CRISPR screening identifies nucleolar RPL22 as a heterochromatin destabilizer and senescence driver

Dysfunction of the ribosome manifests during cellular senescence and contributes to tissue aging, functional decline, and development of aging-related disorders in ways that have remained enigmatic. Here, we conducted a comprehensive CRISPR-based loss-of-function (LOF) screen of ribosome-associated genes (RAGs) in human mesenchymal progenitor cells (hMPCs). Through this approach, we identified ribosomal protein L22 (RPL22) as the foremost RAG whose deficiency mitigates the effects of cellular senescence. Consequently, absence of RPL22 delays hMPCs from becoming senescent, while an excess of RPL22 accelerates the senescence process. Mechanistically, we found in senescent hMPCs, that RPL22 accumulates within the nucleolus. This accumulation triggers a cascade of events, including heterochromatin decompaction with concomitant degradation of key heterochromatin proteins, specifically heterochromatin protein 1? (HP1?) and heterochromatin protein KRAB-associated protein 1 (KAP1). Subsequently, RPL22-dependent breakdown of heterochromatin stimulates the transcription of ribosomal RNAs (rRNAs), triggering cellular senescence. In summary, our findings unveil a novel role for nucleolar RPL22 as a destabilizer of heterochromatin and a driver of cellular senescence, shedding new light on the intricate mechanisms underlying the aging process. Overall design: For our CRISPR screen, we first constructed a RAG library containing single guide RNAs (sgRNAs) targeting ribosome-related genes and based on the lentiCRISPRv2 plasmid (Addgene # 52961) as previously described. Then, we obtained a lentivirus library and infected senescent hMPCs at a low multiplicity of infection (MOI) (0.3) to ensure that one cell receives no more than one sgRNA. At the same time, we set up a control in which hMPCs were transduced with non-targeting control (NTC) at the same MOI. After two days, the cells transduced with sgRNA were put under puromycin selection (1 µg/ml) (InvivoGen) for six days, after which the cells switched to fresh medium without puromycin and passaged until the control cells underwent growth arrest. We harvested cells at different time points and extracted the genomic DNA using TIANamp Genomic DNA Kit (TIANGEN), according to the manufacturer's protocol.