Dynamic Ribosomal RNA Methylation Regulates Translation in the Hematopoietic System and is Essential for Stem Cell Fitness

Rabany, O.; Ben-Dror, S.; Arafat, M.; Aharoni, H.; Halperin, Y.; Marchand, V.; Romanovski, N.; Ussishkin, N.; Livneh, M.; Reches, A.; Wexler, J.; Mayorek, N.; Monderer-Rothkoff, G.; Shifman, S.; Mammer, W.; VanInsberghe, M.; Pauli, C.; Muller-Tidow, C.; Karmi, O.; Livneh, Y.; van Oudenaarden, A.; Motorin, Y.; Nachmani, D.

doi:10.1101/2025.09.29.679281

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bioRxiv · 10.1101/2025.09.29.679281

Dynamic Ribosomal RNA Methylation Regulates Translation in the Hematopoietic System and is Essential for Stem Cell Fitness

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Abstract

Self-renewal and differentiation are at the basis of hematopoiesis. While it is known that tight regulation of translation is vital for hematopoietic stem cells (HSCs) biology, the mechanisms underlying translation regulation across the hematopoietic system remain obscure. Here we reveal a novel mechanism of translation regulation in the hematopoietic hierarchy, which is mediated by ribosomal RNA (rRNA) methylation dynamics. Using ultra-low input ribosome-profiling, we characterized cell-type-specific translation capacity during erythroid differentiation. We found that translation efficiency changes progressively with differentiation and can distinguish between discrete cell populations as well as to define differentiation trajectories. To reveal the underlying mechanism, we performed comprehensive mapping of the most abundant rRNA modification - 2-O-methyl (2OMe). We found that, like translation efficiency, 2OMe dynamics followed a distinct trajectory during erythroid differentiation. Genetic perturbation of individual 2OMe sites demonstrated their distinct roles in modulating proliferation and differentiation. By combining CRISPR screening, molecular and functional analyses, we identified a specific methylation site, 28S-Gm4588, which is progressively lost during differentiation, as a key regulator of HSC self-renewal. We showed that low methylation at this site led to translational skewing, mediated mainly by codon frequency, which promoted differentiation. Functionally, HSCs with diminished 28S-Gm4588 methylation exhibited impaired self-renewal capacity ex-vivo, and loss of fitness in-vivo in bone marrow transplantations. Extending our findings beyond the hematopoietic system, we also found distinct dynamics of 2OMe profiles during differentiation of non-hematopoietic stem cells. Our findings reveal rRNA methylation dynamics as a general mechanism for cell-type-specific translation, required for cell function and differentiation. KEY POINTSO_LIHematopoietic differentiation is associated with rRNA methylation dynamics to control cell-type-specific translation. C_LIO_LITranslation efficiency can distinguish discrete cell types and define differentiation trajectories. C_LIO_LIHSC fitness is regulated by a single rRNA methylation. C_LI

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Rabany, O., Ben-Dror, S., Arafat, M., Aharoni, H., Halperin, Y., Marchand, V., Romanovski, N., Ussishkin, N., Livneh, M., Reches, A., Wexler, J., Mayorek, N., Monderer-Rothkoff, G., Shifman, S., Mammer, W., VanInsberghe, M., Pauli, C., Muller-Tidow, C., Karmi, O., Livneh, Y., van Oudenaarden, A., Motorin, Y., Nachmani, D.. 2025-09-30. Dynamic Ribosomal RNA Methylation Regulates Translation in the Hematopoietic System and is Essential for Stem Cell Fitness. https://doi.org/10.1101/2025.09.29.679281

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