@article{6fab4828bf8947a1aa7f045b584f97f9,
title = "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.",
keywords = "CRL4, DCAF15, RBM23, RBM39, sulfonamides",
author = "Ting, {Tabitha C.} and Maria Goralski and Katherine Klein and Baiyun Wang and Jiwoong Kim and Yang Xie and Deepak Nijhawan",
note = "Funding Information: The authors thank Nicholas Gaskill for his optimizations for the in vitro ubiquitination reaction, including buffer composition, protein reagent preparation, design of constructs for insect cell expression, and protocol development. We also thank Thu Nguyen for helpful discussions regarding RBM23 and David McFadden for reviewing the manuscript. T.C.T. is supported by a McKnight graduate student fellowship. This work was supported by grants issued to D.N. from the Welch Foundation (I-1879) and the National Cancer Institute (R37CA226771 and RO1CA217333). D.N. and T.C.T. designed experiments and wrote the manuscript. T.C.T. performed protein purifications from SF9 insect cells, in vitro ubiquitination assays, RBM39 ubiquitination site analysis, RBM39- and RBM23-binding site analyses, endogenous tagging of RBM23-AID-3xFLAG cell lines, and RNA sequencing experiments. M.G. performed degradation analyses for DCAF15 mutants and RBM23, generated baculoviruses for SF9 expression of DCAF15 mutants, carried out immunoprecipitations (IPs) for DCAF15 mutant binding analyses, and generated endogenously tagged RBM23-3xFLAG cell lines in the background of parental HCT116, DCAF15 single guide RNA (sgRNA), and RBM39 G268V cells. K.K. performed the DCAF15 error-prone mutagenesis screen and performed initial validation analyses of DCAF15 mutants. B.W. purified RBM23 and RBM39 minimal binding site mutants from E. coli and performed FRET assays. J.K. performed statistical analyses of data from the DCAF15 mutagenesis screen and RNA sequencing. The authors declare no competing interests. Funding Information: The authors thank Nicholas Gaskill for his optimizations for the in vitro ubiquitination reaction, including buffer composition, protein reagent preparation, design of constructs for insect cell expression, and protocol development. We also thank Thu Nguyen for helpful discussions regarding RBM23 and David McFadden for reviewing the manuscript. T.C.T. is supported by a McKnight graduate student fellowship. This work was supported by grants issued to D.N. from the Welch Foundation ( I-1879 ) and the National Cancer Institute ( R37CA226771 and RO1CA217333 ). Publisher Copyright: {\textcopyright} 2019 The Authors",
year = "2019",
month = nov,
day = "5",
doi = "10.1016/j.celrep.2019.09.079",
language = "English (US)",
volume = "29",
pages = "1499--1510.e6",
journal = "Cell Reports",
issn = "2211-1247",
publisher = "Cell Press",
number = "6",
}