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Appendix F: Ideation Challenge Commissioned Papers
Pages 192-238

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From page 192... ... THE IMPACT OF RNA MODIFICATIONS ON CELLULAR FUNCTION VIA ALTERATIONS TO STRUCTURE AND MACROMOLECULAR INTERACTIONS Author List3 Ulf Andersson Vang Ørom - Aarhus University Nikos Tapinos - Brown University Matthew G Blango - Leibniz Institute for Natural Product Research and Infection Biology: Hans Knöll Institute Jennifer Strasburger - National Human Genome Research Institute Mark Adams - The Jackson Laboratory CHALLENGE RNA structure and RNA modifications are aspects central to RNA function. Read the entire page →
From page 193... ... with experimental and computational approaches. We encourage the community to incorporate these data into a global model that can predict RNA structure, mRNA–protein interactions, and how these are affected by differential RNA modifications. Read the entire page →
From page 194... ... Hence, changing the stoichiometry of modifications may offer another approach to generate functional diversity of mRNA. While current RNA modification profiling methods can map the modification locations, they do not quantify the relative fraction of modified and unmodified RNA for a given transcript or phase modifications along a single RNA strand. Read the entire page →
From page 195... ... 4. Direct RNA sequencing: Several approaches have been described using nanopore direct RNA sequencing technology coupled with artificial intelligence (AI) Read the entire page →
From page 196... ... specific RNA motifs harboring individual modifications, like those implemented in eTAM-seq. Another example comes from the bacterial (Escherichia coli, 196 Prepublication Copy Read the entire page →
From page 197... ... These techniques, although they avoid the false positive signals obtained by MazF and the limited MazF cleavage to ACA motifs, cannot discriminate between m6Am and m1A, are more expensive than traditional MeRIP-seq and are limited to m6A. Development of similar enzyme-assisted approaches that discriminate between other modified and unmodified nucleotides at single-base resolution will significantly enhance our understanding of the biological importance and role of RNA modifications in mammalian systems. Read the entire page →
From page 198... ... . Bioinformatic strategies for processing scRNA-seq and direct RNA sequencing have greatly improved, but to understand RNA modifications of a single molecule within a single cell, work remains (86) Read the entire page →
From page 199... ... . These examples hint at regulation of biological function, but we also know that RNA modifications can influence RNA structure. Read the entire page →
From page 200... ... Specific Actions. Should our dream come to fruition, we expect to have the tools to produce a detailed description of RNA modifications on a single molecule of RNA in a single cell, which would 200 Prepublication Copy Read the entire page →
From page 201... ... . We believe that the strategic, specific, and reliable installation of RNA modifications on synthetic RNA molecules is a technology that is being advanced in many ways, but still needs additional effort to realize a robust method to create useful standards for the types of experiments we are proposing. Read the entire page →
From page 202... ... Beneficiaries: The beneficiaries of biomedical research would be wide ranging, from basic researchers, to patients, to local and national governments, in terms of potential economic impact. In the short term, other basic researchers would benefit the most quickly from improved technologies in RNA structure prediction, molecular interaction detection, and direct RNA sequencing. Read the entire page →
From page 203... ... A more challenging aspect to assess is the prediction of RNA structure and the influence of RNA modifications on structure and interactions with macromolecules. While the field is expected to continuously progress, when can we really claim that we have reached our goal? Read the entire page →
From page 204... ... DirectRMDB: a database of post transcriptional RNA modifications unveiled from direct RNA sequencing technology. Nucleic Acids Res. Read the entire page →
From page 205... ... :208–16. Prepublication Copy 205 Read the entire page →
From page 206... ... :1440–6. 206 Prepublication Copy Read the entire page →
From page 207... ... Identification of differential RNA modifications from nanopore direct RNA sequencing with xPore. Nat Biotechnol. Read the entire page →
From page 208... ... EpiNano: Detection of m6A RNA Modifications Using Oxford Nanopore Direct RNA Sequencing. Methods Mol Biol Clifton NJ. Read the entire page →
From page 209... ... RNA modifications stabilize the tertiary structure of tRNAfMet by locally increasing conformational dynamics. Nucleic Acids Res. Read the entire page →
From page 210... ... 210 Prepublication Copy Read the entire page →
From page 211... ... 4. A lack of availability, sensitivity, multiplexing capability, quantitative accuracy, and/or sample throughput in current methods and technologies for mapping RNA modifications. Read the entire page →
From page 212... ... Through these efforts, the EpiC project would curate and maintain a programmatically accessible database of RNA modification structure, function, and position data. A successful EpiC would be an organization like the existing ENCODE and ENSEMBL projects, drawing funding and leadership from federal sources, while collecting, maintaining, and reporting comprehensive information on all known or newly discovered RNA modifications. Read the entire page →
From page 213... ... . FIGURE F-3 Current methods for mapping RNA modifications categorized according to the analytical technique being used to generate the reporting signal of RNA modifications. Read the entire page →
From page 214... ... Both mass spectrometry and nanopore-based methods can detect RNA modifications directly, and therefore are free from non-specific antibody recognition, incompletion or errors from reverse transcriptase or ligase reactions, or cross-reactivity of some chemical reagents that are utilized in next generation sequencing (NGS) methods. Read the entire page →
From page 215... ... Information should encompass all forms of RNA modifications, across all types of RNA, and spanning all organisms regularly involved in epitranscriptomics research. The information should be presented in an approachable, easy-to-find format, with informative educational resources about RNA modifications and Prepublication Copy 215 Read the entire page →
From page 216... ... Yet, both types of RNA modifications should be documented and deposited in the EpiC Database. A current lack of standardization, data-file formats, and nomenclature prevents consistency in data deposition, notation, and labeling, as well as the universal application of web-based analytic tools to all available or newly generated datasets. Read the entire page →
From page 217... ... Based on the experience from the Human Genome Project, it will certainly be beneficial to many epitranscriptomic studies if regional core facilities for mapping RNA modifications are available, which can also be coordinated and managed by EpiC. The EpiC-participating laboratories will benefit if housed within multidisciplinary research centers. Read the entire page →
From page 218... ... 218 Prepublication Copy Read the entire page →
From page 219... ... Similarly, experts should come to a consensus on optimized protocols that future researchers can use with confidence to ensure reproducibility among experimental results from mapping RNA modifications. Read the entire page →
From page 220... ... IMPACT OF THE EPIC PROJECT By establishing standards, mapping the epitranscriptome, exploring disease associations, and fostering collaboration, the EpiC project will significantly contribute to RNA biology and its translational applications. In particular, the creation of the EpiC database will impact the RNA community by providing an integrated resource of the different types of RNA modifications across all species, tissues, transcripts, developmental stages, and disease states. Read the entire page →
From page 221... ... Expanding the Epitranscriptomic RNA Sequencing and Modification Mapping Mass Spectrometry Toolbox with Prepublication Copy 221 Read the entire page →
From page 222... ... Nucleotide resolution profiling of m3C RNA modification by HAC-seq. Nucleic Acids Res. Read the entire page →
From page 223... ... DirectRMDB: a database of post transcriptional RNA modifications unveiled from direct RNA sequencing technology. Nucleic Acids Res. Read the entire page →
From page 224... ... There has also been substantial growth in the awareness of the multifaceted control these modifications exert over various aspects of RNA biology, including processing, stability, localization, and translation of mRNA into functional proteins. Nevertheless, it is important to recognize that the study of RNA modifications is still in its early stages due to their formidable complexity and dynamic nature. Read the entire page →
From page 225... ... 2. Considering the vast amount of data present at the epitranscriptomic level, the best path forward may be to begin by examining one or a few well-defined human experimental cell Prepublication Copy 225 Read the entire page →
From page 226... ... The database's core structure and features need to accommodate the highly dynamic nature of the epitranscriptome. By their nature, RNA modifications are capable of rapidly adjusting how individual RNA molecules are utilised within cells, operating on much shorter timescales than conventional genetic or epigenetic regulatory mechanisms. Read the entire page →
From page 227... ... By collaborating and sharing standardised protocols, pipelines, and resources, this can help cut costs to individual laboratories, while maintaining the quality and integrity of the research. Therefore, the objectives are twofold: an initial human epitranscriptome will be generated and published as a publicly available resource; and standardised protocols will be established and distributed for any researchers wishing to map RNA modifications in their cell system of choice. Read the entire page →
From page 228... ... CURRENT TECHNOLOGIES AND METHODOLOGIES We are proposing using a combination of validated biochemical methodologies to map individual RNA modifications at single nucleotide resolution. The 2 predominant methods for single nucleotide resolution mapping are shown in Figure F-7 [19] Read the entire page →
From page 229... ... . This method is primarily for the Prepublication Copy 229 Read the entire page →
From page 230... ... . With the establishment of robust biochemical mapping techniques for individual RNA modifications through this consortium, it is entirely realistic that some of these could be combined with current single-cell RNA sequencing methodologies to allow researchers to quantify nuanced differences in the epitranscriptomes of individual cells. Read the entire page →
From page 231... ... Spatial transcriptomics for RNA modifications may be, then, a useful tool to uncover the differential epigenetic modifications and to link them to the tumour microenvironment. While direct RNA mapping, as described above could easily be utilised to sequence spatially barcoded RNA transcripts, other modifications of the technique for mapping modifications may be the use of m6A antibodies, m6A PLA, m6A seqFISH coupled to spatial transcriptomics. Read the entire page →
From page 232... ... . By mapping all RNA modifications, the consortium will lay the foundation for researchers to understand the role of specific modifications (by organism, by tissue, by 232 Prepublication Copy Read the entire page →
From page 233... ... . This builds off works originally pioneering by Dr Karikó and colleagues, who demonstrated that particular RNA modifications, already found on cellular RNAs, can reduce the antagonism of the innate immune response when incorporated into in vitro transcribed RNA prior to transfection [45] Read the entire page →
From page 234... ... 5-Methylcytosine (M5C) RNA Modification Controls the Innate Immune Response to Virus Infection by Regulating Type I Interferons. Read the entire page →
From page 235... ... AlkAniline-Seq: Profiling of M7G and M3C RNA Modifications at Single Nucleotide Resolution. Angewandte Chemie International Edition 2018, 57, 16785–16790, doi:10.1002/ANIE.201810946. Read the entire page →
From page 236... ... Direct Nanopore Sequencing for the 17 RNA Modification Types in 36 Locations in the E Coli Ribosome Enables Monitoring of Stress-Dependent Changes . Read the entire page →
From page 237... ... Prepublication Copy 237 Read the entire page →

From page 192...

... THE IMPACT OF RNA MODIFICATIONS ON CELLULAR FUNCTION VIA ALTERATIONS TO STRUCTURE AND MACROMOLECULAR INTERACTIONS Author List3 Ulf Andersson Vang Ørom - Aarhus University Nikos Tapinos - Brown University Matthew G Blango - Leibniz Institute for Natural Product Research and Infection Biology: Hans Knöll Institute Jennifer Strasburger - National Human Genome Research Institute Mark Adams - The Jackson Laboratory CHALLENGE RNA structure and RNA modifications are aspects central to RNA function.

Appendix F: Ideation Challenge Commissioned Papers Pages 192-238

Appendix F: Ideation Challenge Commissioned Papers
Pages 192-238