Genome-wide RNAi screen for human 60S subunit biogenesis factors
Genome-wide RNAi screen identifies novel players in human 60S subunit biogenesis including key enzymes of polyamine metabolism
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2022-02-03
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Abstract
Ribosome assembly is an essential process that is linked to human congenital diseases and tumorigenesis. While great progress has been made in deciphering mechanisms governing ribosome biogenesis in eukaryotes, an inventory of factors that support ribosome synthesis in human cells is still missing, in particular regarding the maturation of the large 60S subunit. Here, we performed a genome-wide RNAi screen using an imaging-based, single cell assay to unravel the cellular machinery promoting 60S subunit assembly in human cells. Two genome wide libraries from different vendors (Qiagen and Ambion) were screened in HeLa cells expressing RPL29-GFP under a tetracycline inducible promoter. Screening plates were imaged by automated microscopy (9 sites per well). Images were subjected to an automated pipeline for illimitation correction, segmentation and feature extraction. Phenotypes of individual cells were then classified using a supervised machine learning algorithm and the rate of cells displaying 60S ribosome biogenesis defects (hitrate) was calculated. Hitrates of individual siRNAs were combined by the redundant siRNA analysis (RSA) algorithm. Genes which ranked high in the genome-wide screen, but were previously not associated with ribosome assembly, were validated using a custom-made library containing pools of 30 siRNAs per gene.
This dataset includes all raw images obtained in the genome-wide screening approach and in the validation screen.
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Contact person : Dörner, Kerstin
Contact person: Kutay, Ulrike
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Publisher
ETH Zurich
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Date collected
2014-2019
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Subject
ribosome biogenesis; 60S ribosomal subunit; Nucleolus; RNAi screening
Organisational unit
03543 - Kutay, Ulrike / Kutay, Ulrike
02891 - ScopeM / ScopeM