Exploring the landscape of ribosome heterogeneity in human embryonic stem cells during differentiation

Ribosomes are universally conserved molecular machines responsible for protein synthesis. Over recent years, evidence from different model systems has revealed that ribosome composition can be heterogeneous, which generates subpopulations of ribosomes that have the potential for regulating translation in different cellular processes. One process that involves a large change in translational profile is cell fate decisions, in particular, gastrulation of embryonic stem cells into the three primary germ layers. Using in vitro trilineage differentiation of human embryonic stem cells as a model, ribosome heterogeneity was characterised by measuring the changes in ribosomal proteins (RPs) mRNA expression, snoRNA expression, and rRNA modification stoichiometry. Analysis of RP mRNA expression revealed that the expression of an RP paralog, eL39L, is enriched during pluripotency, suggesting that eL39L-containing ribosomes may mediate specialised translation of pluripotency-associated mRNAs. To investigate the functions of eL39L, an epitope tag was introduced to the N-terminus of eL39L by genome editing, which would enable isolation of eL39L-containing ribosomes by immunoprecipitation. Cell lines containing edited eL39L were generated, but tagged eL39L expression was not evident, meaning that the cell lines were not suitable for downstream analyses. To explore rRNA modification heterogeneity during cell fate decisions, small RNA sequencing, and direct RNA sequencing were carried out to investigate the changes in snoRNA expression and rRNA pseudouridylation (pseU) and 2′-O-methylation (2OMe), respectively. pseU and 2OMe modification levels across all known sites in the rRNA were quantified. Distinct patterns of pseU and 2OMe stoichiometric changes between pluripotent and differentiated cells were also observed. Integrating rRNA modification and guide snoRNA expression data revealed sites where expression changes were correlated, which may indicate functional relevance during cell fate decisions. In summary, aspects of RP and rRNA modification heterogeneity were explored, but the potential translational regulation from heterogeneous ribosomes for cell fate decisions remains to be elucidated.