Integrated sequencing approach to probe rRNA modification landscape during human embryonic stem cell differentiation

Abstract

The ribosome, long regarded as a passive, uniform machine, has only recently been recognised as a direct regulator of translation. Mass spectrometry and sequencing approaches have shown that heterogeneity in ribosome composition exists, which can actively regulate the translational process. One source of this heterogeneity is the modification of ribosomal RNA (rRNA), primarily pseudouridylation (pseU) and 2′-O-methylation (2OMe), mediated by specific H/ACA and C/D box small nucleolar RNAs (snoRNAs). Here, we investigate how the stoichiometry of rRNA modifications varies during embryonic stem cell differentiation. Using the modification basecalling capability of Nanopore direct RNA sequencing, we have identified distinct stoichiometric changes in modification patterns between pluripotent and differentiated cells, revealing highly dynamic, site-specific regulation. Further, profiling of snoRNA expression during trilineage differentiation revealed differential expression of H/ACA and C/D box snoRNAs responsible for a subset of these dynamic modifications. By integrating rRNA and snoRNA sequencing approaches, we have built a comprehensive profile of rRNA modification dynamics during early embryonic cell fate decisions, highlighting potential regulatory mechanisms for ribosome heterogeneity during development. GRAPHICAL ABSTRACT