Consensus Study Report

NATIONAL ACADEMIES PRESS 500 Fifth Street, NW Washington, DC 20001

This activity was supported by contracts between the National Academy of Sciences, National Institutes of Health, and The Warren Alpert Foundation. Any opinions, findings, conclusions, or recommendations expressed in this publication do not necessarily reflect the views of any organization or agency that provided support for the project.

International Standard Book Number-13: 978-0-309- 70695-7
International Standard Book Number-10: 0-309- 70695-5
Digital Object Identifier: https://doi.org/10.17226/27165
Library of Congress Control Number: 2024940945

This publication is available from the National Academies Press, 500 Fifth Street, NW, Keck 360, Washington, DC 20001; (800) 624-6242 or (202) 334-3313; http://www.nap.edu.

Copyright 2024 by the National Academy of Sciences. National Academies of Sciences, Engineering, and Medicine and National Academies Press and the graphical logos for each are all trademarks of the National Academy of Sciences. All rights reserved.

Printed in the United States of America.

Cover credit: Dr. Janet Iwasa designed the cover art using nucleotides 112–122 of 18S rRNA from the cryo-electron microscopy structure of ribosomal frameshifting during translation of the SARS-CoV-2 RNA genome (PDB 7O7Y) (see Bhatt, P. R., A. Scaiola, G. Loughran, M. Leibundgut, A. Kratzel, R. Meurs, R. Dreos, K. M. O’Connor, A. McMillan, J. W. Bode, V. Thiel, D. Gatfield, J. F. Atkins, and N. Ban. 2021. “Structural basis of ribosomal frameshifting during translation of the SARS-CoV-2 RNA genome.” Science 372 (6548): 1306-1313. https://doi.org/10.1126/science.abf3546). Teal color highlights RNA modifications: a nitrogen in a pseudouridine and a ribose methylation of uridine.

Suggested citation: National Academies of Sciences, Engineering, and Medicine. 2024. Charting a Future for Sequencing RNA and its Modifications: A New Era for Biology and Medicine. Washington, DC: The National Academies Press. https://doi.org/10.17226/27165.

The National Academy of Sciences was established in 1863 by an Act of Congress, signed by President Lincoln, as a private, nongovernmental institution to advise the nation on issues related to science and technology. Members are elected by their peers for outstanding contributions to research. Dr. Marcia McNutt is president.

The National Academy of Engineering was established in 1964 under the charter of the National Academy of Sciences to bring the practices of engineering to advising the nation. Members are elected by their peers for extraordinary contributions to engineering. Dr. John L. Anderson is president.

The National Academy of Medicine (formerly the Institute of Medicine) was established in 1970 under the charter of the National Academy of Sciences to advise the nation on medical and health issues. Members are elected by their peers for distinguished contributions to medicine and health. Dr. Victor J. Dzau is president.

The three Academies work together as the National Academies of Sciences, Engineering, and Medicine to provide independent, objective analysis and advice to the nation and conduct other activities to solve complex problems and inform public policy decisions. The National Academies also encourage education and research, recognize outstanding contributions to knowledge, and increase public understanding in matters of science, engineering, and medicine.

Learn more about the National Academies of Sciences, Engineering, and Medicine at www.nationalacademies.org.

Consensus Study Reports published by the National Academies of Sciences, Engineering, and Medicine document the evidence-based consensus on the study’s statement of task by an authoring committee of experts. Reports typically include findings, conclusions, and recommendations based on information gathered by the committee and the committee’s deliberations. Each report has been subjected to a rigorous and independent peer-review process and it represents the position of the National Academies on the statement of task.

Proceedings published by the National Academies of Sciences, Engineering, and Medicine chronicle the presentations and discussions at a workshop, symposium, or other event convened by the National Academies. The statements and opinions contained in proceedings are those of the participants and are not endorsed by other participants, the planning committee, or the National Academies.

Rapid Expert Consultations published by the National Academies of Sciences, Engineering, and Medicine are authored by subject-matter experts on narrowly focused topics that can be supported by a body of evidence. The discussions contained in rapid expert consultations are considered those of the authors and do not contain policy recommendations. Rapid expert consultations are reviewed by the institution before release.

For information about other products and activities of the National Academies, please visit www.nationalacademies.org/about/whatwedo.

TOWARD SEQUENCING AND MAPPING OF RNA MODIFICATIONS COMMITTEE¹

BRENDA L. BASS (Co-Chair), University of Utah School of Medicine

TAEKJIP HA (Co-Chair), Harvard Medical School and Boston Children’s Hospital

NICHOLAS MORGAN ADAMS, Thermo Fisher Scientific

JUAN D. ALFONZO, Brown University

JEFFREY C. BAKER, National Institute for Innovation in Manufacturing Biopharmaceuticals

SUSAN J. BASERGA, Yale University and the Yale School of Medicine

LYDIA MARIA CONTRERAS, The University of Texas at Austin

MARKUS HAFNER, National Institute of Arthritis and Musculoskeletal and Skin Diseases

SARATH CHANDRA JANGA, Indiana University Indianapolis

PATRICK ALAN LIMBACH, University of Cincinnati

JULIUS BEAU LUCKS, Northwestern University

MARY ANDERLIK MAJUMDER, Baylor College of Medicine

NICOLE M. MARTINEZ, Stanford University

KATE D. MEYER, Duke University School of Medicine

KEITH ROBERT NYKAMP, Genetic Insight Group, Invitae Corporation

TAO PAN, The University of Chicago

Study Staff

TRISHA TUCHOLSKI, Study Director (from August 2023), Program Officer, Board on Life Sciences

STEVEN MOSS, Study Director (until August 2023), Senior Program Officer, Board on Life Sciences

KAVITA M. BERGER, Director, Board on Life Sciences

KATHRYN ASALONE, Associate Program Officer, Board on Health Sciences Policy

LYLY G. LUHACHACK, Program Officer, Board on Life Sciences

JESSICA DE MOUY, Research Associate, Board on Life Sciences

HOANG-NAM VU, Senior Program Assistant, Board on Life Sciences

Consultants

MICHAEL ZIERLER, RedOx Scientific Editing

___________________

¹ See Appendix F, Disclosure of Unavoidable Conflict(s) of Interest.

BOARD ON LIFE SCIENCES

ANN ARVIN (Chair), Stanford University

DENISE N. BAKEN, Shield Analysis Technology, LLC

TANYA Y. BERGER-WOLF, The Ohio State University

VALERIE H. BONHAM, Kennedy Krieger Institute

PATRICK M. BOYLE, Ginkgo Bioworks

DOMINIQUE BROSSARD, University of Wisconsin–Madison

MAURO COSTA-MATTIOLI, Baylor University

GERALD L. EPSTEIN, Johns Hopkins Center for Health Security

ROBERT J. FULL, University of California, Berkeley

INDIA G. HOOK-BARNARD, Engineering Biology Research Consortium

BERONDA MONTGOMERY, Michigan State University

LOUIS J. MUGLIA, University of Cincinnati College of Medicine

ROBERT NEWMAN, Aspen Institute

LUCILA OHNO-MACHADO, University of California, San Diego

SUDIP S. PARIKH, American Association for the Advancement of Science

NATHAN D. PRICE, University of Washington

SUSAN R. SINGER, Rollins College

DAVID R. WALT, Harvard Medical School

PHYLLIS M. WISE, University of Colorado

Staff

KAVITA BERGER, Director

ANDREW BREMER, Program Officer

JESSICA DE MOUY, Research Associate

LAYLA GARYK, Program Assistant

CYNTHIA GETNER, Senior Financial Business Partner

NIA JOHNSON, Program Officer

LYLY LUHACHACK, Program Officer

DASIA MCKOY, Senior Program Assistant

CHRISTL SAUNDERS, Program Coordinator

AUDREY THEVENON, Senior Program Officer

TRISHA TUCHOLSKI, Program Officer

SABINA VADNAIS, Research Associate

HOANG-NAM VU, Senior Program Assistant

BOARD ON HEALTH SCIENCES POLICY

SHARON TERRY (Chair), Genetic Alliance

DAVID BLAZES, Bill and Melinda Gates Foundation

ARAVINDA CHAKRAVARTI, New York University Grossman School of Medicine

AMANDER CLARK, University of California, Los Angeles

STEVEN K. GALSON, Amgen (retired)

M. EHSAN HOQUE, University of Rochester

FRANCES E. JENSEN, University of Pennsylvania

FRANK R. LIN, Johns Hopkins School of Medicine and Bloomberg School of Public Health

SUZET M. MCKINNEY, Sterling Bay

DIETRAM A. SCHEUFELE, University of Wisconsin–Madison

MATTHEW K. WYNIA, University of Colorado, Anschutz Medical Campus

Staff

CLARE STROUD, Senior Board Director

KATHRYN ASALONE, Associate Program Officer

KELSEY BABIK, Associate Program Officer

SARAH BEACHY, Senior Program Officer

MICHAEL BERRIOS, Research Associate

ASHLEY BOLOGNA, Senior Program Assistant

KATHERINE BOWMAN, Senior Program Officer

LISA BROWN, Senior Program Officer

KYLE CAVAGNINI, Associate Program Officer

EVA CHILDERS, Program Officer

EMILY PACKARD DAWSON, Program Officer

AUTUMN DOWNEY, Senior Program Officer

MICHELLE DREWRY, Associate Program Officer

REBECCA ENGLISH, Senior Program Officer

ALEX HELMAN, Senior Program Officer

BRITTANY HSIAO, Associate Program Officer

MELVIN JOPPY, Senior Program Assistant

EESHAN KHANDEKAR, Program Officer

ANDREW MARCH, Program Officer

MATTHEW MASIELLO, Associate Program Officer

CHANEL MATNEY, Program Officer

EMILY MCDOWELL, Research Associate

CHRISTA NAIRN, Program Coordinator

KIMBERLY OGUN, Senior Program Assistant

NOAH ONTJES, Associate Program Officer

ASHLEY PITT, Senior Program Assistant

ANDREW POPE, Advisor

SHEENA POSEY NORRIS, Senior Program Officer

SAMANTHA SCHUMM, Program Officer

CAROLYN SHORE, Senior Program Officer

RAYANE SILVA-CURRAN, Senior Program Assistant

SHALINI SINGARAVELU, Program Officer

CARSON SMITH, Research Associate

GAYATRI SOMAIYA, Senior Program Assistant

LYDIA TEFERRA, Research Associate

MAYA THIRKILL, Associate Program Officer

JOSEPH TUMFOUR, Associate Program Officer

SCOTT WOLLEK, Senior Program Officer

TEQUAM WORKU, Program Officer

OLIVIA YOST, Program Officer

Acknowledgments

The study committee and project staff would like to thank the study sponsors—The Warren Alpert Foundation, National Institute of Environmental Health Sciences (NIEHS), and National Human Genome Research Institute (NHGRI)—for providing essential leadership on the topic of sequencing RNA and its modifications. We thank Vivian Cheung, whose vision and advocacy were instrumental in commissioning the report. We thank Fred Tyson of NIEHS and Carolyn Hutter and Jennifer Strasburger of NHGRI for organizing a highly informative workshop on RNA modifications in 2022 with the National Institutes of Health, which heavily influenced the report writing process and laid a solid foundation for the committee to reference and build upon. This consensus study report was greatly enhanced by discussions with speakers, moderators, and participants of our workshop, webinars, and ideation challenge (full participant lists can be found in Appendixes B and C). We are especially thankful to them for providing up-to-date and, often, privileged information during the information-gathering activities. We also thank the individuals listed in Appendix F for authoring commissioned reports on their ideas and perspectives regarding this topic. Clean, visually appealing, and scientifically accurate graphics are important, and we are grateful to Janet Iwasa and Rachel Torrez for designing the report cover and many of the figures. We thank Tucker Nelson, Julie Eubank, and Chris King of the National Academies of Sciences, Engineering, and Medicine Office for Congressional and Government Affairs for providing guidance on how to best communicate key conclusions and recommendations to potential partners and policy makers. We thank Heidi Schweingruber and Kerry Brenner from the National Academies Board on Science Education, who provided essential feedback for the section on workforce development. Finally, the staff and committee would like to thank the National Academies Research Center for providing expertise and assistance with fact-checking this report.

This page intentionally left blank.

Reviewers

This consensus study report was reviewed in draft form by individuals chosen for their diverse perspectives and technical expertise. The purpose of this independent review is to provide candid and critical comments that will assist the National Academies of Sciences, Engineering, and Medicine in making each published report as sound as possible and to ensure that it meets the institutional standards for quality, objectivity, evidence, and responsiveness to the study charge. The review comments and draft manuscript remain confidential to protect the integrity of the deliberative process.

We thank the following individuals for their review of this report:

Although the reviewers listed above provided many constructive comments and suggestions, they were not asked to endorse the conclusions or recommendations of this report, nor did they see the final draft before its release. The review of this report was overseen by PAUL AHLQUIST

(NAS), University of Wisconsin–Madison, and DAN BLAZER (NAM), Duke University School of Medicine. They were responsible for making certain that an independent examination of this report was carried out in accordance with the standards of the National Academies and that all review comments were carefully considered. Responsibility for the final content rests entirely with the authoring committee and the National Academies.

Preface

On the morning of October 9, 2023, we learned that Drs. Karikó and Weissman shared the 2023 Nobel Prize in Physiology or Medicine for their research on RNA modifications that enabled the development of effective mRNA vaccines against SARS-CoV-2, the virus responsible for causing the COVID-19 pandemic. The Nobel announcement came on the day of our final scheduled committee meeting and gave us all a boost and a feeling that our year-long effort to write a report evaluating the present status and future prospects for mapping and sequencing RNA modifications had been prescient.

For some of us, the in-person meeting of the committee in March of 2023 was the first time in years being in a small room with dozens of people not wearing masks—a reminder of just how disruptive the COVID-19 pandemic had been and how timely the work of the committee would be. As we began our work, we were inspired by the Human Genome Project and its impact on shaping our modern-day knowledge of the sequence of each gene, and in some cases, variants that correlate with disease. But each gene gives rise to dozens, sometimes thousands, of RNA molecules that combine the information passed from our DNA genomes in multiple ways; importantly, these RNA molecules are also subject to biological processes that chemically alter, or modify, their sequences.

We realized that understanding this “epitranscriptome” would enhance our understanding of health and disease immeasurably, but we also quickly realized that our task was very different from determining a genome sequence. While each organism typically has one primary genome sequence, every cell in every organism has a different set of modified RNA molecules that make up its epitranscriptome; further, every epitranscriptome is constantly changing with, among other things, developmental stage and environment. We came to the consensus that our task should not be evaluating whether efforts should be directed to determining an epitranscriptome, but rather, evaluating the importance of developing technologies and infrastructure that would enable the determination of any epitranscriptome of interest.

The 16-member committee of diverse expertise was shepherded to stay on track and on time in the amazingly capable hands of Steven Moss and Trisha Tucholski of the National Academies of Sciences, Engineering, and Medicine. In addition, Kathryn Asalone, Kavita Berger, Jessica De

Mouy, Lyly Luhachack, Nam Vu, and Michael Zierler supported the committee’s work marvelously and helped to write this report. We were impressed by the National Academies’ ability to recruit an outstanding slate of committee members with diverse viewpoints and differing degrees of proximity to the core area of RNA modifications. The committee included two scientists from industry, which we considered important because the development of scalable and reliable next-generation technologies would need strong buy-in from the private sector. We cherished the intellectual exchanges and the comradeship we built through many hours of intense yet collegial debate. In this report, we chart a path forward for sequencing RNA and its modifications, and present guidelines that will foster the technology and infrastructure needed to enable, for any cell type or organism, the complete end-to-end sequencing of its epitranscriptome, so that researchers may then choose their own adventures. We envision a day when basic and clinical researchers, patients, and the interested public, can use a smartphone to bring up an accurate representation of any RNA to enable their research or understand their disease.

Taekjip Ha and Brenda Bass, Co-Chairs
Toward Sequencing and Mapping of RNA Modifications Committee

Contents

PREFACE

SUMMARY

1 INTRODUCTION

The Life of an RNA

A Brief History of RNA Modifications Research

Key Components of a Roadmap to Unlock Any Epitranscriptome

Study Scope and Approach

Organization of the Report

References

2 IMPORTANCE AND IMPACTS OF RNA MODIFICATIONS IN BIOLOGY, DISEASE, MEDICINE, AND SOCIETY

What are RNA Modifications?

RNA Modifications in Biology

RNA Modifications in Disease

RNA Modifications for Preventing and Treating Disease

RNA Modifications Beyond Human Health

Major Challenges and Scientific Gaps

Conclusions

References

3 CURRENT AND EMERGING TOOLS AND TECHNOLOGIES FOR STUDYING RNA MODIFICATIONS

Current Approaches for Studying RNA Modifications

Major Challenges and Scientific Gaps

Emerging Tools and Technologies

Conclusions

Roadmap for Advancing Tools and Technologies

References

4 STANDARDS AND DATABASES FOR RNA MODIFICATIONS

The Current State of Standards

The Current State of Databases

Major Challenges and Scientific Gaps

Conclusions

Roadmap for Developing Standards and Databases

References

5 DRIVING INNOVATION TO STUDY RNA MODIFICATIONS

Important Drivers of Innovation

Accelerators of Innovation

A Large-Scale Research Initiative

Conclusions

Roadmap For Education, Training, and Workforce Development

References

6 A BOLD VISION FOR THE FUTURE OF SEQUENCING RNA AND ITS MODIFICATIONS: CONCLUSIONS, RECOMMENDATIONS, AND A PATH FORWARD

Key Components of a Roadmap to Unlock Any Epitranscriptome

Launching A Large-Scale Initiative

Broader Impact

References

APPENDIXES

A TABLES OF COMPUTATIONAL TOOLS

B PUBLIC MEETING AGENDAS

C VIRTUAL MEETINGS

D STUDY COMMITTEE BIOGRAPHIES

E DISCLOSURE OF UNAVOIDABLE CONFLICTS OF INTEREST

F IDEATION CHALLENGE COMMISSIONED PAPERS

Boxes, Figures, and Tables

BOXES

1-1 DNA and RNA Base-Pairing

1-2 tRNA Modopathies: The Importance of Modifications in Health and Disease

1-3 Statement of Task

2-1 The Critical Role of RNA Modifications in Vaccines Against COVID-19

3-1 Nanopore Sequencing

3-2 Bioinformatics of Nanopore Sequencing

4-1 Types of Standards and Their Uses

5-1 Comparing the Human Genome Project with Possible Efforts Related to RNA Modifications

FIGURES

S-1 Key efforts needed to unlock any epitranscriptome

S-2 Roadmap for advancing tools and technologies in the pursuit of mapping epitranscriptomes including milestones along the path for achieving the 15-year vision

S-3 Roadmap for developing standards and databases

S-4 Roadmap for education, training, and workforce development

1-1 DNA transfers its information to RNA

1-2 rRNA and tRNA are necessary for translation of mRNAs by the ribosome

1-3 Watson-Crick base pairs

1-4 RNA nucleosides and examples of known chemical modifications

1-5 RNA modifications located on the human mitochondrial tRNA^His

1-6 Next-generation sequencing methods

1-7 Key efforts needed to unlock any epitranscriptome

2-1 RNA modifications can be used to distinguish cellular (self) RNAs from exogenous (nonself) RNAs

2-2 How the N1-methyl-pseudouridine (Ψ) modification of the nucleoside uridine permits the COVID-19 mRNA vaccines to succeed in protecting against infection and serious disease

2-3 New antisense oligonucleotide (ASO) therapy that modulates pre-mRNA splicing treats spinal muscular atrophy

2-4 Expression of human FTO in field-grown potatoes increases their biomass

3-1 Modification measurements using liquid chromatography–tandem mass spectrometry (LC-MS/MS)

3-2 Nanopore RNA sequencing

3-3 Mass spectrometry approaches to identify sites of modification (bottom up vs. top down)

3-4 Mass spectrometry fragmentation nomenclature for oligonucleotides and RNAs

3-5 Nanopore sequencing computational workflow and applications

3-6 Roadmap for advancing tools and technologies

4-1 Example usage of physical standards

4-2 RNA biotypes represented across 15 databases, showing overlap and unique aspects of RNA modification database resources

4-3 Roadmap for developing standards and databases

5-1 Roadmap for education, training, and workforce development

6-1 Key efforts needed to unlock any epitranscriptome

6-2 Roadmap for advancing tools and technologies

6-3 Roadmap for developing standards and databases

6-4 Roadmap for education, training, and workforce development

F-1 RNA modifications dictate RNA structure, intermolecular interactions, and function

F-2 Vision for the Unified Epitranscriptomics Consortium

F-3 Current methods for mapping RNA modifications categorized according to the analytical technique being used to generate the reporting signal of RNA modifications

F-4 A summary of the components, features, and vision of the proposed EpiC database

F-5 Timeline of Milestones for EpiC

F-6 Timeline for the comprehensive mapping of the human epitranscriptome

F-7 Single nucleotide resolution mapping can be performed using either modification-specific biochemical approaches or nanopore sequencing

TABLES

2-1 Human Diseases Caused by Aberrant tRNA Modifications

2-2 RNA Therapeutics Approved by the U.S. Food and Drug Administration (FDA) and/or European Medicines Agency (EMA)

3-1 Summary of Indirect Sequencing Techniques Applied to Specific RNA Modifications

3-2 Specificity and Limitations of Enzymes Used for RNA and Oligonucleotide Digestion

3-3 Overview of Various Tools for Identifying RNA Modifications in Direct RNA Sequencing Data

4-1 Databases for RNA Modifications

A-1 State-of-the-Art Nanopore Basecalling Tools with Principle of Analysis, Input, and Output Along with Resources

A-2 Various Quality Control Tools Used with Nanopore Sequencing Data

A-3 ONT Assemblers for Long-Reads

A-4 Aligners for RNA-seq data

This page intentionally left blank.

Acronyms and Abbreviations