Sde2 is an intron‐specific pre‐ mRNA splicing regulator activated by ubiquitin‐like processing
2017; Springer Nature; Volume: 37; Issue: 1 Linguagem: Inglês
10.15252/embj.201796751
ISSN1460-2075
AutoresPoonam Thakran, Prashant Arun Pandit, Sumanjit Datta, Kiran Kumar Kolathur, Jeffrey A. Pleiss, Shravan Kumar Mishra,
Tópico(s)RNA and protein synthesis mechanisms
ResumoArticle25 September 2017free access Source DataTransparent process Sde2 is an intron-specific pre-mRNA splicing regulator activated by ubiquitin-like processing Poonam Thakran orcid.org/0000-0001-7348-9677 Max Planck – DST Partner Group, Department of Biological Sciences, Centre for Protein Science Design and Engineering, Indian Institute of Science Education and Research (IISER) Mohali, Mohali, Punjab, India Search for more papers by this author Prashant Arun Pandit Max Planck – DST Partner Group, Department of Biological Sciences, Centre for Protein Science Design and Engineering, Indian Institute of Science Education and Research (IISER) Mohali, Mohali, Punjab, India Search for more papers by this author Sumanjit Datta orcid.org/0000-0001-9476-7162 Max Planck – DST Partner Group, Department of Biological Sciences, Centre for Protein Science Design and Engineering, Indian Institute of Science Education and Research (IISER) Mohali, Mohali, Punjab, India Search for more papers by this author Kiran Kumar Kolathur Max Planck – DST Partner Group, Department of Biological Sciences, Centre for Protein Science Design and Engineering, Indian Institute of Science Education and Research (IISER) Mohali, Mohali, Punjab, India Search for more papers by this author Jeffrey A Pleiss Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY, USA Search for more papers by this author Shravan Kumar Mishra Corresponding Author [email protected] [email protected] orcid.org/0000-0003-3899-0495 Max Planck – DST Partner Group, Department of Biological Sciences, Centre for Protein Science Design and Engineering, Indian Institute of Science Education and Research (IISER) Mohali, Mohali, Punjab, India Search for more papers by this author Poonam Thakran orcid.org/0000-0001-7348-9677 Max Planck – DST Partner Group, Department of Biological Sciences, Centre for Protein Science Design and Engineering, Indian Institute of Science Education and Research (IISER) Mohali, Mohali, Punjab, India Search for more papers by this author Prashant Arun Pandit Max Planck – DST Partner Group, Department of Biological Sciences, Centre for Protein Science Design and Engineering, Indian Institute of Science Education and Research (IISER) Mohali, Mohali, Punjab, India Search for more papers by this author Sumanjit Datta orcid.org/0000-0001-9476-7162 Max Planck – DST Partner Group, Department of Biological Sciences, Centre for Protein Science Design and Engineering, Indian Institute of Science Education and Research (IISER) Mohali, Mohali, Punjab, India Search for more papers by this author Kiran Kumar Kolathur Max Planck – DST Partner Group, Department of Biological Sciences, Centre for Protein Science Design and Engineering, Indian Institute of Science Education and Research (IISER) Mohali, Mohali, Punjab, India Search for more papers by this author Jeffrey A Pleiss Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY, USA Search for more papers by this author Shravan Kumar Mishra Corresponding Author [email protected] [email protected] orcid.org/0000-0003-3899-0495 Max Planck – DST Partner Group, Department of Biological Sciences, Centre for Protein Science Design and Engineering, Indian Institute of Science Education and Research (IISER) Mohali, Mohali, Punjab, India Search for more papers by this author Author Information Poonam Thakran1,‡, Prashant Arun Pandit1,‡, Sumanjit Datta1, Kiran Kumar Kolathur1, Jeffrey A Pleiss2 and Shravan Kumar Mishra *,*,1 1Max Planck – DST Partner Group, Department of Biological Sciences, Centre for Protein Science Design and Engineering, Indian Institute of Science Education and Research (IISER) Mohali, Mohali, Punjab, India 2Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY, USA ‡These authors contributed equally to this work *Corresponding author. Tel: +91 856 694 8924; E-mail: [email protected], [email protected] EMBO J (2018)37:89-101https://doi.org/10.15252/embj.201796751 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract The expression of intron-containing genes in eukaryotes requires generation of protein-coding messenger RNAs (mRNAs) via RNA splicing, whereby the spliceosome removes non-coding introns from pre-mRNAs and joins exons. Spliceosomes must ensure accurate removal of highly diverse introns. We show that Sde2 is a ubiquitin-fold-containing splicing regulator that supports splicing of selected pre-mRNAs in an intron-specific manner in Schizosaccharomyces pombe. Both fission yeast and human Sde2 are translated as inactive precursor proteins harbouring the ubiquitin-fold domain linked through an invariant GGKGG motif to a C-terminal domain (referred to as Sde2-C). Precursor processing after the first di-glycine motif by the ubiquitin-specific proteases Ubp5 and Ubp15 generates a short-lived activated Sde2-C fragment with an N-terminal lysine residue, which subsequently gets incorporated into spliceosomes. Absence of Sde2 or defects in Sde2 activation both result in inefficient excision of selected introns from a subset of pre-mRNAs. Sde2 facilitates spliceosomal association of Cactin/Cay1, with a functional link between Sde2 and Cactin further supported by genetic interactions and pre-mRNA splicing assays. These findings suggest that ubiquitin-like processing of Sde2 into a short-lived activated form may function as a checkpoint to ensure proper splicing of certain pre-mRNAs in fission yeast. Synopsis Sde2 is translated as an inactive precursor protein, whose processing by two deubiquitinating enzymes (DUBs) serves as a checkpoint for its timely incorporation into the spliceosome, to ensure proper splicing of a subset of mRNAs in Schizosaccharomyces pombe. The inactive Sde2 precursor contains an N-terminal domain with low homology to ubiquitin. Cleavage of the ubiquitin-fold domain by DUBs Ubp5 and Ubp15 is critical for spliceosomal incorporation of the Sde2 C-terminal domain. Sde2 is involved in the excision of selected introns from a subset of pre-mRNAs. Sde2 facilitates Cactin association with spliceosomes. Sde2 mutants show defects in telomeric silencing and genomic stability. Introduction Protein-coding genes in eukaryotes are often interrupted by non-coding introns. Following their transcription into precursor messenger RNAs (pre-mRNAs), the splicing process removes the introns and joins the exons to generate translatable mRNAs. The pre-mRNAs can also undergo alternative RNA splicing to generate more than one protein-coding mRNAs from a gene. The majority of human genes have introns and undergo constitutive as well as alternative RNA splicing (Ast, 2004; Kim et al, 2006). The spliceosome is a large ribonucleoprotein (RNP) complex that assembles with five small-nuclear RNAs (snRNAs) and nearly two hundred proteins to perform pre-mRNA splicing. The snRNP complexes form the spliceosomal core, while RNA-binding proteins, RNP modifying enzymes (ATPases, helicases) and modifications of splicing factors (phosphorylation, methylation) regulate activity of this complex (Wahl et al, 2009). Ubiquitin and ubiquitin-like proteins (collectively referred to as UBLs) share the ubiquitin-fold and act as key regulators of a variety of cellular processes including protein homoeostasis, signalling, trafficking and DNA transactions (Welchman et al, 2005; Hochstrasser, 2009; van der Veen & Ploegh, 2012). The canonical ubiquitin is covalently conjugated to proteins by a set of enzymes in a process termed ubiquitination. Ubiquitin itself is not encoded as a single polypeptide, but rather is translated as a precursor, fused either to ribosomal protein genes, or with multiple ubiquitin moieties in tandem (Finley et al, 1989; Hochstrasser, 2009; Kimura & Tanaka, 2010). These precursors are processed by ubiquitin-specific hydrolases (also referred to as deubiquitinating enzymes or DUBs) immediately after di-glycine (GG) motifs to generate functional ubiquitin (Turcu et al, 2009). Whereas several UBLs follow ubiquitin's mode of processing and conjugation by employing respective UBL-specific enzymes, Hub1/UBL5 lacks the di-glycine motif, and acts through a non-covalent process. Hub1 binds to the HIND domain-containing splicing factors Snu66 and/or Prp38 and functions in pre-mRNA splicing by promoting the usage of non-canonical 5′ splice sites in introns. Hub1 thereby promotes alternative splicing of the SRC1 pre-mRNA in Saccharomyces cerevisiae (Mishra et al, 2011). Specific roles of Hub1 in pre-mRNA splicing have also been reported in both Schizosaccharomyces pombe and mammalian cells (Wilkinson et al, 2004; Mishra et al, 2011; Ammon et al, 2014). Whereas HUB1 is a non-essential gene in S. cerevisiae, its orthologs in S. pombe and mammalian cells are essential for viability, perhaps because of the increased prevalence of introns and alternative splicing in S. pombe and humans. The process of pre-mRNA splicing has been linked to heterochromatin formation at the centromeres and telomeres in S. pombe (Bayne et al, 2008; Huang & Zhu, 2014; Kallgren et al, 2014; Wang et al, 2014). Earlier splicing factors were thought to provide a platform for the generation of small interfering siRNAs in the RNAi pathway of heterochromatin formation, since their mutants were defective in heterochromatin formation, but appeared normal in pre-mRNA splicing (Bayne et al, 2008). Recent reports however connect the heterochromatin defects observed in splicing factor mutants with reduced splicing of mRNAs encoding bona fide components of the heterochromatin pathway (Bayne et al, 2014; Kallgren et al, 2014; Wang et al, 2014). Schizosaccharomyces pombe lacking the sde2 gene, named for silencing defective, showed defective telomeric silencing (Sugioka-Sugiyama & Sugiyama, 2011). Strains lacking sde2 also showed defects in centromeric silencing and defects in splicing of cytoskeleton constituents and centromeric outer repeat transcripts (Bayne et al, 2014). The human ortholog of Sde2, C1orf55, was present in spliceosomal preparations (Bessonov et al, 2008), and recently, the S. pombe protein was also shown to associate with splicing factors (Bayne et al, 2014; Chen et al, 2014). In this study, we report that sde2 genetically interacts with hub1 in S. pombe. The Sde2 protein has a ubiquitin-fold at its N-terminus, which must be cleaved by the ubiquitin-specific proteases (USPs) Ubp5 and Ubp15. After cleavage, the C-terminal domain of Sde2, which starts with a lysine, gets incorporated into the spliceosome. Loss of Sde2 results in inefficient removal of selected introns from a subset of genes having functions in telomeric silencing, DNA replication and transcription. Furthermore, the N-end rule pathway of proteasomal degradation controls the level of Sde2 protein in the cell. The intron-specific pre-mRNA splicing activity of the ubiquitin-fold-containing Sde2 protein becomes critical for chromatin silencing and genomic stability in S. pombe. Results Sde2 is processed like ubiquitin precursors We have previously reported the role of the UBL Hub1 in alternative RNA splicing through usage of non-canonical 5′ splice sites in the budding yeast S. cerevisiae (Mishra et al, 2011). Orthologs of Hub1 in S. pombe and humans are essential for viability and play specific roles in pre-mRNA splicing (Mishra et al, 2011; Ammon et al, 2014). To identify spliceosomal regulators in an intron prevalent organism, we screened for genetic interactors of hub1 by combining S. pombe hub1-1 mutant (Yashiroda & Tanaka, 2004) with the haploid deletion library of non-essential genes (Kim et al, 2010). As expected from Hub1's role in pre-mRNA splicing, deletion mutants of multiple splicing factors were synthetically sick with hub1-1 (Fig EV1A). Among them, the deletion of sde2 gene was also synthetically sick with hub1-1. We confirmed this interaction by generating double mutants of hub1-1 with Δsde2 independently (Fig EV1B). As reported previously in high-throughput studies (Kennedy et al, 2008; Zhang et al, 2013), the Δsde2 strain grew slowly under standard conditions, and the growth defect was more pronounced at elevated temperatures and under genotoxic stress conditions (hydroxyurea, valproic acid, sodium butyrate, cadmium) (Appendix Fig S1A). Click here to expand this figure. Figure EV1. Sde2 is processed like ubiquitin and genetically interacts with ubiquitin-like protein Hub1 Genetic interactors of hub1. hub1-1 is the temperature-sensitive hub1(I42S) mutant reported by Yashiroda and Tanaka (2004). A genetic screen was performed with hub1-1 and the haploid deletion library of non-essential genes in Schizosaccharomyces pombe. hub1-1 was synthetically sick with the deletion mutants of given genes including sde2. Among the top hits, genes with relevance to pre-mRNA splicing are shown. Confirmation of the negative genetic interaction between hub1-1 and Δsde2 mutants. sde2 gene, from ATG to the stop codon, was deleted in a S. pombe Δhub1 strain, which was kept viable with a ura4+ marked hub1 expression plasmid with its own promoter and terminator. The resultant strain was transformed with leu2+ marked expression plasmids expressing wild-type (WT) hub1 or the hub1-1 mutant. Fivefold serial dilutions of cells from these transformations were spotted on indicated agar plates. Plates were incubated at 25°C. 5-fluoroorotic acid (FOA) (1 g/l of media) was used to shuffle-out ura4+ plasmid. Thus, cells growing on −Leu + FOA plates will have hub1 mutants in Δsde2 background. Alignment of Sde2 protein orthologs from different eukaryotes. Abbreviations used are (respective NCBI protein accession numbers are given in parentheses) as follows: Dh, Debaryomyces hansenii (XP_458854); An, Aspergillus niger (XP_001391007); Sp, Schizosaccharomyces pombe (NP_594019); At, Arabidopsis thaliana (NP_192009); Ce, Caenorhabditis elegans (NP_506378); Ag, Anopheles gambiae (XP_321833); Dm, Drosophila melanogaster (NP_651207); Xl, Xenopus laevis (NP_001084858); Dr, Danio rario (AAH93198); Hs, Homo sapiens (NP_689821). The sequence marked in orange indicates most conserved region in Sde2 orthologs. Expression of human Sde2 (C1orf55) protein in U2OS cells. Constructs with sequences encoding 3FLAG epitope tag at the N-terminus of HsSDE2 gene and single MYC epitope tag at its C-terminus under CMV (cytomegalovirus) promoter were used. With WT Sde2, Sde2-C would start with lysine (KGG…), whereas with Sde2 GGAGG mutant, Sde2-C would start with alanine (AGG…). Asterisk indicates antibody cross reactivity signal. Arrows indicate the HsSde2-C protein formed after processing of the GGAGG mutant precursor accumulated to higher levels than the protein formed from the wild-type precursor. Download figure Download PowerPoint Putative orthologs of Sde2 exist in organisms from yeast to humans (Fig EV1C). However, in the Fungi subphylum Saccharomycotina, an Sde2-like protein is observed in Debaryomyces, but is absent in S. cerevisiae, Candida albicans and Pichia pastoris. The protein structure prediction program i-TASSER (Yang et al, 2015) predicted the presence of a ubiquitin-fold at the N-terminus and a C-terminal helical domain in Sde2 (henceforth referred to as Sde2UBL and Sde2-C, respectively). An invariant signature motif, GGKGG, separates the moderately conserved Sde2UBL and Sde2-C (Figs 1A and EV1C). This motif flanks Sde2UBL and resembles the di-glycine (GG) motif typical of UBL precursors processed by the UBL-specific proteases. Figure 1. Sde2 is a conserved protein with ubiquitin-fold Predicted structure of Schizosaccharomyces pombe Sde2 protein shows the presence of a ubiquitin-fold (Sde2UBL) and a helical C-terminal domain (Sde2-C). The alignment on the left shows the linker GGKGG motif between Sde2UBL and Sde2-C in Sde2 orthologs. Dh, Debaryomyces hansenii; An, Aspergillus niger; Sp, Schizosaccharomyces pombe; At, Arabidopsis thaliana; Ce, Caenorhabditis elegans; Ag, Anopheles gambiae; Dm, Drosophila melanogaster; Xl, Xenopus laevis; Dr, Danio rario; Hs, Homo sapiens. Expression and processing of Sde2 protein in S. pombe detected by immunoblot analysis (Western blot, WB) using epitope tag-specific antibodies. Underlined residues mark changes from wild-type (WT) Sde2. Asterisk indicates antibody cross reactivity signal. Complementation of S. pombe Δsde2 by GGKGG mutants of Sde2. Constructs are as in (B). Fivefold serial dilution spotting was done on indicated agar plates. Plates were incubated at 30°C and 37°C until growth appeared. Complementation of S. pombe Δsde2 by Sde2 domains. The experiment is as in (C). Expression constructs encoding Sde2 WT, the processing-defective mutant sde2(AAKGG), Sde2UBL, Sde2-C, and Sde2UBL and Sde2-C together were used. Source data are available online for this figure. Source Data for Figure 1 [embj201796751-sup-0006-SDataFig1.pdf] Download figure Download PowerPoint We generated a tagged version of Sde2, which contained epitope tags at both its N- and C-termini, to detect the protein in S. pombe by immunoblot assays. These experiments revealed that the full-length protein was cleaved, separating it into Sde2UBL and Sde2-C (Fig 1B). After processing, Sde2UBL was diffusely localized in S. pombe whereas Sde2-C was predominantly nuclear (Appendix Fig S1B). Processing of Sde2 is presumed to occur at the GG^KGG sequence as alanine substitutions of the first GG residues abolished cleavage (Fig 1B) and a processing-defective Sde2 was nuclear (Appendix Fig S1B). Importantly, these alanine substitutions also failed to rescue growth defects of a Δsde2 strain (Fig 1C), suggesting that cleavage of Sde2 is essential for its function. By contrast, alanine mutations of the lysine or the second GG residues had negligible effects on processing and these mutants also rescued growth defects of Δsde2, albeit to a lesser extent than the wild type. Sde2 must be synthesized in the precursor form that should get processed for function, as de novo expressions of Sde2UBL did not complement growth defects in Δsde2 strain whereas Sde2-C complemented the defects partially after overexpression (level of Sde2-C when expressed from a plasmid using methionine codon was higher than Sde2-C generated from its chromosomal location; Appendix Fig S1C). Co-expression of the two domains did not improve growth of the deletion strain (Fig 1D). An extension with a non-cleavable peptide upstream of Sde2-C did not result in any complementation (Appendix Fig S1D). Thus, Sde2-C is the functional unit whereas Sde2UBL plays a regulatory role. To examine evolutionary conservation of Sde2 processing, an epitope-tagged version of the human ortholog, C1orf55 (herein referred to as HsSde2) was generated. Similar to S. pombe, HsSde2 precursor was also cleaved into HsSde2UBL and HsSde2-C in mammalian cells, alanine mutations of the first GG residues in the GGKGG motif of HsSde2 abolished its processing, and mutation of the lysine did not have any visible effect on processing (Fig EV1D). Interestingly, the HsSde2-C protein formed after processing of the GGAGG mutant precursor accumulated to higher levels than the protein formed from the wild-type precursor (Fig EV1D; see below). Nevertheless, despite the similar post-translational processing of Sde2 in S. pombe and humans, these orthologs retain organism-specific features, as expression of HsSde2 in S. pombe Δsde2 strain failed to complement its growth defects. Furthermore, the two domains of HsSde2 were not interchangeable with the S. pombe counterparts, as a domain-swapped S. pombe–human Sde2 chimera could not complement Δsde2 (Appendix Fig S1E). Ubiquitin-specific proteases Ubp5 and Ubp15 process Sde2 To identify Sde2 processing enzymes, we monitored accumulation of its precursor in selected mutants of S. pombe proteases by immunoblot assays. The precursor accumulated in a strain lacking ubp15, a USP domain-containing DUB (Appendix Fig S2A). Residual Sde2-C in the Δubp15 strain suggested involvement of additional enzymes for the processing. It was previously reported that ubp15 genetically interacts with ubp5, with the double mutant showing synthetic sickness (Richert et al, 2002). Processing of Sde2 was completely abolished in a Δubp5 Δubp15 double mutant (Fig 2A and B). To monitor sub-cellular location of Sde2 processing, we generated Sde2UBL–GFP chimera, with a nuclear localization signal (NLS) or a nuclear export signal (NES) at the C-terminus of GFP. An inefficient processing of the Sde2UBL–GFP–NES chimera in S. pombe suggests that Sde2 processing takes place in the nucleus (Fig 2C). Ubp5 and Ubp15 are generally involved in processing of ubiquitin and ubiquitin conjugates (Kouranti et al, 2010), and clearly, Sde2 is not the only substrate of Ubp5 and Ubp15; accordingly, the Δubp5 Δubp15 strain showed more severe growth defects than the Δsde2 strain (Appendix Fig S2B). Figure 2. Ubiquitin-specific proteases Ubp5 and Ubp15 process Sde2 Sde2 processing in Schizosaccharomyces pombe. Constructs from Fig 1B were expressed in S. pombe WT, ∆ubp5, ∆ubp15 and ∆ubp5 ∆ubp15 strains. The protein expression was analysed by anti-FLAG immunoblotting. Endogenous S. pombe Sde2-C and the full-length Sde2 precursor in ∆ubp5 ∆ubp15 strain. Sde2 protein from indicated strains was immunoprecipitated using a polyclonal antibody against recombinant Sde2-C, followed by Western blot analysis using same antibody. Sde2 is processed in the nucleus. Anti-GFP immunoblot analysis of S. pombe expressing Sde2UBL–GFP, and its variants with a nuclear localization signal (NLS) or a nuclear export signal (NES). The non-nuclear NES version was poorly processed. Processing of S. pombe Sde2 in bacteria. Expression constructs harbouring indicated cDNAs were co-transformed in Escherichia coli BL21(DE3) strain. Following protein expression, total cell lysates were processed by immunoblotting using anti-Sde2-C antibody. Anti-HIS immunoblotting was done to monitor expression of the proteases and its mutants. Source data are available online for this figure. Source Data for Figure 2 [embj201796751-sup-0007-SDataFig2.pdf] Download figure Download PowerPoint To further test whether Ubp5 and Ubp15 acted directly as the proteases, we examined processing of SpSde2 by these DUBs in the recombinant system of Escherichia coli. Efficient processing of Sde2 was readily observed in E. coli upon co-expression of Ubp5 and Ubp15, but not their catalytically inactive cysteine mutants (Fig 2D). Activity of these proteases on Sde2 is highly specific, as another USP domain-containing DUB, Ubp16, did not process Sde2 (Fig EV2A). Although S. cerevisiae lacks Sde2, it contains an Ubp15 ortholog. Nevertheless, SpSde2 was poorly processed in wild-type S. cerevisiae, presumably because SpUbp15 and ScUbp15 share only 44% identity. Overexpression of ScUbp15 enabled detection of processed SpSde2-C in S. cerevisiae. Importantly, increased efficiency of SpSde2 processing was apparent upon co-expression of either SpUbp5 or SpUbp15 in S. cerevisiae, highlighting their roles in processing of SpSde2 (Appendix Fig S2C). Click here to expand this figure. Figure EV2. Replacement of Sde2 UBL fold with other ubiquitin-like folds A USP domain-containing DUB Ubp16 does not process Sde2. Experiment similar to Fig 2D. Processing of ubiquitin–Sde2-C fusion by the proteases was used as control. Anti-HIS immunoblotting was used to monitor expression of the proteases. Sde2UBL can be replaced with the UBLs ubiquitin and Ned8. Complementation of growth defects of Schizosaccharomyces pombe Δsde2 strain with indicated fusion constructs. All constructs with a C-terminal 3FLAG tag were expressed under the thiamine-repressible nmt81 promoter. Expressed proteins are N-terminal fusions of the ubiquitin-folds of various UBLs (Ubiquitin/Ubi4, Ned8 or Hub1) with Sde2-C. The GGKGG motif was kept at the junctions in the fusion proteins. After processing of the UBLs, Sde2-C would start with a lysine. Anti-FLAG immunoblot assay of fusion proteins shown in (B). Processing of ubiquitin- and Ned8–Sde-C chimeras was not affected in Δubp5 Δubp15 double mutant. Expression of the clones used in (B) and indicated with numerical was monitored by immunoblotting using anti-FLAG antibody. Source data are available online for this figure. Download figure Download PowerPoint We reasoned that if Sde2UBL was processed at the GG motif mainly for generating LysSde2-C, then it should be replaceable with UBLs that are similarly processed. Therefore, we generated fusions of Sde2-C with S. pombe ubiquitin, Ned8 (NEDD8 ortholog) or Hub1 (used here as a control UBL that is not processed). The GGKGG sequence was kept at the junction of the chimera. Ubiquitin and Ned8, but not Hub1, chimeras of LysSde2-C complemented growth defects of the Δsde2 strain (Fig EV2B). Ubiquitin- and Ned8-specific proteases processed respective fusions to form the functional LysSde2-C (Fig EV2C). Processing of ubiquitin–Sde2-C and Ned8–Sde2-C fusions was not affected in Δubp5 Δubp15 double mutant (Fig EV2D). Since Hub1 does not get processed, its chimera was unable to generate functional Sde2-C. Similar to the processing-defective mutants of Sde2, alanine mutations of the GG in the chimeras were not processed and remained non-functional. Sde2-C is a substrate of the N-end rule pathway To monitor steady-state levels of wild-type and mutant Sde2 proteins, sequence encoding 6HA epitope tag was inserted chromosomally at the C-terminus of sde2 variants. MetSde2-C formed after processing of the GGMGG mutant precursor accumulated to higher levels than the LysSde2-C formed from the wild-type GGKGG protein (Fig 3A and B). Interestingly, the level of MetSde2-C after de novo translation using methionine codon was lower than the MetSde2-C formed after processing of the GGMGG mutant precursor. However, the differences in protein levels were not due to variations in transcription, as all strains showed comparable levels of sde2 mRNA in reverse transcription quantitative PCR (RT–qPCR) assays (Appendix Fig S3A). Thus, the presence of Sde2UBL facilitates the expression of Sde2-C protein. Both strains with MetSde2-C showed strong growth defects; however, growth of the strain with MetSde2-C formed after processing of the GGMGG mutant was better than with MetSde2-C from de novo translation (Fig 3C). Figure 3. Sde2-C is made short-lived by the N-end rule pathway Chromosomal 6HA epitope-tagged sde2 variants. The sde2 promoter is common to all variants. Western blot with anti-HA antibody to detect steady-state levels of wild-type and mutant Sde2 proteins. Growth phenotype of strains in (A). Strains with MetSde2-C proteins, formed either after processing of GGMGG mutant or translated de novo, show ∆sde2-like growth defects. Constructs used to monitor steady-state level and turnover of the proteins. Plasmids contain 3FLAG epitope tags at the C-termini of Sde2 under nmt81 promoter. Protein turnover assays. The Sde2 variants with C-terminal 3FLAG tag were expressed in Δsde2 strain from a plasmid under nmt81 promoter. In Sde2 WT, after processing Sde2-C starts with lysine, in GGMGG mutant with methionine and Met–Sde2-C is de novo translated. Total proteins from 1.0 OD600 cells for the given time points were run on SDS–PAGE followed by anti-FLAG Western blotting. WT Sde2 is stable in the proteasome mutant mts3-1. Assay is similar to (B). Higher molecular weight adducts detected with anti-FLAG antibody likely represent ubiquitinated Sde2. Denaturing Ni-NTA pull-down (Pd) of ubiquitin conjugates from Schizosaccharomyces pombe expressing Sde2–3FLAG. Ubiquitination of Sde2 is detected by anti-FLAG immunoblotting. Source data are available online for this figure. Source Data for Figure 3 [embj201796751-sup-0008-SDataFig3.pdf] Download figure Download PowerPoint The lower levels of S. pombe and human LysSde2-C (Figs 3D and EV1D) could be because lysine residue at the amino-terminus of proteins is descriptive of a type 1 degron in the N-end rule pathway (Bachmair et al, 1986; Sriram et al, 2011). Thus, we tested whether Sde2-C was a substrate of this pathway by measuring its turnover rates. Indeed, the LysSde2-C protein was short-lived, whereas the MetSde2-C proteins were significantly stabilized (Fig 3D). Schizosaccharomyces pombe deletion of the N-end rule ligase ubr11 (Fujiwara et al, 2013) partially stabilized LysSde2-C (Appendix Fig S3B). The mutation of the 19S proteasome regulatory particle subunit mts3 (Gordon et al, 1996) stabilized LysSde2-C, and higher molecular weight adducts of Sde2 observed in this mutant (Fig 3E) were confirmed to be ubiquitin conjugates of Sde2 by pull-down assays of the conjugates under denaturing conditions (Fig 3F). Ubiquitination of Sde2 was diminished but not abolished in a Δubr11 strain (Appendix Fig S3C), indicating involvement of additional ligase(s) for degradation of Sde2-C. To elucidate role of the conserved lysine in GGKGG motif in Sde2 orthologs, we generated substitution mutations with each of the other 19 amino acids and measured the processing efficiency, half-life and complementation capacity of each of the resulting proteins. All amino acid variants, excluding the version with a proline GGPGG, underwent efficient processing (Fig EV3; Appendix Fig S3D and E), indicating that the lysine is not required for processing. In assays to measure proteins half-life, the MetSde2-C variant was found to be most
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