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Transcriptomic m1A - a new key player in the epitranscriptome arena

Periodic Reporting for period 2 - m1ARNA (Transcriptomic m1A - a new key player in the epitranscriptome arena)

Reporting period: 2019-01-01 to 2020-06-30

Reversible epigenetic modifications regulate gene expression to define cell fate and response to environmental stimuli. While DNA and chromatin modifications are well studied, the roles of RNA modifications in regulation of gene expression is only starting to be revealed. RNA modifications were previously known to occur in highly abundant RNA species, such as rRNA and tRNA. However, in recent years, a growing number of modifications have been identified and characterized in low-abundance species of RNA such as mRNA and long non-coding RNA. These modifications, collectively known as the epitranscriptome, regulate RNA processing events such as splicing, transport, translation and turnover, by embedding RNA transcripts with information additional to that carried in their sequence of bases. The discovery of dedicated cellular machineries that deposit, remove and recognize mRNA modifications (known as writers, erasers and readers, respectively) has helped to reveal the essential roles of these modifications in cellular, developmental and disease processes. This study focuses on two major epitranscriptome marks; N6-methyladenosine (m6A) and N1-methyladenosine (m1A).
The information regarding roles of epitranscriptomic modifications in regulation of gene expression was acquired mostly from m6A studies. m6A, the most prevalent internal (non-cap) mRNA modification in eukaryotes, was first discovered in mRNA decades ago, but its study was accelerated only recently by three principal breakthrough discoveries: (1) Development of a high throughput mapping tool, m6A-seq, which exposed its unique and evolutionarily conserved profile; (2) Discovery of specific m6A demethylases, FTO and ALKBH5, that exposed its dynamic nature and established connections to human diseases and developmental defects; (3) Discovery of dedicated m6A readers, belonging to the YTH-domain containing family of RNA binding proteins. Recruitment of m6A readers to methylated transcripts generates a new layer of gene expression regulation that affects many steps in mRNA metabolism, from transcription, splicing and export in the nucleus to translation, degradation and localization in the cytoplasm.
Four years ago, we identified a new epitranscriptomic mark - m1A. m1A mapping uncovered the main features of this methylome: It is greatly enriched around the start codon, suggesting its involvement in regulation of translation; It is dynamically modulated under different physiological stimuli and stress conditions; and it is positively correlated with protein levels. These features are well conserved between human and mouse, alluding to its biological importance.
The main challenge of this research project is to identify the proteins involved in m1A deposition, removal and reading, and through them to uncover the roles of m1A in control of gene expression, particularly in translation regulation. As epigenetic modifications operate in a context-dependent, concerted way, and since both m6A and m1A exhibit a distinct topology, marking the beginnings and ends of coding sequences, respectively, we will decipher their interplay in regulating gene expression via a putative “epitranscriptomic RNA code”. The proposed study will shed light on a new fundamental translational regulatory layer involving m1A. This regulatory mode of action is expected to be involved in central cellular functions, and may be essential to the ability of cells to respond and survive stress conditions. Understanding this mechanism may help elucidate the fine translation regulation of genes in normal and pathological conditions and identify targets amenable to therapeutic manipulations.
The field of m1A is still largely unknown. As the study of m6A was largely impeded until an unbiased transcriptome-wide mapping tool was developed and its erasers and readers were discovered, advancing m1A research depends on accurate mapping tool and on discovery of writers, erasers and readers. We were able to make progress in three out of four of the outstanding questions.
We developed an improved m1A mapping tool with single-nucleotide and single-transcript resolution by incorporating experimental and bioinformatic changes. The new mapping tool identified 824 m1A sites that occur mostly in the 5’UTRs, 59 of them occur in 5’-cap-associated nucleotides, and 416 of the sites were mapped at single transcript resolution.
We identified ALKBH3 as an m1A eraser and generated knockout (KO) cells. Depletion of ALKBH3 increased mRNA m1A levels by ~30%, raised the number of sites by ~19% and increased m1A stoichiometry. Our results are supported by two recent publications. We are currently trying to generate Alkbh3 KO in mESCs for generation of a mouse model.
Reader proteins reveal the cellular machineries involved. RNA affinity chromatography and protein arrays identified 7 putative m1A readers including YTHDF2 and YTHDF3. YTHDF2 was recently reported as an m1A reader affecting transcript stability. We are currently mapping the distribution of YTHDF3 target sites (by eCLIP) and correlating them with m1A sites. Preliminary results show 309 m1A sites overlapping YTHDF3 target sequences. We are also assessing the effect of YTHDF3 depletion on protein levels of m1A- but not m6A-methylated transcripts.
Stress conditions such as starvation (to amino acids, serum or glucose) and heat shock alter m1A mRNA levels. Currently we are integrating the data obtained from m1A profiles, transcriptome sequencing, splicing patterns, RNA stability and differential nuclear/cytoplasmic distribution of methylated transcripts in normal and stress conditions. The different layers of information are integrated to generate a multi-layer correlation analysis and identify differential m1A methylation patterns that affect mRNA metabolism. Special attention is given to the effect of m1A on translation regulation. We also identified m1A-associated differential translation patterns from ribosomal and polysomal profiling, translation efficiency measures and proteomic profiles.
Quantitative analyses of m1A levels in unbound, monosome- and polysome-bound mRNA (dividing mRNA to fractions of increased expression) showed that m1A levels were highest in the polysome-bound and lowest in unbound mRNA fractions. m1A levels in the three fractions increased in ALKBH3 KO compared to WT cells, mostly at the polysomal-bound fraction. Our results clearly indicate that m1A positively regulates translation.
"The objective of this proposal is to study m1A, a new epitranscriptomic mark. Similar to the cracking of other major epitranscriptomic and epigenetic marks, the decoding of m1A is expected to have far-reaching implications on cell and organismal development, homeostasis and disease.
Accumulating data of mRNA modifications suggest that these marks enable cells to promptly respond and adapt to different stimuli and environmental perturbations. It may be postulated 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 decorate different fractions of the same gene transcript population, may result in different outcomes. Such crosstalk can involve different mechanisms, in a manner similar to the crosstalk between DNA methylation and histone modifications. Our research explores the possibility that m1A acts in concert with m6A through decorating mainly the beginnings and ends of the coding region. The study of a possible collaboration of these two epitranscriptomic marks may provide a new regulatory dimension.
Uncovering the roles and regulations of m1A is expected to be important for key physiological and developmental processes. The development of high resolution mapping tools and the identification of key regulatory proteins are expected to facilitate studies aiming at identification of deranged m1A methylation in disease states such as cancer and neurodegeneration. The characterization of the enzymes regulating this modification may provide a basis for a systematic search for compounds that modulate m1A decoration and the downstream processes it mediates.
To summarize, our study extends the knowledge regarding m1A, an important new mRNA modification, to uncover a new layer of regulation and to integrate this it with the massive data available about the well-studied m6A modification in gene expression control. This will enable us to study the interplay between these two central mRNA modifications and to explore their roles in the evolving multilayer epigenetic network that controls gene expression. 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 proposed study is highly multidisciplinary."