Analysis of ribosomes from the Wild-type and FMR1 knockout human embryonic stem cells

Fragile X Messenger Ribonucleoprotein 1 (FMRP) is a multifunctional, multidomain RNA-binding protein whose loss causes Fragile X syndrome. It is also known to associate with ribosomes and modulate translation. In human embryonic stem cells (hESCs), knockout (KO) of the FMR1 gene results in significantly increased protein translation rates and alterations in the 2-O-methylation patterns of rRNA. To understand the structural underpinnings of the process, we performed electron cryomicroscopy analysis of ribosomes isolated from both wild-type (WT) and FMR1 KO hESCs that revealed a subpopulation of dormant ribosomes in the FMR1 KO cells, in addition to ribosomes with tRNAs. This dormant subpopulation, absent in the WT hESCs, is characterized by the binding of SERPINE-mRNA binding protein 1 in the mRNA tunnel and eukaryotic elongation factor 2 near the A-site, preventing translation. The presence of elevated protein translation in FMR1 KO cells, alongside a subpopulation of inactive ribosomes, suggests that FMRP can function as a translational brake. However, due to the high cost of ribosome recycling, the cell appears to adopt a strategy of maintaining a subset of dormant ribosomes. Additionally, we analysed the 2-O-methylation patterns in the 28S rRNA, in both the WT and FMR1 KO hESCs, identifying few potential differentially methylated sites. Thus, these findings provide insights into the mechanisms of ribosome dormancy in the absence of FMRP and lay the groundwork for understanding the role of rRNA methylation in translational regulation.