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1、LETTERdoi:10.1038/nature14978Cell-fate determination by ubiquitin-dependent regulation of translationAchim Werner1,2, Shintaro Iwasaki2, Colleen A. McGourty2, Sofia Medina-Ruiz2, Nia Teerikorpi1,2, Indro Fedrigo2, Nichol

2、as T. Ingolia2 molecular weight is given in kDa. c, KBTBD8-depleted hESCs were subjected to neural conversion and analysed by immuno- fluorescence microscopy (mean of 3 biological replicates, 6s.e.m; ,1,500 cells per co

3、ndition). d, Xenopus tropicalis embryos injected with translation-blocking morpholinos against KBTBD8 were analysed by in situ hybridization. e, Model of the CUL3KBTBD8-controlled developmental switch.1Howard Hughes Medi

4、cal Institute, University of California, Berkeley, California 94720, USA. 2Department of Molecular and Cell Biology, University of California, Berkeley, California 94720, USA.2 4 S E P T E M B E R 2 0 1 5 | V O L 5 2 5 |

5、 N A T U R E | 5 2 3G2015 Macmillan Publishers Limited. All rights reserved(Fig. 3a, b and Extended Fig. 6a, b). The same aberrant differentiation program was observed if we depleted TCOF1 or NOLC1 (Fig. 3a, c and Extend

6、ed Data Fig. 6a, c, d), but not other KBTBD8-binding partners (Fig. 3a and Extended Data Fig. 6e, f). Demonstrating that these pro- teins act in a common pathway, co-depletion of KBTBD8 and TCOF1 or NOLC1,respectively,mi

7、rrored thedifferentiation program of singly depleted hESCs (Fig. 3d). We therefore conclude that TCOF1 and NOLC1 are critical monoubiquitylation substrates of CUL3KBTBD8during neural crest specification. Consistent with

8、this notion, muta- tions in TCOF1 cause Treacher Collins syndrome, a craniofacial dis- order characterized by loss of cranial neural crest cells2,3. To understand how CUL3KBTBD8 drives neural crest specification, we iden

9、tified proteins that selectively recognized ubiquitylated, but not unmodified, TCOF1 using cells that were reconstituted with either wild-type KBTBD8, inactive KBTBD8(Y74A), or empty vector. Notably, NOLC1 emerged as the

10、 major effector that was recruited to ubiquitylated TCOF1 (Fig. 4a), an observation that was confirmed by affinity purification coupled to western blot analysis (Fig. 4b). Monoubiquitylation often stabilizes binding part

11、ners, and depletion of KBTBD8 caused degradation of both TCOF1 and NOLC1 at later stages of neural conversion (Fig. 3c and Extended Data Fig. 7a, b). On the basis of these results, we established a sequential affinity pu

12、rification protocol to determine the composition of ubiquitylation- dependentTCOF1–NOLC1complexes.WefoundthatTCOF1–NOLC1 assembliesengaged RNA polymerase I; the H/ACA complex catalysing rRNA pseudouridylation; and the SS

13、U processome controllingmaturation and modification of the small ribosomal subunit (Fig. 4c, d and Extended Data Fig. 7c). Accordingly, ubiquitylation by CUL3KBTBD8 brought endogenous RNA polymerase I into com- plexes wi

14、th the SSU processome (Fig. 4e), which required TCOF1 and NOLC1 (Extended Data Fig. 7d). Similar observations were made in hESCs, where a robust interaction between RNA polymerase I and SSU processome was lost upon deple

15、tion of KBTBD8 (Fig. 4f). Thus, CUL3KBTBD8 induces the ubiquitin-dependent formation of TCOF1–NOLC1 complexes that serve as a platform to connect RNA polymerase I with enzymes responsible for ribosomal processing and mod

16、ification (Fig. 4g). This observation supports a role of ubiquityla- tion in neural crest specification, as mutations in RNA polymerase I also cause Treacher Collins syndrome19. Although KBTBD8 targets proteins linked to

17、 ribosome biogenesis, its depletion did not affect the abundance of rRNAs or mRNAs encoding ribosomal proteins; levels of ribosomal proteins; processing of precursor rRNAs; nucleolar integrity; export of the small riboso

18、mal subunit; ribosome binding to mRNA judged by polysome gradient analysis; global mRNA translation detected by metabolic labelling; or cell survival (Fig. 5a, d and Extended Data Fig. 8a–h). Accordingly, a global reduct

19、ion in translation caused by rapamycin did not pheno- copy the loss of KBTBD8 (Extended Data Fig. 9a, b). Depletion of TCOF1 also did not affect rRNA synthesis, p53 activation, or cell survival at the time of neural cres

20、t specification (Extended Data Fig. 9c–e), although consistent with previous work3,20, it reduced rRNA levels and triggered cell death at late stages of neural conversionNucleosomeDKC1RPAC1RPA1RPA2 RPA49RPA2RPA2 RPA34RNA

21、 Pol I complexRPA2 RPA43RPABC1SSU processomeNOP56Box C/D snoRNPcomplexH/ACA snoRNPcomplexRibosome maturationUTP-C–CKII complexrRNA transcriptionrRNA modificationTCOF1 NOLC1ARRB1/2H2AE3 ligaseUbNHP2NOP10 NOP58FBL TCOF1–

22、NOLC1 complexCSK2A1 CSK2A2CSK2BNCLH2BH3 H4H1KBTBD8Fold enrichment>20 fold>10 fold>5 folda cdRPA1KBTBD8RPA2RPB1DKC1NHP2NOP58NOP56HA–NOLC1HA–NOLC1Flag–TCOF1Flag–TCOF1ARRB1ARRB2Input (1%)1st IP: anti-HA (10%)Wester

23、n250100755025025010050157537752nd IP: anti-FlagCSK2ACSK2AWestern37NOP58 75RPA2KBTBD8 75RPA1 250100IP: anti-IgGW579AWTY74A–KBTBD8–FlagIP: anti-RPA1IP: anti-IgGIP: anti-RPA1W579AWTY74A–Input e75 NOP5837CSK2A IgG75 KBTBD8RP

24、A2 100Western250 RPA1InputshKBTBD8 shCtrlIP: anti-IgG/RPA1gCUL3KBTBD8(Ubi)SSU proc. (PO4) H/ACA (Ψ)Ub Ub Ub UbRNA Pol I76854321–12 4 6 8 10Fold change TSCs (WT-control)TCOF1 interacting proteinsFold change TSCs (WT-Y74A)

25、KBTBD8 ubiquitylation- dependentKBTBD8 binding- dependentKBTBD8- independentNOLC1 00fbNOLC1KBTBD8Western2501507550 ARBB1 ARBB250 ARBB1 ARBB2NOLC1 15075 KBTBD8IP: anti-Flag250 Flag–TCOF1Flag–TCOF1W579AWTY74AKBTBD8InputTC

26、OF1NOLC1Figure 4 | Ubiquitylation-dependent TCOF1–NOLC1 complexes couple RNA polymerase I to ribosome modification enzymes. a, Interactors of TCOF1 in 293T cells reconstituted with KBTBD8 or KBTBD8(Y74A) (sum of 3 biolog

27、ical replicates per condition). b, Validation of CUL3KBTBD8-dependent formation of TCOF1–NOLC1 complexes. c, CompPASS mass spectrometry analysis of sequential immunoprecipitation of Flag–TCOF1/HA–NOLC1 complexes. d, Vali

28、dation of sequential affinity purification of KBTBD8-dependent TCOF1–NOLC1 complexes (full scans available in Supplemen- tary Fig. 1). e, Immunoprecipitation of RNA polymerase I from 293T cells reconstituted with KBTBD8

29、variants. f, Immunoprecipitation of RNA polymerase I from hESCs depleted of KBTBD8. g, Model of ubiquitin- dependent formation of a TCOF1–NOLC1 platform. Molecular weight is given in kDa.2 4 S E P T E M B E R 2 0 1 5 | V

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