Neddylation modification of the U3 snoRNA-binding protein RRP9 by Smurf1 promotes tumorigenesis
2021; Elsevier BV; Volume: 297; Issue: 5 Linguagem: Inglês
10.1016/j.jbc.2021.101307
ISSN1083-351X
AutoresMengge Du, Fan Liu, Yan Chang, Shuai Tong, Wei Liu, Yujiao Chen, Ping Xie,
Tópico(s)Cancer-related gene regulation
ResumoNeddylation is a posttranslational modification that attaches ubiquitin-like protein Nedd8 to protein targets via Nedd8-specific E1-E2-E3 enzymes and modulates many important biological processes. Nedd8 attaches to a lysine residue of a substrate, not for degradation, but for modulation of substrate activity. We previously identified the HECT-type ubiquitin ligase Smurf1, which controls diverse cellular processes, is activated by Nedd8 through covalent neddylation. Smurf1 functions as a thioester bond-type Nedd8 ligase to catalyze its own neddylation. Numerous ubiquitination substrates of Smurf1 have been identified, but the neddylation substrates of Smurf1 remain unknown. Here, we show that Smurf1 interacts with RRP9, a core component of the U3 snoRNP complex, which is involved in pre-rRNA processing. Our in vivo and in vitro neddylation modification assays show that RRP9 is conjugated with Nedd8. RRP9 neddylation is catalyzed by Smurf1 and removed by the NEDP1 deneddylase. We identified Lys221 as a major neddylation site on RRP9. Deficiency of RRP9 neddylation inhibits pre-rRNA processing and leads to downregulation of ribosomal biogenesis. Consequently, functional studies suggest that ectopic expression of RRP9 promotes tumor cell proliferation, colony formation, and cell migration, whereas unneddylated RRP9, K221R mutant has no such effect. Furthermore, in human colorectal cancer, elevated expression of RRP9 and Smurf1 correlates with cancer progression. These results reveal that Smurf1 plays a multifaceted role in pre-rRNA processing by catalyzing RRP9 neddylation and shed new light on the oncogenic role of RRP9. Neddylation is a posttranslational modification that attaches ubiquitin-like protein Nedd8 to protein targets via Nedd8-specific E1-E2-E3 enzymes and modulates many important biological processes. Nedd8 attaches to a lysine residue of a substrate, not for degradation, but for modulation of substrate activity. We previously identified the HECT-type ubiquitin ligase Smurf1, which controls diverse cellular processes, is activated by Nedd8 through covalent neddylation. Smurf1 functions as a thioester bond-type Nedd8 ligase to catalyze its own neddylation. Numerous ubiquitination substrates of Smurf1 have been identified, but the neddylation substrates of Smurf1 remain unknown. Here, we show that Smurf1 interacts with RRP9, a core component of the U3 snoRNP complex, which is involved in pre-rRNA processing. Our in vivo and in vitro neddylation modification assays show that RRP9 is conjugated with Nedd8. RRP9 neddylation is catalyzed by Smurf1 and removed by the NEDP1 deneddylase. We identified Lys221 as a major neddylation site on RRP9. Deficiency of RRP9 neddylation inhibits pre-rRNA processing and leads to downregulation of ribosomal biogenesis. Consequently, functional studies suggest that ectopic expression of RRP9 promotes tumor cell proliferation, colony formation, and cell migration, whereas unneddylated RRP9, K221R mutant has no such effect. Furthermore, in human colorectal cancer, elevated expression of RRP9 and Smurf1 correlates with cancer progression. These results reveal that Smurf1 plays a multifaceted role in pre-rRNA processing by catalyzing RRP9 neddylation and shed new light on the oncogenic role of RRP9. Ribosome synthesis is highly conserved and regulated. It requires the coordinated cooperation of DNA-dependent RNA polymerases, transcription factors, small nucleolar ribonucleoproteins (snoRNPs), and ribosomal proteins (1Henras A.K. Soudet J. Gerus M. Lebaron S. Caizergues-Ferrer M. Mougin A. Henry Y. The post-transcriptional steps of eukaryotic ribosome biogenesis.Cell Mol. Life Sci. 2008; 65: 2334-2359Crossref PubMed Scopus (431) Google Scholar, 2Tafforeau L. Zorbas C. Langhendries J.L. Mullineux S.T. 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Xie P. Zhang L. Itch promotes the neddylation of JunB and regulates JunB-dependent transcription.Cell Signal. 2016; 28: 1186-1195Crossref PubMed Scopus (21) Google Scholar). Smurf1 functions as a thioester bond–type Nedd8 ligase to catalyze its own neddylation. Smurf1, Nedd8, NAE1, and Ubc12 are positively correlated with progression and poor prognosis in human colorectal cancer (29Xie P. Zhang M. He S. Lu K. Chen Y. Xing G. Lu Y. Liu P. Li Y. Wang S. Chai N. Wu J. Deng H. Wang H.R. Cao Y. et al.The covalent modifier Nedd8 is critical for the activation of Smurf1 ubiquitin ligase in tumorigenesis.Nat. Commun. 2014; 5: 3733Crossref PubMed Scopus (129) Google Scholar). Although numerous ubiquitination substrates of Smurf1, such as Smad1/5/8, MEKK2, and RhoA (31Zhu H. Kavsak P. Abdollah S. Wrana J.L. Thomsen G.H. A SMAD ubiquitin ligase targets the BMP pathway and affects embryonic pattern formation.Nature. 1999; 400: 687-693Crossref PubMed Scopus (672) Google Scholar, 32Yamashita M. 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Smurf1 deletion decreases the efficiency of pre-rRNA processing, and this effect is reversed by reintroduction of wild-type RRP9 but not its neddylation-depleted K221R mutant. Therefore, RRP9 neddylation mediated by Smurf1 has an important function in pre-rRNA synthesis. Furthermore, increased expression of Smurf1 and RRP9 shows a positive correlation with human colon cancer progression. These findings characterize the functional diversity of neddylation and reveal a new mechanism of Smurf1 in tumorigenesis. High-throughput mapping of the dynamic TGF-β signaling network identified a series of potential Smurf1-interacting proteins, among which RRP9 was included (35Barrios-Rodiles M. Brown K.R. Ozdamar B. Bose R. Liu Z. Donovan R.S. Shinjo F. Liu Y. Dembowy J. Taylor I.W. Luga V. Przulj N. Robinson M. Suzuki H. Hayashizaki Y. et al.High-throughput mapping of a dynamic signaling network in mammalian cells.Science. 2005; 307: 1621-1625Crossref PubMed Scopus (564) Google Scholar). To further support the interaction, affinity purification and mass spectrometry were first employed to investigate the Smurf1 interactome. Whole-cell extracts were prepared and subjected to affinity purification on an anti-Smurf1 affinity gel. Mass spectrometry analysis indicated that Smurf1 was copurified with RRP9 (Figs. 1A and S1A). Endogenous RRP9 was efficiently coimmunoprecipitated with Smurf1 (Figs. 1B and S1B). Smurf1 was shown to interact with endogenous RRP9 (Fig. 1C). The glutathione S-transferase pull-down assay indicated that the interaction between RRP9 and Smurf1 seemed to be direct (Fig. 1D). In addition, Smurf1 colocalized with RRP9 in the nuclei of HCT116 cells (Fig. 1E). Then, full-length Myc-tagged RRP9 and the 1 to 144 aa and 145 to 475 aa truncations were transfected into HCT116 cells. Immunoprecipitation with an antibody against Myc-tagged proteins followed by immunoblotting with antibodies against Smurf1 showed that Smurf1 was efficiently coimmunoprecipitated with the WD40 domain (aa 145–475) of RRP9. (Fig. 1, G and H). Collectively, these data indicated that RRP9 was a Smurf1-specific interacting partner. Considering that Smurf1 is an E3 ligase for both ubiquitin and Nedd8, we first intended to investigate whether RRP9 can be ubiquitinated by Smurf1. MG132, a proteasome inhibitor, did not affect the protein stability of RRP9 compared with that of Smurf1 (Fig. 2A). In addition, knockout of Smurf1 did not affect the protein stability of RRP9 in HCT116 cells or mice (Fig. 2, B and C). The C426A mutation abolishes the catalytic activity of the Smurf1 neddylation ligase (29Xie P. Zhang M. He S. Lu K. Chen Y. Xing G. Lu Y. Liu P. Li Y. Wang S. Chai N. Wu J. Deng H. Wang H.R. Cao Y. et al.The covalent modifier Nedd8 is critical for the activation of Smurf1 ubiquitin ligase in tumorigenesis.Nat. Commun. 2014; 5: 3733Crossref PubMed Scopus (129) Google Scholar). Thus, we intended to detect the expression of RRP9 in Smurf1−/− and Smurf1C426A knock-in (KI) mice. The data showed that the protein level of RRP9 in Smurf1−/− and Smurf1C426A KI mice did not differ from that in Smurf1+/+ mice (Fig. S2A), and the same result was found for its mRNA level (Fig. S2B). These data strongly indicated that Smurf1 does not affect the protein stability of RRP9 via the ubiquitin–proteasome pathway. Next, we investigated whether RRP9 can be neddylated by Smurf1. To date, RRP9 neddylation has not been reported in the literature. To further evaluate this modification, immunoprecipitation (IP) under partial denaturing conditions was performed with an anti-RRP9 antibody in HCT116 cells, and the precipitates were detected as smeared bands. The densities of the smeared bands were clearly reduced by treatment with the E1 inhibitor MLN4924 (Fig. 2D). When Nedd8 was ectopically expressed, RRP9 migrated as a high-molecular-weight band, but the smeared bands were not produced from the lysate of cells expressing Nedd8-ΔGG, a mutant incapable of covalent conjugation to its substrates (Fig. 2E), suggesting that the smeared bands correspond to Nedd8-conjugated RRP9. Moreover, knockout of NEDP1 by sgRNA increased RRP9 neddylation (Fig. 2F), which indicated that NEDP1 is the deneddylase for RRP9. These data suggested that RRP9 was conjugated with Nedd8. We next determined whether Smurf1 is involved in RRP9 neddylation. Immunoprecipitation under partial denaturing conditions was performed with an anti-RRP9 antibody, and the precipitates were analyzed. The results revealed that endogenous RRP9 was covalently conjugated to Nedd8 but not ubiquitin in the colon tissues of Smurf1+/+ mice (Fig. 2G, Lane 2; Fig. 2H, Lane 2). We found that RRP9 neddylation was almost completely abolished in Smurf1−/− and Smurf1C426A KI mice (Fig. 2G, Lane 3; Fig. 2H, Lane 3). In addition, RRP9 neddylation was markedly reduced with deletion of UBA3 (neddylation E1), Ubc12 (neddylation E2), and Smurf1 using target sgRNAs (Fig. 2I). In addition, ectopic expression of Smurf1 markedly enhanced the neddylation of RRP9 (Fig. 2J). Finally, in vitro neddylation assays were performed with purified RRP9, Smurf1, Nedd8, NAE (E1), and Ubc12 (E2). The results showed that Smurf1 catalyzed RRP9 neddylation in vitro (Fig. 2K). Collectively, these data supported the hypothesis that Smurf1 is likely the major Nedd8 E3 ligase for RRP9. Analysis of the RRP9 sequence showed that it contained a total of 29 lysines: 7 lysines in the N-terminal domain, 8 lysines in the Glu-rich region, 13 lysines in the WD40 domain, and 1 lysine in the C-terminal domain (Fig. 3A). We generated RRP9 truncations to identify the neddylation regions in RRP9. The results in HCT116 cells showed that the 1 to 144 aa region in RRP9 could not be neddylated, indicating that these 15 lysines were not RRP9 neddylation sites (Fig. 3B). Then, individual K-R mutations in the region from 145 aa to 475 aa, which contains 14 lysines in RRP9, were introduced to identify the sites of Nedd8 covalent conjugation. The K221R mutant exhibited almost no neddylation compared with the other K-R mutants (Fig. 3C). Further in vitro neddylation assays confirmed that Smurf1 could not catalyze RRP9 K221R neddylation (Fig. 3D). Therefore, these data suggested that K221 was the major site of RRP9 neddylation. The role of RRP9 in tumor prognosis remains unknown. Next, we explored the role of RRP9 in tumorigenesis. We generated stably transduced HCT116 colon cancer cells by performing lentiviral transduction with Lenti-sgNC (negative control) and Lenti-sgRRP9 (Fig. S3A). The CCK-8 and colony formation assay results indicated that deletion of RRP9 notably inhibited cell proliferation (Fig. 4, A and B). Knockout of RRP9 decreased tumor cell migration (Fig. 4C). Then, we intended to determine whether RRP9 affects cellular senescence in tumors. SA-β-Gal staining showed that deletion of RRP9 increased tumor cell senescence (Fig. 4D). Furthermore, loss of RRP9 obviously inhibited tumor growth in xenografted nude mice (Fig. 4, E–G). Consistent with previous findings, knockout of RRP9 led to a reduction in the total 18S rRNA level (Fig. 4H). Ribosome assembly is coupled with irreversible pre-rRNA processing, which is a classical hallmark of cell growth and proliferation. Therefore, interference with ribosomal biogenesis is often associated with cancer. RRP9 is reported to be essential for U3 snoRNP assembly and ribosomal RNA maturation (36Newman D.R. Kuhn J.F. Shanab G.M. Maxwell E.S. Box C/D snoRNA-associated proteins: Two pairs of evolutionarily ancient proteins and possible links to replication and transcription.RNA. 2000; 6: 861-879Crossref PubMed Scopus (110) Google Scholar, 37Rothé B. Manival X. Rolland N. Charron C. Senty-Ségault V. Branlant C. Charpentier B. Implication of the box C/D snoRNP assembly factor Rsa1p in U3 snoRNP assembly.Nucleic Acids Res. 2017; 45: 7455-7473Crossref PubMed Scopus (13) Google Scholar). Enlarged nucleoli are typically associated with activation of ribosome biogenesis. Silver staining was used for the identification of argyrophilic nucleolar organizer regions (AgNORs). RRP9 knockout resulted in a lower average AgNOR area of positive silver staining. This suggested that knockout of RRP9 leads to downregulation of ribosomal biogenesis (Fig. 4I). Collectively, the aforementioned data suggested that loss of RRP9 inhibited tumor cell growth and migration. To gain insight into the role of RRP9 neddylation in cancer progression, we reintroduced sgRNA-resistant RRP9-WT and K221R into HCT116 cells with RRP9 deletion (Fig. S3B). Then, RNA was labeled with biotin and analyzed by fluorography. Consistent with previous findings, the data revealed that the amount of 18S rRNA was reduced in RRP9-knockout cells (Fig. 5A, Lane 2). Notably, reintroduction of wild-type RRP9 rescued this effect, whereas reintroduction of the neddylation-deficient mutant of RRP9 (K221R) did not restore processing (Fig. 5A, Lanes 3 and 4). Next, AgNORs were identified to evaluate alterations in nucleolar morphology. Silver staining showed that RRP9 knockout resulted in a reduction in the average AgNOR area, while ectopic expression of RRP9-WT markedly reversed these effects. RRP9-K221R overexpression had no such effect (Fig. 5B). This suggested that RRP9 neddylation leads to upregulation of ribosomal biogenesis. Considering that elevated ribosomal biogenesis is typically associated with stimulation of cell proliferation (38Pelletier J. Thomas G. Volarevic S. Ribosome biogenesis in cancer: New players and therapeutic avenues.Nat. Rev. Cancer. 2018; 18: 51-63Crossref PubMed Scopus (289) Google Scholar, 39Kampen K.R. Sulima S.O. Vereecke S. Keersmaecker K.D. Hallmarks of ribosomopathies.Nucleic Acids Res. 2020; 48: 1013-1028Crossref PubMed Scopus (9) Google Scholar), we then investigated tumor cell proliferation changes resulting from RRP9 neddylation. The growth curves showed a reduced cell growth rate resulting from RRP9 deletion (Fig. 5, C and D). Reintroduction of RRP9-WT significantly promoted cell proliferation. In contrast, reintroduction of RRP9-K221R into these cells caused a nonsignificant increase in tumor growth (Fig. 5, C and D). Moreover, ectopic expression of RRP9 markedly enhanced tumor cell migration, but overexpression of RRP9-K221R had no such effect (Fig. 5E). Collectively, these data indicated that RRP9 neddylation promoted pre-rRNA processing and activation of ribosomal biogenesis, which resulted in enhanced tumor cell proliferation and migration. If Smurf1-mediated RRP9 neddylation is required for proper 18S rRNA processing, depletion of either RRP9 or Smurf1 should cause similar defects in pre-rRNA processing. In fact, the function of Smurf1 in pre-rRNA synthesis remains unknown. We generated a cell line with stable deletion of Smurf1 to investigate pre-rRNA synthesis (Fig. 6A). Fluorographic analysis indicated that the amount of 18S rRNA was reduced in Smurf1-knockout cells (Fig. 6, B–D). Total RNA was extracted and resolved on a 1% formaldehyde-agarose gel. These results also showed that knockout of RRP9 led to a decrease in the total 18S rRNA abundance (Fig. S3C). Furthermore, Smurf1 knockout resulted in a decrease in nucleoli (Fig. 6E). Therefore, it was concluded that deletion of Smurf1 inhibited pre-rRNA processing. Smurf1 plays an oncogenic role in colon cancer (29Xie P. Zhang M. He S. Lu K. Chen Y. Xing G. Lu Y. Liu P. Li Y. Wang S. Chai N. Wu J. Deng H. Wang H.R. Cao Y. et al.The covalent modifier Nedd8 is critical for the activation of Smurf1 ubiquitin ligase in tumorigenesis.Nat. Commun. 2014; 5: 3733Crossref PubMed Scopus (129) Google Scholar). We intended to explore whether Smurf1 promotes tumorigenesis by regulating pre-rRNA processing. Consistent with previous findings, Smurf1 knockout resulted in marked decreases in the tumor cell growth rate (Fig. 6, F and G) and cell migration (Fig. 6H), and Smurf1-knockout cells displayed indications of severe senescence defects (Fig. 6I). Next, sgSmurf1-cotransfected RRP9-WT and RRP9-K221R cell lines were generated (Fig. S3D). Silver staining showed that Smurf1 knockout resulted in a reduction in the average AgNOR area. Ectopic expression of RRP9-WT markedly reversed these effects, but overexpression of RRP9-K221R had almost no effect (Fig. 6J), indicating that neddylation of RRP9 might be important but not essential for Smurf1 to accelerate pre-rRNA processing. Moreover, deletion of Smurf1 markedly suppressed cell proliferation, colony formation, and cell migration, whereas introduction of wild-type RRP9 rescued the processing defect (Fig. 6, K–M). However, introduction of the neddylation-deficient mutant of RRP9 (K221R) did not restore processing (Fig. 6, K–M). These data demonstrated that the oncogenic role of Smurf1 was involved in the positive regulation of pre-rRNA synthesis by catalyzing RRP9 neddylation. Previous data indicated that Nedd8-mediated Smurf1 activation promotes colon cancer progression (29Xie P. Zhang M. He S. Lu K. Chen Y. Xing G. Lu Y. Liu P. Li Y. Wang S. Chai N. Wu J. Deng H. Wang H.R. Cao Y. et al.The covalent modifier Nedd8 is critical for the activation of Smurf1 ubiquitin ligase in tumorigenesis.Nat. Commun. 2014; 5: 3733Crossref PubMed Scopus (129) Google Scholar). Human colorectal cancer tissue samples and matched adjacent normal tissue samples were evaluated to investigate the potential clinical relevance of RRP9 in colon cancer. Compared with that in distal and adjacent tissues, RRP9 expression was notably increased in tumor tissues (Fig. 7, A and B). The samples were divided into groups based on the tumor stage to better understand the correlation of RRP9 with colon cancer progression. The results showed that the expression of RRP9 was lower in low-TNM stage tumors and higher in high-TNM stage tumors (Fig. 7, C and D). Consistent with previous findings, these results also indicated that Smurf1 was significantly upregulated in colorectal cancer tissues compared with matched adjacent tissues (Fig. 7, E and F), in addition to the high expression of RRP9 in tumor tissues (Fig. 7, E and G). Importantly, a positive correlation of Smurf1 with RRP9 was observed among patients with tumors (Fig. 7H). Both the expression and neddylation of RRP9 were clearly upregulated in patients with colorectal cancer (Fig. 7, I–K). Taken together, these data suggested that RRP9 was highly expressed in colon cancer, concomitant with the enhancement of Smurf1-catalyzed RRP9 neddylation, to promote tumor progression. Previous studies reported that Nedd8 ligases such as Dcn1, Roc1/Rbx1, IAPs, c-Cbl, RNF111, RNF168, and MDM2 are all RING finger scaffold-type E3s. Smurf1 is the first identified HECT-type Nedd8 ligase to supplement the classification of Nedd8 ligases. Smurf1 integrates the ubiquitination and neddylation pathways as a dual E3 ligase. Smurf1 forms a Nedd8-thioester intermediate and then catalyzes its own neddylation on multiple lysine residues. This neddylation enhances Smurf1-mediated ubiquitination (29Xie P. Zhang M. He S. Lu K. Chen Y. Xing G. Lu Y. Liu P. Li Y. Wang S. Chai N. Wu J. Deng H. Wang H.R. Cao Y. et al.The covalent modifier Nedd8 is critical for the activation of Smurf1 ubiquitin ligase in tumorigenesis.Nat. Commun. 2014; 5: 3733Crossref PubMed Scopus (129) Google Scholar, 40He S. Cao Y. Xie P. Dong G. Zhang L. The Nedd8 non-covalent binding region in the Smurf HECT domain is critical to its ubiquitn ligase function.Sci. Rep. 2017; 7: 41364Crossref PubMed Scopus (17) Google Scholar). A large number of Smurf1 ubiquitination substrates have been identified and include Smad1/5, MEKK2, and RhoA (31Zhu H. Kavsak P. Abdollah S. Wrana J.L. Thomsen G.H. A SMAD ubiquitin ligase targets the
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