Saturation mutagenesis of the human 5S rRNA gene defines expression- and ribosome-incorporation-defective variants

Cells were fractionated on sucrose gradients and 5S rRNA sequenced from the input (total) RNA and the assembled 60S large ribosomal subunit, alongside the input plasmid DNA library. Comparing the plasmid library to total RNA measures per-variant expression/stability; comparing total RNA to the 60S fraction measures incorporation of each variant into the assembling large subunit, the step monitored by the 5S RNP-p53 surveillance pathway.