which target double-stranded regions, A-to-I editing in mature transcripts is often found in 3′ UTRs but can also occur in coding sequences, where it can lead to protein recoding. Jarmoskaite and Li provide an insightful history of research progress in our understanding of ADAR function and its role in innate immunity. They also review the pathways through which dsRNA editing by ADAR enzymes impacts self versus non-self recognition and the promise of ADAR targeting for the treatment of cancer and other diseases. Mendoza and Beal provide a structural and functional perspective of I-modifications in mRNA. They introduce numerous functions of I-modification in RNA biology such as mRNA stability, splicing, translation, and protein binding, as well as the impact of the A-to-I conversion on the structure and stability of RNA structures. They also highlight specific open questions that further relate I-modification to immune responses, for example, via the innate immune regulator MDA5. Finally, Bass provides a historical and current perspective on the adenosine deaminases that act on RNA. Her personal recollections of her own research on A-to-I editing and ADAR enzymes during the early days of this field is a thrilling read on the excitement and thought process involved in making new discoveries. She also discusses the many outstanding questions of the A-to-I field, as well as a brief introduction to the NASEM report on RNA modifications which she co-chaired.

Pseudouridine is the most abundant RNA modification in total cellular RNA and is also an abundant modification in eukaryotic mRNA. Gilbert outlines recent progress in the study of pseudouridine in mRNA, highlighting the technical developments in Ψ mapping and quantification as well as recent updates to our understanding of Ψ function. She stresses the importance of obtaining precise, quantitative maps of Ψ and suggests potential paths forward toward achieving this goal. She also shares her thoughts on future areas of focus and challenges that remain for the field, including the importance of establishing defined roles for individual pseudouridine sites in cellular mRNAs. Focusing on Ψ and m¹A, Zhang, Zhang, Ma, and Yi review recent research with a focus on the methods used to map these modifications and studies revealing their functional roles. They also provide their perspective on important areas of focus for future studies, in particular, efforts for single-molecule and single-cell modification mapping and stoichiometry measurement. They also discuss the promise of targeted installation of Ψ and m¹A to improve understanding of the function of these modifications as well as for potential therapeutic applications.

Yi and colleagues also discuss how inconsistencies in the results of m¹A mapping studies have led to discrepancies over the abundance of m¹A in mRNA. In their article, Dai, He, and colleagues summarize these studies and then present new results showing that chemical reduction of m¹A can improve the readthrough and mutation rate at m¹A sites for a variety of reverse transcriptases. They also develop improved conditions for Dimroth rearrangement of m¹A to m⁶A for the purpose of generating control samples. Using these improvements, they present a new method, red-m¹A-seq, which they use to quantitatively map m¹A in tRNAs and rRNAs from cultured human cells. It will be exciting to see in future studies if this method will lead to better and more consistent maps of m¹A in mRNA and help settle some of the debates regarding m¹A prevalence in cytoplasmic mRNAs.

5-methyl-C in mRNA was first described in the 1970s, around the same time as the discovery of m⁶A in mRNA. Guarnacci and Preiss describe the various mapping methods currently used for m⁵C mapping, the known writers, potential erasers, and readers of this mRNA modification, and its known functions. They also provide a perspective on current challenges and suggestions on how to address them in the future.

2′-O-methylation (Nm) is a well-known modification close to the 5′ end of eukaryotic mRNA caps and plays a role in marking the mRNA as self for the innate immune sensors. Internal 2′-O-methyl modifications in mRNA have also been described. Zhou, Pecot, and Holley describe the progress, challenges, and future directions of Nm in mRNAs. The current Nm mapping methods for mRNA still require significant improvements for precision. The functional consequences of Nm modifications have been shown to impact translation, splicing, and innate immune response. They also describe the implications of Nm in numerous diseases, and the current challenges to be addressed and future directions for this modification.

Schiffers and Oberdoerffer discuss one of the lesser-known mRNA modifications: ac⁴C. Initially characterized in tRNA and rRNA, this modification has now been shown to exist in cellular mRNAs. The authors provide a highly comprehensive overview of the current methods used to map ac⁴C and discuss their inherent limitations. Highlighting strengths and weaknesses of various approaches, they share their insights into how improvements in ac⁴C detection will facilitate studies of its function and ultimately enable a better understanding of how this modification is regulated and its contribution to physiology and disease.

We extend our sincere thanks to all the authors for their contributions and for making this, in our opinion, a truly exceptional special issue. A common theme that emerged in nearly every article was the importance of mRNA modifications for cellular function across diverse systems ranging from plants to viruses to humans. At the same time, though, we come away with the recognition that there is still much work to be done: For abundant modifications such as m⁶A, tool development and efforts to understand and exploit modification function have far outpaced other mRNA modifications. Even so, there are many gaps in m⁶A knowledge that need to be filled. For other modifications, there is universal recognition of the need to expand our efforts to understand how these modifications are regulated and what their roles are in controlling gene expression and organism function. These sentiments are echoed in the recent NASEM report, further illustrating the urgent need to make epitranscriptomics a high priority. At the same time, though, the articles in this special issue provide an excellent overview of just how much progress has been made in the past decade, demonstrating that this is truly an exciting time for epitranscriptomics research. We hope that the readers will come away with the same impression.

We would like to conclude by extending our extreme gratitude to Tim Nilsen for orchestrating this special issue and for keeping us on task. Special thanks also go to Ann Marie Micenmacher for coordinating with us at every step and making our lives so much easier.

• © 2024 Meyer and Pan; Published by Cold Spring Harbor Laboratory Press for the RNA Society