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SLFN11 Induces Apoptosis Through Ribosome Biogenesis Impairment

ABSTRACT: Impairment of ribosome biogenesis (RiBi) is triggered by the inhibition of rRNA synthesis and processing and leads to various biological effects. We discovered that Schlafen 11 (SLFN11)-dependent sensitization to replication stress produced by chemotherapeutic drugs induces RiBi impairment. Mechanistically, SLFN11 alters the chromatin structure of rDNA in response to replication stress, sequestering RNA polymerase I on chromatin while inhibiting rRNA synthesis and nucleolar R-loop formation. SLFN11-induced RiBi impairment inhibits translation overall, depleting extremely short-lived proteins, particularly MCL1, an ubiquitous anti-apoptotic protein. Consequently, SLFN11 activates apoptosis in an MCL1-dependent but TP53-independent manner. Comparable effects were observed with RNA polymerase I inhibitors and other RiBi inhibitory conditions regardless of SLFN11. These findings were validated across 34 diverse human cancer cell lines. Thus, we demonstrate that RiBi impairment is a robust inactivator of MCL1 and an activator of apoptosis and identify an additional mechanism by which SLFN11 sensitizes cancer cells to chemotherapeutic agents.

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Junko Murai

PROVIDER: PXD052471 | JPOST Repository | Thu May 22 00:00:00 GMT+01:00 2025

REPOSITORIES: jPOST

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			Action	DRS
	230313mm_003_CEM_Control_%231a.d.zip	Other
	230313mm_004_CEM_CPT-100nM_6hr_%232a.d.zip	Other
	230313mm_005_CEM_Chk1-i-100nM_6hr_%233a.d.zip	Other
	230313mm_007_CB45_Control_%235a.d.zip	Other
	230313mm_008_CB45_CPT-100nM_6hr_%236a.d.zip	Other

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Project description:Epigenetic methyl-CpG silencing of the ribosomal RNA (rRNA) genes is thought to down-regulate rRNA synthesis in mammals. In contrast, we now show that CpG methylation in fact positively influences rRNA synthesis and processing. Human HCT116 cells, inactivated for DNMT1 and DNMT3b or treated with aza-dC, lack CpG methylation and reactivate a large fraction of normally silent rRNA genes. Unexpectedly, these cells display reduced rRNA synthesis and processing, and accumulate unprocessed 45S rRNA. Reactivation of the rRNA genes is associated with their cryptic transcription by RNA polymerase II. Ectopic expression of cryptic rRNA gene transcripts recapitulates the defects associated with loss of CpG methylation. The data demonstrate that rRNA gene silencing prevents cryptic RNA polymerase II transcription of these genes. Lack of silencing leads to the partial disruption of rRNA synthesis and rRNA processing, providing an unexpected explanation for the cytotoxic effects of loss of CpG methylation. Agilent custom microarray analyses of transcripts from sense and antisense strands of the human rRNA genes present in total nuclear RNA from HCT116-DNMT1-/-;DNMT3b-/- (DKO) and HCT116 (PS) cells. The DKO/PS RNA ratio was normalized within the 5â region of the 45S ribosomal RNA, since this sense coding region was found by quantitative Northern and S1-mapping analyses to equally present in the RNA pools from both cells (as compared to total RNA levels and 18S and 28S levels). The distribution for sense RNAs from the coding region is strongly influenced by the predominant RPI 45S rRNA transcript and hence peaks at 0, the DKO and PS 45S rRNA signals having been normalized. 60-mer probes were designed to tile Human rDNA (GenBank accession U13369) on both strands using ArrayOligoSelector (Wardle et al., 2006). A total of 1,146 different non-overlapping probes (573 for each DNA strand) were designed to cover the 43 kb of human rDNA and each printed in triplicate on the array. The experiment was performed using a dye-swap duplicate analysis. The microarrays were scanned on a G2565CA DNA microarray scanner (Agilent Technologies) and the quantification performed using the Feature Extraction software (Agilent technologies). The data were analyzed using the LIMMA software package in R (Smyth and Speed, 2003).

Project description:Myriad of craniofacial disorders are caused by heterozygous mutations in general regulators of housekeeping cellular functions such as ribosome biogenesis. While it is understood that many of these highly tissue-specific malformations are a consequence of defects in cranial neural crest cells (cNCCs), an embryonic cell group that gives rise to most of the facial structures during embryogenesis, the mechanism underlying cell type-selectivity of these effects remains largely unknown. Here we show that DDX21, a DEAD-box RNA helicase involved in control of both RNA polymerase (Pol) I and Pol II dependent transcriptional arms of ribosome biogenesis, is a key mediator of the nucleolar stress response. Using an in vitro model of Treacher Collins Syndrome (TCS), a craniofacial disorder caused by heterozygous mutations in components of the Pol I transcriptional machinery, we demonstrate that perturbations in ribosomal RNA (rRNA) transcription induce DDX21-mediated surveillance mechanism in cNCCs, leading to DDX21 relocalization from the nucleolus to the nucleoplasm, its eviction from Pol I and Pol II chromatin targets and inhibition of rRNA processing. Importantly, these effects are cell type-selective, cell-autonomous and involve activation of the p53 pathway. We further show that cNCCs are characterized by the inherently high reservoir of p53 mRNA and sensitivity to p53-mediated apoptosis. Remarkably, preventing the loss of DDX21 from nucleolus and chromatin can rescue both the sensitivity to apoptosis and TCS-associated craniofacial phenotypes in vivo. This mechanism is not restricted to TCS, as gene function perturbations linked to other ribosomopathies with craniofacial manifestations also lead to the activation of DDX21 surveillance. Lastly, we provide evidence that inhibition of rRNA synthesis leads to rDNA damage, and furthermore, that rDNA damage is sufficient to activate DDX21 surveillance and elicits tissue-selective and dosage-dependent effects on craniofacial development in vivo.

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SLFN11 Induces Apoptosis Through Ribosome Biogenesis Impairment

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