4866–4880 Nucleic Acids Research, 2017, Vol. 45, No. 8 Published online 14 January 2017 doi: 10.1093/nar/gkw1365 USP15 regulates dynamic protein–protein interactions of the spliceosome through deubiquitination of PRP31 Tanuza Das1, Joon Kyu Park2, Jinyoung Park1,EunjiKim2, Michael Rape3,4, Eunice EunKyeong Kim2,* and Eun Joo Song1,* 1Molecular Recognition Research Center, Korea Institute of Science and Technology, Hwarangno 14-gil 5, Seongbuk-gu, Seoul 02792, Korea, 2Biomedical Research Institute, Korea Institute of Science and Technology, Hwarangno 14-gil 5, Seongbuk-gu 02792, Seoul, Korea, 3Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA and 4Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, CA 94720, USA Received June 05, 2016; Revised December 26, 2016; Editorial Decision December 27, 2016; Accepted January 02, 2017 ABSTRACT ing the catalytically activated spliceosome. The activated spliceosome then catalyzes the excision of introns and the Post-translational modifications contribute to the ligation of exons. As such, the spliceosome undergoes rapid spliceosome dynamics by facilitating the physical re- but tightly regulated changes in its composition during its arrangements of the spliceosome. Here, we report catalytic cycle, with distinct proteins and RNAs associating USP15, a deubiquitinating enzyme, as a regulator of and dissociating at defined stages of the splicing reaction protein–protein interactions for the spliceosome dy- (3,4). Proper pre-mRNA splicing is one of the most criti- namics. We show that PRP31, a component of U4 cal steps in gene expression, and defects in splicing are well snRNP, is modified with K63-linked ubiquitin chains known as a common disease-causing mechanism in humans by the PRP19 complex and deubiquitinated by USP15 (5). Although the detailed mechanism is not yet known, cell and its substrate targeting factor SART3. USP15SART3 cycle progression is closely related to RNA splicing. For ex- makes a complex with USP4 and this ternary com- ample, splicing regulators like SON and SR factor have been shown to contribute to the precise splicing of cell cycle reg- plex serves as a platform to deubiquitinate PRP31 ulators (6–9). Moreover, recently, it has been reported that and PRP3. The ubiquitination and deubiquitination many genes undergo cell cycle dependent alternative splic- status of PRP31 regulates its interaction with the ing changes and that periodic alternative splicing is con- U5 snRNP component PRP8, which is required for trolled by CLK1 (10). the efficient splicing of chromosome segregation re- Post-translational modifications contribute to the lated genes, probably by stabilizing the U4/U6.U5 tri- spliceosome dynamics by facilitating the physical rear- snRNP complex. Collectively, our data suggest that rangements of the spliceosome (4,11–13), for example, USP15 plays a key role in the regulation of dynamic phosphorylation has an important role in the regulation of protein–protein interactions of the spliceosome. the spliceosome (12,14). Ubiquitin modification is involved in diverse cellular processes such as protein degradation, regulation of cellular activity, localization and interaction INTRODUCTION (15). Ubiquitin has seven lysine residues, K6, K11, K27, K29, K33, K48 and K63 which result in the formation of Pre-mRNA splicing is catalyzed by one of the most complex polyubiquitin chains. These chains are diverse in structure and dynamic macromolecular machines called the spliceo- and function depending on the lysine residue used. For some which is assembled from five small nuclear RNAs instance, K11- or K48-linked chains promote degradation (snRNAs) and numerous proteins. This large ribonucleo- of ubiquitinated proteins by 26S proteasome (16). On protein (RNP) is assembled onto intron-containing mR- the other hand, K63-linked chains are not responsible NAs by the recognition of the 5 splice site by U1 snRNP for proteolysis. Instead, they regulate protein localization and the branch point by U2 snRNP (1,2). Upon formation and assemble of DNA repair complexes as well as are / of the A complex, the U4 U6.U5 tri-snRNP complex is re- involved in signal transduction or kinase activation (17,18). cruited to generate the B complex. After rearrangements in Recently, reversible ubiquitination has been shown to have RNA-RNA and RNA-protein interactions, the U1 and U4 a critical role in regulating the spliceosome dynamics. snRNAs and their associated proteins are released yield- *To whom correspondence should be addressed. Tel: +82 2 958 5086; Fax:+ 82 2 958 5170; Email: [email protected] Correspondence may also be addressed to Eunice EunKyeong Kim. Tel: +82 2 958 5937; Fax: +82 2 958 5909; Email: [email protected] C The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact [email protected] Nucleic Acids Research, 2017, Vol. 45, No. 8 4867 For example, it has been shown in yeast extracts that The following antibodies were purchased and used for ubiquitination is required to assemble and disassemble the immunostaining and western blotting: antibodies against U4/U6.U5 tri-snRNP complex through ubiquitin conju- USP15 (Bethyl Laboratories Inc.), USP4 (Bethyl Labo- gation of PRP8 which is a component of the U5 snRNP ratories Inc.), SART3 (Bethyl Laboratories Inc.), ␤-actin (11). Also we have reported that the modification of PRP3, (Santa Cruz Biotechnology Inc.), Myc (Santa Cruz Biotech- which is a component of the U4 snRNP, with K63-linked nology), HA (Santa Cruz Biotechnology), Flag (Sigma- ubiquitin chains by the PRP19 complex and USP4 with its Aldrich), PRP31 (Sigma-Aldrich) and Alexa-488 or Alexa- substrate targeting factor SART3 guides the rearrangement 546 conjugated secondary antibodies (Invitrogen). of the complex resulting in an active spliceosome, and the loss of USP4 impairs mitotic progression by interfering Protein purification and gel filtration chromatography with mRNA splicing, for example, of ␣-tubulin and Bub1 (19). The human SART3 HAT domains (residues 1–691) and Because the spliceosome consists of >100 proteins and DUSP-UBL domain of USP15 (residues 1–226) were needs tight regulation for its dynamics, we thought that cloned into pET-28a (Novagen) to generate N-terminal other proteins beside PRP3 might be reversibly modified histidine-tagged proteins. The N-domain (residues 1–333) by ubiquitination and deubiquitination. Therefore, we have and C-domain (residues 85–499) of human PRP31 were been screening for other spliceosomal proteins that re- cloned into pET-32a (Novagen) as N-terminal histidine- quire ubiquitin-dependent regulation in mitotic progres- tagged thioredoxin fusion proteins. All proteins were ex- sion. Here, we report that PRP31, which is a component pressed in Escherichia coli Rosetta (DE3) cells with induc- ◦ of the U4 snRNP, is another spliceosomal protein. It is tion by 0.5 mM isopropyl-␤-D-thiogalactoside at 18 Cfor modified with K63-linked ubiquitin chains by the PRP19 18 h. Proteins in 50 mM Tris–HCl (pH 8.0) and 150 mM complex and deubiquitinated by USP15 and its substrate NaCl buffer were purified with His-Trap affinity column targeting factor SART3. SART3 mediates complex forma- (GE Healthcare) eluted with 25–500 mM imidazole. Affin- tion with USP15 and USP4, and this complex leads to si- ity tags were removed with thrombin, and the resultant pro- multaneous deubiquitination of the substrates PRP31 and teins were then further purified with a Superdex 200 26/60 PRP3. In addition, the depletion of USP15 and USP4 in- gel filtration column (GE Healthcare) which had been pre- terferes with proper mRNA splicing of Bub1 and ␣-tubulin equilibrated with 50 mM Tris–HCl (pH 8.0) and 150 mM and chromosome segregation. We propose that PRP31 and NaCl. PRP3 serve as regulatory proteins in the rearrangements For the separation of protein complexes, cell lysates over- of the spliceosome components by reversible ubiquitination expressed with HA-SART3, Myc-USP4 and Flag-USP15 and deubiquitination. were co-fractionated by gel filtration with a Superdex 200 10/300 column (GE Healthcare) in a buffer containing 50 MATERIALS AND METHODS mM Tris–HCl (pH 7.4), 150 mM NaCl and 1 mM EDTA. Molecular weight markers (Sigma-Aldrich) with a range of Cell culture and transfections 66 000–443 000 Da were used for size calibration. Eluted HeLa and HEK 293T cells were maintained in Dulbecco’s fractions (0.5 ml) were collected and aliquots were subject modified Eagle’s medium (DMEM) supplemented with to the SDS-PAGE and detected by western blotting. 10% heat inactivated fetal bovine serum, penicillin (10 U/ml) and streptomycin (100 ␮g/ml). All cells were incu- GST pull down assay bated at 37◦Cin5%CO. Plasmids were transfected in 2 1–226 1–230 HEK 293T cells, using the calcium phosphate/DNA co- GST-USP15 and GST-USP4 were coupled to glu- tathione beads (Sigma) and incubated with SART31–691 for precipitation method (20). Transfection in HeLa cells was ◦ performed with the Effectene transfection reagent (Qiagen) 3hat4C. Beads were washed with buffer containing 50 according to the manufacturer’s instructions. mM HEPES, pH 7.5, 150 mM NaCl, 1.5 mM MgCl2,5mM KCl and 0.1% Tween-20, and eluted with 2× SDS sample buffer. Samples were separated by SDS-PAGE and detected Cloning and antibodies by Coomassie staining. USP15 and PRP31 genes were amplified by PCR and cloned into the pCS2 expression plasmid, incorporating siRNA knockdown experiments HA, Myc or Flag tag on the N terminus. The catalyti- cally inactive mutant of USP15C269A was made by mutat- Three different siRNAs targeting three different regions of ing the Cysteine (TGT) at position 269 into an Alanine the USP15 gene were used for the knockdown.