Charting a Future for Sequencing RNA and Its Modifications A New Era for Biology and Medicine (2024) / Chapter Skim
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1 Introduction
1 Introduction
Pages 13-28
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From page 13... ...
, the nascent RNA transcript from a single gene2F2 is subject to a cut-and-paste method called splicing, whereby a single gene can give rise to hundreds, in some cases thousands, of distinct RNA molecules, or isoforms, each of which contains subsets of the original RNA sequence. Thus, genetic information is pieced together in diverse ways (Marasco and Kornblihtt, 2023; Wright, Smith, and Jiggins, 2022)
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(C) Alternative splicing combines exonic sequences in many distinct ways and together with differential RNA modification creates diverse RNA isoforms.
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base-pairs with the three-nucleotide codon of the mRNA, the ribosome will catalyze the addition of its amino acid to the growing chain of amino acids on the adjacent tRNA; this process will continue along the mRNA until the mature protein is made. Prepublication Copy 15
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All RNAs -- coding and noncoding -- are decorated with chemical modifications, many of which have essential functions and several of which perform multiple functional roles in the cell. Understanding the full picture of the biological functions of RNA modifications will be impossible without a complete determination of many epitranscriptomes representing different cell types, tissues, and organisms.
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In addition, early methods did not provide "positional" information about where in the linear RNA sequence the modifications occurred. That began to change in the mid-1960s, when Robert Holley, who shared the Nobel Prize in Physiology or Medicine for this work, determined the linear structure of a tRNA, including a subset of its modified nucleotides (Holley et al., 1965)
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It is now overwhelmingly clear that tRNA modifications are important for stabilizing its structure, as they ensure that the tRNA is charged with the correct amino acid and maintain accuracy in codon6F7 recognition 7 See https://www.genome.gov/genetics-glossary/Codon (accessed October 27, 2023)
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. The method of comparing a genomic DNA sequence with the DNA sequence copied from RNA using an enzyme called reverse transcriptase led to discovery of the dramatic example of uridine insertion and deletion in the Prepublication Copy 19
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, in most cases it is still not known what combination of modified nucleotides exists together in a single, complete RNA transcript. The State of RNA Sequencing Technologies Technologies for DNA sequencing took a huge leap forward with the HGP, which spurred the further advances and automation of the technology known as Sanger sequencing.
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An exciting recent development is third-generation sequencing (TGS) , which involves sequencing single RNA molecules directly from one end to the other without requiring fragmentation or PCR amplification.
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The HGP aimed to provide complete reference genomes for humans and model organisms; similarly, reference epitranscriptomes that represent specific cell types and conditions in both humans and a set of model organisms will be invaluable for future epitranscriptome studies (see Chapter 3)
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NOTES: Expanding research; advancing experimental and computational tools and technology; developing references and standards of various types; establishing stable, integrated, and centralized data resources; and cultivating innovation across several dimensions, including workforce development, are necessary to fully reveal any epitranscriptome. STUDY SCOPE AND APPROACH The central purpose of this report is to develop a roadmap to guide the development of tools, technologies, and infrastructure that are needed to realize the capability of sequencing any RNA, including all of its modifications, in a robust and accessible way.
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• Scientific and technological hurdles including computational and analytic technologies that need to be overcome to achieve direct sequencing of RNA modifications. • Computational and analytic technologies needed to analyze modified RNA.
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Chapter 3 examines the current state of tools and technologies that exist for identifying, quantifying, and sequencing RNA modifications, including approaches for global modification measurement, indirect sequencing, direct sequencing, and associated computational tools. The chapter goes into further depth by laying out the key gaps and challenges that are not addressed by the currently available technologies and explores future areas of technology development that would impact the RNA modifications field.
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1957. "Ribonucleic acids from yeast which contain a fifth nucleotide." Journal of Biological Chemistry 227 (2)
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2021. "The expanding world of tRNA modifications and their disease relevance." Nature Reviews Molecular Cell Biology 22 (6)
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1959. "Studies of an isomer of uridine isolated from ribonucleic acids." Biochimica et Biophysica Acta 32: 393-406.
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