CPSF3-dependent pre-mRNA processing as a druggable node in AML and Ewing’s sarcoma

Ross, Nathan T.; Lohmann, Felix; Carbonneau, Seth; Fazal, Aleem; Weihofen, W.A.; Gleim, Scott; Salcius, Michael; Sigoillot, Frederic; Hénault, Martin; Carl, Sarah H.; Rodríguez-Molina, Juan B; Miller, Howard L.; Brittain, Scott M.; Murphy, Jason; Zambrowski, M.; Boynton, Geoffrey M.; Wang, Yuan; Chen, Aye; Molind, Gregory J; Wilbertz, Johannes; Artus‐Revel, Caroline G; Jia, Min; Akinjiyan, Favour A.; Turner, Jonathan; Knehr, Judith; Carbone, Walter; Schuierer, Sven; Reece‐Hoyes, John S.; Xie, Kevin; Saran, Chitra; Williams, Eric T.; Roma, Guglielmo; Spencer, Matt; Jenkins, Jeremy L.; George, Elizabeth; Thomas, Jason R.; Michaud, Gregory A.; Schirle, Markus; Tallarico, John A.; Passmore, Lori A.; Chao, Jeffrey A.; Beckwith, Rohan E. J.

doi:10.1038/s41589-019-0424-1

Cited by 73 publications

(99 citation statements)

References 57 publications

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“…It is likely that the predominant cytoplasmic and nucleolar staining of S9.6 observed here and in other studies reflects large pools of RNAs with duplexed structural features for which S9.6 has an affinity. We note that lower cytoplasmic S9.6 staining has been observed in some studies [8,35,46], differing from the observations made here. These differences can be attributed to the use of various pre-extractions, chemical treatments, and washing steps prior to immunolabeling that we did not employ.…”

Section: Discussioncontrasting

confidence: 99%

Recognition of cellular RNAs by the S9.6 antibody creates pervasive artefacts when imaging RNA:DNA hybrids

Chédin

Smolka

Sanz

et al. 2020

Preprint

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The contribution of RNA:DNA hybrid metabolism to cellular processes and disease states has become a prominent topic of study. The S9.6 antibody recognizes RNA:DNA hybrids with a subnanomolar affinity, making it a broadly used tool to detect and study RNA:DNA hybrids. However, S9.6 also binds double-stranded RNA in vitro with significant affinity. Though frequently used in immunofluorescence microscopy, the possible reactivity of S9.6 with non-RNA:DNA hybrid substrates in situ, particularly RNA, has not been comprehensively addressed. Furthermore, S9.6 immunofluorescence microscopy has been methodologically variable and generated discordant imaging datasets. In this study, we find that the majority of the S9.6 immunofluorescence signal observed in fixed human cells arises from RNA, not RNA:DNA hybrids. S9.6 staining was quantitatively unchanged by pre-treatment with the human RNA:DNA hybrid-specific nuclease, RNase H1, despite experimental verification in situ that S9.6 could recognize RNA:DNA hybrids and that RNase H1 was active. S9.6 staining was, however, significantly sensitive to pre-treatments with RNase T1, and in some cases RNase III, two ribonucleases that specifically degrade singlestranded and double-stranded RNA, respectively. In contrast, genome-wide maps obtained by high-throughput DNA sequencing after S9.6-mediated DNA:RNA Immunoprecipitation (DRIP) are RNase H1-sensitive and RNase T1-and RNase IIIinsensitive. Altogether, these data demonstrate that the S9.6 antibody, though capable of recognizing RNA:DNA hybrids in situ and in vitro, suffers from a lack of specificity that precludes reliable imaging of RNA:DNA hybrids and renders associated imaging data inconclusive in the absence of controls for its promiscuous recognition of cellular RNAs.

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Section: Discussioncontrasting

confidence: 99%

Recognition of cellular RNAs by the S9.6 antibody creates pervasive artefacts when imaging RNA:DNA hybrids

Chédin

Smolka

Sanz

et al. 2020

Preprint

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“…(b). Structure of human CPSF73 catalytic module (in color) in complex with the acid form of JTE-607 (in black) [95], overlaid with that in complex with sulfate in a closed form [17]. (c).…”

Section: Future Perspectivesmentioning

confidence: 99%

Recent molecular insights into canonical pre-mRNA 3’-end processing

2020

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The majority of eukaryotic messenger RNA precursors (pre-mRNAs) undergo cleavage and polyadenylation at their 3′ end. This canonical 3′-end processing depends on sequence elements in the pre-mRNA as well as a mega-dalton protein machinery. The cleavage site in mammalian pre-mRNAs is located between an upstream poly(A) signal, most frequently an AAUAAA hexamer, and a GU-rich downstream sequence element. This review will summarize recent advances from the studies on this canonical 3′-end processing machinery. They have revealed the molecular mechanism for the recognition of the poly(A) signal and provided the first glimpse into the overall architecture of the machinery. The studies also show that the machinery is highly dynamic conformationally, and extensive rearrangements are necessary for its activation. Inhibitors targeting the active site of the CPSF73 nuclease of this machinery have anti-cancer, anti-inflammatory and anti-protozoal effects, indicating that CPSF73 and pre-mRNA 3′-end processing in general are attractive targets for drug discovery.

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“…It is noteworthy that the Y328 mutations significantly impacted the overall growth fitness ( Figure 3 C), suggesting a default in Tg CPSF3 Y328H/C activity. As the G456S mutation in T. gondii is equivalent to the G330S mutation found in the human CPSF3 counterpart conferring resistance against the anti-cancer agent JTE-607 ( Ross et al., 2020 ), it is likely that the mechanism of resistance is shared. Possibly, the G330S and G456S mutations can only be effective for elongated molecules to clash with the compound thereby impeding binding without affecting recognition of the substrate.…”

Section: Discussionmentioning

confidence: 99%

Target Identification of an Antimalarial Oxaborole Identifies AN13762 as an Alternative Chemotype for Targeting CPSF3 in Apicomplexan Parasites

et al. 2020

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Summary Boron-containing compounds represent a promising class of molecules with proven efficacy against a wide range of pathogens, including apicomplexan parasites. Following lead optimization, the benzoxaborole AN13762 was identified as a preclinical candidate against the human malaria parasite, yet the molecular target remained uncertain. Here, we uncovered the parasiticidal mechanisms of AN13762, by combining forward genetics with transcriptome sequencing and computational mutation discovery and using Toxoplasma gondii as a relevant model for Apicomplexa. AN13762 was shown to target Tg CPSF3, the catalytic subunit of the pre-mRNA cleavage and polyadenylation complex, as the anti-pan-apicomplexan benzoxaborole compound, AN3661. However, unique mutations within the Tg CPSF3 catalytic site conferring resistance to AN13762 do not confer cross-protection against AN3661, suggesting a divergent resistance mechanism. Finally, in agreement with the high sequence conservation of CPSF3 between Toxoplasma and Cryptosporidium , AN13762 shows oral efficacy in cryptosporidiosis mouse model, a disease for which new drug development is of high priority.

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CPSF3-dependent pre-mRNA processing as a druggable node in AML and Ewing’s sarcoma

Cited by 73 publications

References 57 publications

Recognition of cellular RNAs by the S9.6 antibody creates pervasive artefacts when imaging RNA:DNA hybrids

Recognition of cellular RNAs by the S9.6 antibody creates pervasive artefacts when imaging RNA:DNA hybrids

Recent molecular insights into canonical pre-mRNA 3’-end processing

Target Identification of an Antimalarial Oxaborole Identifies AN13762 as an Alternative Chemotype for Targeting CPSF3 in Apicomplexan Parasites

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