Biology subjects

Shachar, R.

Publications and source records attributed to Shachar, R..

3 recordsLinked to original sources

bioRxiv2025

Single-Nucleotide m6A Mapping Uncovers Redundant YTHDF Function in Planarian Progenitor Fate Selection

Cell fate decisions require tight regulation of gene expression. In planarians--highly regenerative flatworms--the mRNA modification N6-methyladenosine (m6A) modulates progenitor production and fate. However, the mechanisms governing m6A deposition in the planarian transcriptome--and the role of their expanded family of YTHDF m6A reader proteins in orchestrating biological functions--remain unclear. Here, we generated the first single-nucleotide resolution map of m6A in planarians, and revealed that simple sequence rules guide m6A deposition, facilitating the flexible evolutionary gain and loss of these marks. Functional analyses of the five planarian m6A readers revealed that while individual reader expression is dispensable, together, the planarian readers regulate the production of specific progenitor lineages and overall body size. Collectively, our findings uncover a robust, redundant regulatory architecture, characterized by multiple m6A sites per gene and coordinated m6A reader expression. This architecture is essential for proper lineage resolution and provides insights into the evolutionary dynamics of the m6A landscape.

developmental biology↗

bioRxiv2024

Passive shaping of intra- and intercellular m6A dynamics via mRNA metabolism

m6A is the most widespread mRNA modification and is primarily implicated in controlling mRNA stability. Fundamental questions pertaining to m6A are the extent to which it is dynamically modulated within cells and across stimuli, and the forces underlying such modulation. Prior work has focused on investigating active mechanisms governing m6A levels, such as recruitment of m6A writers or erasers leading to either global or site-specific modulation. Here, we propose that changes in m6A levels across subcellular compartments and biological trajectories may result from passive changes in gene-level mRNA metabolism. To predict the intricate interdependencies between m6A levels, mRNA localization, and mRNA decay, we establish a differential model m6ADyn encompassing mRNA transcription, methylation, export, and m6A-dependent and independent degradation. We validate the predictions of m6ADyn in the context of intracellular m6A dynamics, where m6ADyn predicts associations between relative mRNA localization and m6A levels, which we experimentally confirm. We further explore m6ADyn predictions pertaining to changes in m6A levels upon controlled perturbations of mRNA metabolism, which we also experimentally confirm. Finally, we demonstrate the relevance of m6ADyn in the context of cellular heat stress response, where genes subjected to altered mRNA product and export also display predictable changes in m6A levels, consistent with m6ADyn predictions. Our findings establish a framework for dissecting m6A dynamics and suggest the role of passive dynamics in shaping m6A levels in mammalian systems.

systems biology↗

bioRxiv2019

Deciphering the ‘m6A code’ via quantitative profiling of m6A at single-nucleotide resolution

N6-methyladenosine (m6A) is the most abundant modification on mRNA, and is implicated in critical roles in development, physiology and disease. The ability to map m6A using immunoprecipitation-based approaches has played a critical role in dissecting m6A functions and mechanisms of action. Yet, these approaches are of limited specificity, unknown sensitivity, and unable to quantify m6A stoichiometry. These limitations have severely hampered our ability to unravel the factors determining where m6A will be deposited, to which levels (the m6A code), and to quantitatively profile m6A dynamics across biological systems. Here, we used the RNase MazF, which cleaves specifically at unmethylated RNA sites, to develop MASTER-seq for systematic quantitative profiling of m6A sites at 16-25% of all m6A sites at single nucleotide resolution. We established MASTER-seq for orthogonal validation and de novo detection of m6A sites, and for tracking of m6A dynamics in yeast gametogenesis and in early mammalian differentiation. We discover that antibody-based approaches severely underestimate the number of m6A sites, and that both the presence of m6A and its stoichiometry are hard-coded via a simple and predictable code within the extended sequence composition at the methylation sites. This code accounts for ~50% of the variability in methylation levels across sites, allows excellent de novo prediction of methylation sites, and predicts methylation acquisition and loss across evolution. We anticipate that MASTER-seq will pave the path towards a more quantitative investigation of m6A biogenesis and regulation in a wide variety of systems, including diverse cell types, stimuli, subcellular components, and disease states.

molecular biology↗