Periodic Reporting for period 4 - m1ARNA (Transcriptomic m1A - a new key player in the epitranscriptome arena) Reporting period: 2022-01-01 to 2022-06-30 Summary of the context and overall objectives of the project Reversible epigenetic modifications regulate gene expression to define cell fate and response to environmental stimuli. While DNA and chromatin modifications are well characterized, the study of mRNA modifications in regulation of gene expression was ignited only in the last decade, revealing a growing number of modifications, collectively known as the epitranscriptome, that regulate RNA processing by embedding transcripts with information additional to their sequence. The discovery of dedicated cellular machineries that deposit, remove and recognize mRNA modifications (writers, erasers and readers, respectively) uncovered their essential roles in cellular, developmental and disease states. The information regarding roles of epitranscriptomic marks in regulation of gene expression was mostly obtained from N6-methyladenosine (m6A) studies. m6A was discovered in mRNA decades ago, but its study was accelerated by three principal breakthrough discoveries: (1) Development of a high throughput mapping tools, exposing its unique and evolutionarily conserved profile; (2) Discovery of specific m6A erasers, FTO and ALKBH5, that exposed its dynamic nature; (3) Discovery of dedicated m6A readers. Recruitment of m6A readers to methylated transcripts generates a new layer of gene expression regulation that affects mRNA metabolism from transcription to degradation.Our discovery of N1-methyladenosine (m1A) in mRNA and our initial studies revealed that it is enriched around the start codon, dynamically modulated under different physiological stimuli and stress conditions, and positively correlated with protein levels, suggesting a role for m1A in translation regulation. The main challenge of our study was to uncover how m1A regulates gene expression. As epigenetic modifications operate in a context-dependent, concerted way, and since both m1A and m6A exhibit distinct topologies, marking the beginnings and ends of coding sequences, respectively, we studied their interplay in regulating gene expression via a putative “epitranscriptomic RNA code”. Uncovering and understanding the m1A-mediated regulatory mechanisms, that may be involved in central cellular functions, may help elucidate the fine translation regulation of genes in normal and pathological conditions and identify new targets amenable to therapeutic manipulations. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far The field of m1A is still largely unknown. As the study of m6A was impeded until an unbiased transcriptome-wide mapping tool was developed and its erasers and readers were discovered, advancing m1A research requires accurate mapping tools and discovery of its writers, erasers and readers. We were able to make progress in three out of four of these outstanding needs.We developed a new methodology for mapping m1A obtain single-nucleotide and single-transcript resolution by combining new experimental and bioinformatic approaches. The new method identified hundreds of m1A sites that occur mostly in the 5’UTRs, with a small subset of sites (~7%) occurring in 5’-cap-associated nucleotides. The dynamic nature of m1A reflects activity of eraser proteins. We identified ALKBH3 as an m1A eraser and generated knockout cells. Depletion of ALKBH3 increased m1A levels by ~30%, raised the number of sites by ~19% and increased overall m1A stoichiometry. The effect of m1A on gene expression is more prominent in Hodgkin lymphoma cells, when depletion of ALKBH3 results in elevated m1A levels in mRNA and correlation with poor clinical outcome.A major challenge of our research was to identify m1A readers. Reader proteins execute modification-dependent, gene-expression regulation programs and may help reveal the cellular machineries involved. RNA affinity chromatography and protein arrays identified putative m1A readers including YTHDF2 and YTHDF3. eCLIP experiments revealed YTHDF2 and YTHDF3 targets that overlapped the m1A sites identified by our novel method. Moreover, depletion of ALKBH3 increased the precipitated fraction of YTHDF3-m1A targets, whereas reducing the levels of the m1A writer TRMT6 generated the opposite effect. We integrated the data obtained from: m1A quantifying, mapping, profiling gene expression, and the prevalence of transcripts in translateable pools. Data was collected under normal and stress conditions, to generate a multilayered condition-specific multilayered profiles. We found that m1A in mRNA is correlated with increased translation in a YTHDF3-dependent manner and reduced stability in a YTHDF2-dependent manner. Combining this information with data obtained from m6A studies uncovered reverse correlation between m1A and m6A levels in mRNA under stress conditions such as heat shock. Analysis of transcripts that are marked by both modifications revealed that the number of transcripts that were found to be methylated in m1A and in m6A, exceeded the numbers expected by probability, both in mouse (mESCs) and in human (HepG2), indicating that similar to epigenetic marks, mRNA modifications operate in a context-dependent concerted way. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) The main objective of this research was to study the function of m1A in mRNA with respect to gene expression regulation. Similar to other major epitranscriptomic and epigenetic marks, the decoding of m1A can have far-reaching implications on cell and organismal development, homeostasis and disease. The ability to survive environmental perturbations, differentiate and respond to stimuli depends on the successful switching from one transcriptional program to another. Accumulating data suggest that mRNA modifications enable cells to promptly respond and adapt to stimuli and environmental perturbations by inducing such switching. It is reasonable to speculate that, similarly to the “histone code”, mRNA modifications cooperate as part of an epitranscriptomic network to generate an "epitranscriptomic code”. A crosstalk between different modifications that decorate the same transcript molecule, or different fractions of the same gene transcript population, may dictate different outcomes. Our research explored the possibility that m1A acts in concert with m6A through marking mainly the start and stop of the coding region, to provide a new regulatory dimension. We show that co-modification of these epitranscriptomic marks of the same transcripts is non-random, supporting an interplay between mRNA modifications to finely control gene expression.To summarize, our study extends the knowledge regarding m1A, an important new mRNA modification, to uncover a new layer of regulation and to integrate it with the massive data available about the well-studied m6A modification in gene expression control. Although many regulatory mechanisms were identified over the years it is clear that gene expression patterns at any state reflect the overall result of numerous mechanisms acting in concert to produce each pattern. Providing a scientific snapshot of the interplay between different mRNA modifications, based on solid characterization of each modification individually, is not an easy task as the study is highly multidisciplinary. Although our study revealed major m1A-dependent regulatory mechanisms of gene expression, but more discoveries are needed to fully understand the different mechanisms involved. m1a.jpg
