Nonsense Mediated RNA Decay Promotes Survival of Cells with Defective Splicing

Nonsense mediated RNA decay (NMD) is an RNA surveillance pathway present in all eukaryotes that detects and degrades nonsense mRNAs, which contain pre-mature translation termination codons. Nonsense mRNAs are prevalent when pre-mRNA splicing is altered or defective. Interestingly, defective pre-mRNA splicing is emerging as a major driver of cancer development, including development of myelodysplastic syndrome (MDS), leukemia, and some solid tumors. Moreover, pre-mRNA splicing is also thought to enhance NMD in human cells, although itճ still unclear whether and how splicing or splicing factors promote NMD. The role of NMD in regulating mis-spliced mRNA and the link between NMD and RNA splicing, suggest that understanding the process of NMD in the context of normal and defective splicing may hold some clues on developing therapies to treat cancers with dysregulated splicing. To better understand the process of NMD, we have developed a novel NMD reporter system to measure NMD activity in individual human cells and used it to perform a genome-wide CRISPR/Cas9 KO screen to identify genes that promote NMD. We found that the SF3B spliceosome complex promotes NMD without splicing of the target mRNA, suggesting that recruitment of certain spliceosome factors, but not pre-mRNA splicing per se, promotes NMD. In the context of defective splicing, we found that expression of cancer-associated spliceosome mutants (including mutant SF3B1) attenuate NMD. Importantly, cancer cells harboring spliceosome mutations were remarkably sensitive to inhibition of NMD. Therefore, inhibition of NMD is a novel potential therapeutic strategy to treat cancers with defective splicing. This finding suggests that small molecule inhibitors of NMD are needed to facilitate development of therapies that target the NMD pathway. In this dissertation, we have evaluated the use of two different compounds to inhibit NMD. SMG1i directly targets SMG1, the only kinase in the NMD pathway, while Compound C, a commonly used AMPK inhibitor, inhibits NMD indirectly probably by down-regulating NMD factors. Compound C is, however, non-specific, but its derivatives may generate specific NMD inhibitors. Collectively, our studies shed some new light on the process of NMD in the context of normal or defective splicing, uncover NMD as a novel vulnerability of cancers with defective splicing, and provide promising lead compounds for developing therapies that target NMD for cancer treatment

Compound C inhibits nonsense-mediated RNA decay independently of AMPK

The nonsense mediated RNA decay (NMD) pathway safeguards the integrity of the transcriptome by targeting mRNAs with premature translation termination codons (PTCs) for degradation. It also regulates gene expression by degrading a large number of non-mutant RNAs (including mRNAs and noncoding RNAs) that bear NMD-inducing features. Consequently, NMD has been shown to influence development, cellular response to stress, and clinical outcome of many genetic diseases. Small molecules that can modulate NMD activity provide critical tools for understanding the mechanism and physiological functions of NMD, and they also offer potential means for treating certain genetic diseases and cancer. Therefore, there is an intense interest in identifying small-molecule NMD inhibitors or enhancers. It was previously reported that both inhibition of NMD and treatment with the AMPK-selective inhibitor Compound C (CC) induce autophagy in human cells, raising the possibility that CC may be capable of inhibiting NMD. Here we show that CC indeed has a NMD-inhibitory activity. Inhibition of NMD by CC is, however, independent of AMPK activity. As a competitive ATP analog, CC does not affect the kinase activity of SMG1, an essential NMD factor and the only known kinase in the NMD pathway. However, CC treatment down-regulates the protein levels of several NMD factors. The induction of autophagy by CC treatment is independent of ATF4, a NMD target that has been shown to promote autophagy in response to NMD inhibition. Our results reveal a new activity of CC as a NMD inhibitor, which has implications for its use in basic research and drug development

USP51 deubiquitylates H2AK13,15ub and regulates DNA damage response

Dynamic regulation of RNF168-mediated ubiquitylation of histone H2A Lys13,15 (H2AK13,15ub) at DNA double-strand breaks (DSBs) is crucial for preventing aberrant DNA repair and maintaining genome stability. However, it remains unclear which deubiquitylating enzyme (DUB) removes H2AK13,15ub. Here we show that USP51, a previously uncharacterized DUB, deubiquitylates H2AK13,15ub and regulates DNA damage response. USP51 depletion results in increased spontaneous DNA damage foci and elevated levels of H2AK15ub and impairs DNA damage response. USP51 overexpression suppresses the formation of ionizing radiation-induced 53BP1 and BRCA1 but not RNF168 foci, suggesting that USP51 functions downstream from RNF168 in DNA damage response. In vitro, USP51 binds to H2A–H2B directly and deubiquitylates H2AK13,15ub. In cells, USP51 is recruited to chromatin after DNA damage and regulates the dynamic assembly/disassembly of 53BP1 and BRCA1 foci. These results show that USP51 is the DUB for H2AK13,15ub and regulates DNA damage response

Context-dependent pro- and anti-resection roles of ZKSCAN3 in the regulation of fork processing during replication stress

Uncontrolled resection of replication forks under stress can cause genomic instability and influence cancer formation. Extensive fork resection has also been implicated in the chemosensitivity of BReast CAncer gene BRCA-deficient cancers. However, how fork resection is controlled in different genetic contexts and how it affects chromosomal stability and cell survival remains incompletely understood. Here, we report a novel function of the transcription repressor ZKSCAN3 in fork protection and chromosomal stability maintenance under replication stress. We show disruption of ZKSCAN3 function causes excessive resection of replication forks by the exonuclease Exo1 and homologous DNA recombination/repair protein Mre11 following fork reversal. Interestingly, in BRCA1-deficient cells, we found ZKSCAN3 actually promotes fork resection upon replication stress. We demonstrate these anti- and pro-resection roles of ZKSCAN3, consisting of a SCAN box, Kruppel-associated box, and zinc finger domain, are mediated by its SCAN box domain and do not require the Kruppel-associated box or zinc finger domains, suggesting that the transcriptional function of ZKSCAN3 is not involved. Furthermore, despite the severe impact on fork structure and chromosomal stability, depletion of ZKSCAN3 did not affect the short-term survival of BRCA1-proficient or BRCA1-deficient cells after treatment with cancer drugs hydroxyurea, PARPi, or cisplatin. Our findings reveal a unique relationship between ZKSCAN3 and BRCA1 in fork protection and add to our understanding of the relationships between replication fork protection, chromosomal instability, and chemosensitivity