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

Abstract

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 structural analysis of ribosomes isolated from both the wild-type (WT) and the FMR1 KO hESCs, which revealed populations of ribosomes with distinct protein factors and tRNA in the FMR1 KO ribosome preparations. The bound protein factors are hallmarks of dormant ribosomes, which are currently not observed in the WT hESCs, and are characterized by the binding of SERPINE-mRNA binding protein 1 in the mRNA tunnel and eukaryotic elongation factor 2 near the A-site. The observation of elevated protein translation in FMR1 KO cells, alongside a subpopulation of dormant ribosomes, further substantiates the role of FMRP in translational regulation. Additionally, we analysed the 2'-O-methylation patterns in the 28S rRNA, in both the WT and the FMR1 KO hESCs, identifying a few potential differentially methylated sites. Thus, these observations provide insights into the mechanisms of ribosome dormancy in the absence of FMRP and in understanding the role of rRNA methylation in translational regulation.