Dataset Information

Aryl Sulfonamides Degrade RBM39 and RBM23 by Recruitment to CRL4-DCAF15.

ABSTRACT: Indisulam and related sulfonamides recruit the splicing factor RBM39 to the CRL4-DCAF15 E3 ubiquitin ligase, resulting in RBM39 ubiquitination and degradation. Here, we used a combination of domain mapping and random mutagenesis to identify domains or residues that are necessary for indisulam-dependent RBM39 ubiquitination. DCAF15 mutations at Q232 or D475 prevent RBM39 recruitment by indisulam. RBM39 is recruited to DCAF15 by its RRM2 (RNA recognition motif 2) and is ubiquitinated on its N terminus. RBM23, which is an RBM39 paralog, is also recruited to the CRL4-DCAF15 ligase through its RRM2 domain and undergoes sulfonamide-dependent degradation. Indisulam alters the expression of more than 3,000 genes and causes widespread intron retention and exon skipping. All of these changes can be attributed to RBM39, and none are the consequence of RBM23 degradation. Our findings demonstrate that indisulam selectively degrades RBM23 and RBM39, the latter of which is critically important for splicing and gene expression.

SUBMITTER: Ting TC

PROVIDER: S-EPMC7950731 | biostudies-literature | 2019 Nov

REPOSITORIES: biostudies-literature

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Publications

Aryl Sulfonamides Degrade RBM39 and RBM23 by Recruitment to CRL4-DCAF15.

Ting Tabitha C TC Goralski Maria M Klein Katherine K Wang Baiyun B Kim Jiwoong J Xie Yang Y Nijhawan Deepak D

Cell reports 20191101 6

Indisulam and related sulfonamides recruit the splicing factor RBM39 to the CRL4-DCAF15 E3 ubiquitin ligase, resulting in RBM39 ubiquitination and degradation. Here, we used a combination of domain mapping and random mutagenesis to identify domains or residues that are necessary for indisulam-dependent RBM39 ubiquitination. DCAF15 mutations at Q232 or D475 prevent RBM39 recruitment by indisulam. RBM39 is recruited to DCAF15 by its RRM2 (RNA recognition motif 2) and is ubiquitinated on its N term ...[more]

PMID: 31693891

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Project description:BackgroundVpr is a virion-associated protein that is encoded by lentiviruses and serves to counteract intrinsic immunity factors that restrict infection. HIV-1 Vpr mediates proteasome-dependent degradation of several DNA repair/modification proteins. Mechanistically, Vpr directly recruits cellular targets onto DCAF1, a substrate receptor of Cullin 4 RING E3 ubiquitin ligase (CRL4) for poly-ubiquitination. Further, Vpr can mediate poly-ubiquitination of DCAF1-interacting proteins by the CRL4. Because Vpr-mediated degradation of its known targets can not explain the primary cell-cycle arrest phenotype that Vpr expression induces, we surveyed the literature for DNA-repair-associated proteins that interact with the CRL4-DCAF1. One such protein is SIRT7, a deacetylase of histone 3 that belongs to the Sirtuin family and regulates a wide range of cellular processes. We wondered whether Vpr can mediate degradation of SIRT7 via the CRL4-DCAF1.MethodsHEK293T cells were transfected with cocktails of plasmids expressing DCAF1, DDB1, SIRT7 and Vpr. Ectopic and endogeneous levels of SIRT7 were monitered by immunoblotting and protein-protein interactions were assessed by immunoprecipitation. For in vitro reconstitution assays, recombinant CRL4-DCAF1-Vpr complexes and SIRT7 were prepared and poly-ubiqutination of SIRT7 was monitored with immunoblotting.ResultsWe demonstrate SIRT7 polyubiquitination and degradation upon Vpr expression. Specifically, SIRT7 is shown to interact with the CRL4-DCAF1 complex, and expression of Vpr in HEK293T cells results in SIRT7 degradation, which is partially rescued by CRL inhibitor MNL4924 and proteasome inhibitor MG132. Further, in vitro reconstitution assays show that Vpr induces poly-ubiquitination of SIRT7 by the CRL4-DCAF1. Importantly, we find that Vpr from several different HIV-1 strains, but not HIV-2 strains, mediates SIRT7 poly-ubiquitination in the reconstitution assay and degradation in cells. Finally, we show that SIRT7 degradation by Vpr is independent of the known, distinctive phenotype of Vpr-induced cell cycle arrest at the G2 phase, CONCLUSIONS: Targeting histone deacetylase SIRT7 for degradation is a conserved feature of HIV-1 Vpr. Altogether, our findings reveal that HIV-1 Vpr mediates down-regulation of SIRT7 by a mechanism that does not involve novel target recruitment to the CRL4-DCAF1 but instead involves regulation of the E3 ligase activity.

Dataset Information

Aryl Sulfonamides Degrade RBM39 and RBM23 by Recruitment to CRL4-DCAF15.

Publications

Aryl Sulfonamides Degrade RBM39 and RBM23 by Recruitment to CRL4-DCAF15.

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