FEBS Letters 588 (2014) 3685–3691

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The beta-isoform of the BRCA2 and CDKN1A()-interacting (BCCIP) stabilizes nuclear RPL23/uL14

Emanuel Wyler a,1, Franziska Wandrey a, Lukas Badertscher a,b, Christian Montellese a,b, Daniel Alper a, ⇑ Ulrike Kutay a, a Institute of Biochemistry, ETH Zurich, CH-8093 Zurich, Switzerland b Molecular Life Sciences Ph.D. Program, CH-8057 Zurich, Switzerland article info abstract

Article history: BRCA2 and CDKN1A(p21,CIP1)-interacting protein (BCCIP) is an evolutionary conserved protein Received 18 July 2014 implicated in maintenance of genome stability and cell cycle progression. Two isoforms of BCCIP Revised 6 August 2014 with distinct C-terminal domains exist in humans. We show that mammalian BCCIPb, but not Accepted 11 August 2014 BCCIPa, forms a ternary complex with the ribosomal protein RPL23/uL14 and the pre-60S trans- Available online 19 August 2014 acting factor eIF6. Complex formation is dependent on an intact C-terminal domain of BCCIPb. Edited by Felix Wieland Depletion of BCCIPb reduces the pool of free RPL23, and decreases eIF6 levels in nucleoli. Overex- pression of BCCIPb leads to nucleoplasmic accumulation of extra-ribosomal RPL23 and stabilizes overexpressed RPL23, suggesting that BCCIPb functions as nuclear chaperone for RPL23. Keywords: BCCIP Ó 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Ribosome biogenesis Eukaryotic initiation factor 6 (eIF6) Ribosomal protein RPL23/uL14

1. Introduction and BCCIPb causes a G1/S delay accompanied by a p53-dependent increase in p21 levels [3,4], downregulation of either isoform The protein BCCIP, also called TOK-1, has been originally identi- reduces p21 levels and impairs checkpoint activation in response fied as an interaction partner of the tumor suppressor to DNA damage [4]. p21(CDKN1A,CIP1) and BRCA2 in yeast two-hybrid assays [1,2]. Mouse knockdown experiments have recently demonstrated In humans, BCCIP exists in two isoforms, BCCIPa and BCCIPb, that BCCIP is essential for embryonic development [8]. BCCIP- which are generated by alternative pre-mRNA splicing of the 30 deficient mouse embryonic fibroblasts display severe proliferation exon. While the N-terminal 258 amino acids are identical in both defects accompanied by spontaneous chromatin bridges, and are isoforms, the C-terminal domains comprising 64 and 56 amino sensitized towards UV-induced DNA damage [8]. Consistent with acids in BCCIPa and BCCIPb, respectively, differ. Both BCCIPa and a role of BCCIP in the maintenance of genome stability, reduced BCCIPb are localized to the cell nucleus [1,2]. BCCIP expression has been observed in several cancer types [9–13]. Overexpression and depletion studies in mouse and human cell BCCIP is evolutionary conserved in eukaryotes. The budding lines have implicated BCCIP in cell cycle control, i.e. G1/S transition yeast homolog of BCCIP, Bcp1, is also essential and has been impli- [3–5], homologous recombination and suppression of spontaneous cated in the biogenesis of 60S ribosomal subunits as well as in DNA damage [6,7]. The observed influence of BCCIP on the G1/S nuclear export of the PI4P 5-kinase Mss4 [14,15]. checkpoint has been explained by its ability to regulate expression In the course of characterizing trans-acting factors involved in of the CDK inhibitor p21 [3,4]. Whereas overexpression of BCCIPa ribosome biogenesis in human cells, we found BCCIPb associated with eukaryotic initiation factor 6 (eIF6) in tandem-affinity purifi- cation (TAP) experiments. Using either BCCIPa or BCCIPb as TAP Abbreviations: BCCIP, BRCA2 and CDKN1A(p21,CIP1)-interacting protein; TAP, tandem affinity purification; IMAC, immobilized metal affinity chromatography bait, we identified a complex of RPL23(uL14) and eIF6 as a specific ⇑ Corresponding author. interaction partner of BCCIPb. Further analysis revealed that E-mail address: [email protected] (U. Kutay). BCCIPb is required to maintain a nucleoplasmic, extra-ribosomal 1 Present address: Max-Delbrück-Center for Molecular Medicine, Berlin Institute pool of RPL23. for Medical Systems Biology, Robert-Rössle-Strasse 10, D-13125 Berlin, Germany. http://dx.doi.org/10.1016/j.febslet.2014.08.013 0014-5793/Ó 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. 3686 E. Wyler et al. / FEBS Letters 588 (2014) 3685–3691

2. Materials and methods were analyzed by SDS–PAGE followed by silver staining, Western blotting or mass spectrometric analysis of selected protein bands 2.1. Molecular cloning as described [16].

For expression of TAP constructs in HEK293 cells, pcDNA5/FRT/ 2.4. Protein expression and in vitro binding assays TO-derived vectors encoding for eIF6-StHA, HASt-BCCIPa, HASt- BCCIPb and C-terminal truncations thereof were generated as Proteins were expressed in Escherichia coli Rosetta (Novagen) at described previously [16]. For expression of GFP-BCCIPa and 25 °C. Cells were lysed in lysis buffer (10 mM Tris/HCl pH 7.5, GFP-BCCIPb fusion proteins, the pcDNA5/FRT/TO vector backbone 200 mM NaCl, 2 mM MgCl2, 5% glycerol, 50 mM imidazole, 2 mM (Invitrogen) was modified by insertion of the GFP coding sequence. b-mercaptoethanol) by sonication. To promote complex formation, The pcDNA3-derived FLAG-RPL23 expression vector CMV-FLAG- the lysates were mixed and incubated at 4 °C for 1 h. Then, Ni–NTA L23 has been described [17]. For bacterial expression of zz-eIF6 agarose was added for 1 h. Beads were washed four times in lysis full-length or DC, pET28 (Novagen) was modified by inserting buffer and bound proteins were eluted with 400 mM imidazole. the coding sequence encoding the zz-tag [18], followed by inser- Eluates were analyzed by SDS–PAGE followed by Coomassie stain- tion of an eIF6 full-length or DC-encoding insert into the NdeI ing or Western blotting. and XhoI sites. Co-expression of RPL23-His6 and BCCIPb was driven from a pET-Duet vector (Novagen). 2.5. Transient transfections, RNAi and immunofluorescence 2.2. Antibodies Cells were transiently transfected for 24 h using X-tremeGENE 9 Antibodies against human RPL23, LSG1, PA2G4, eIF6 used for (Roche) and fixed in 4% PFA. For RNAi, cells were transfected with Western blotting and BCCIP were raised in rabbits against purified, 9 nM siRNA using Interferin (Polyplus transfection) for 72 h and recombinant His-tagged proteins. An antibody specifically either fixed in 4% PFA or harvested for sucrose gradient centrifuga- recognizing the BCCIPb isoform was raised in rabbits against a tion or Western blot analysis. The following siRNA were used: All- C-terminal peptide of BCCIPb (C-PGDKMNEIMDKLKEYLSV). Other Stars negative control siRNA (Qiagen); si-BCCIPa GAACCUUCAUG antibodies have been described previously [16,19,20]. Anti-FLAG ACUGUUGG; si-BCCIPb GAGGCAAAUGGUCUUUUGA [4]. Immuno- was obtained from Sigma (F1804), anti-eIF6 (sc-390441) for immu- fluorescence analysis was performed as previously described nofluorescence and anti-MRTO4 (sc-81856) from Santa Cruz, and [20]. Images were taken using a Leica SP2 confocal scanning anti-HA from Covance (MMS-101P). Secondary antibodies for system. immunofluorescence were purchased from Invitrogen (LuBio- Science, Switzerland). 2.6. Sucrose gradient analysis

2.3. Tandem affinity purification and mass spectrometry 400 lg of HeLa cell extract were loaded onto linear 10–45% sucrose gradients in 10 mM Tris/HCl pH 7.5, 100 mM KCl, 2 mM

TAP was performed from stable HEK293 FlpIn TRex cells 24 h MgCl2 and centrifuged for 2 h at 55000 rpm, 4 °C in a TLS55 rotor after induction of expression of the indicated bait proteins with (Beckman Coulter). Gradient fractions were precipitated with 0.5 lg/ml tetracycline as previously described [16]. The eluates TCA and analyzed by Western blotting.

Fig. 1. BCCIPb forms a complex with RPL23 and eIF6. (A) Tandem affinity purification was performed using cell lysates of HEK293 cells expressing tagged forms of eIF6, BCCIPa and BCCIPb. Eluates were analyzed by SDS–PAGE and silver staining. TAP bait proteins are marked with asterisks. Protein band examined by mass spectrometry is indicated by an open circle. (B) Western blot analysis of the TAP eluates using the indicated antibodies. (C) zz-eIF6, zz-eIF6DC, BCCIPb, or BCCIPb and RPL23-His6 were expressed in E. coli. Bacterial lysates (input) were either used individually or mixed (zz-eIF6DC and BCCIPb/RPL23-His6 lysates) for retrieval of His-tagged RPL23 and associated factors by IMAC. Inputs and eluates were analyzed by SDS–PAGE followed by Coomassie blue staining. E. Wyler et al. / FEBS Letters 588 (2014) 3685–3691 3687

3. Results and discussion spectrometry, which altogether identified 22 unique peptides and covered almost the entire C-terminal domain of BCCIPb. 3.1. BCCIPb, RPL23 and eIF6 form a protein complex Reciprocal TAP with either N-terminally HASt-tagged BCCIPa or BCCIPb as bait proteins confirmed that only the b-isoform is part of To identify factors associated with human pre-60S particles, we a protein complex consisting of three components, namely BCCIPb, used the 60S ribosomal subunit biogenesis factor eIF6 [21,22] as eIF6, and RPL23 (Fig. 1A, second and third lane). In contrast to eIF6 bait for TAP. eIF6 was expressed with a C-terminal tandem strepta- TAP, HASt-BCCIPb did not isolate a pre-ribosomal particle, indicat- vidin-binding peptide (St) and hemagglutinin epitope (HA) tag in ing that BCCIPb might be either unable to associate with pre- HEK293 cells using an experimental setup previously employed ribosomal subunits or that the interaction is too transient or for the purification of pre-ribosomal particles [16,23]. Eluates of unstable (Fig. 1B). Notably, appending the TAP tag to the C termi- eIF6-StHA TAP indeed contained pre-60S particles as indicated by nus of BCCIPb yielded a similar pattern of interaction partners the complex pattern of silver stained bands in SDS–PAGE (data not shown). (Fig. 1A, left lane), and as verified by the presence of known 60S Next, we tried to rebuild the trimeric complex using recombi- trans-acting factors like LSG1, NMD3, PA2G4/ARX1 and the ribo- nant proteins expressed in bacteria. Due to the low solubility of somal protein RPL10/uL16 on immunoblots (Fig. 1B, left lane). both RPL23 and eIF6, we started with a dimer of untagged BCCIPb However, the silver stained pattern of interacting partners revealed and RPL23-His6. A complex of BCCIPb and RPL23-His6 could be suc- that one protein of 45 kDa was present at a higher stoichiometry cessfully isolated by immobilized metal affinity chromatography than other proteins (Fig. 1A, left lane, open circle) and almost as (IMAC) after co-expression of both factors from a bicistronic cas- prominent as the bait (Fig. 1A, left lane, asterisks). The over- sette (Fig. 1C). There was no binding of BCCIPb to the Ni–NTA affin- enriched protein was identified as the b-isoform of BCCIP by mass ity matrix in the absence of RPL23-His6 (data not shown).

Fig. 2. Overexpression of BCCIPb leads to nucleoplasmic accumulation of RPL23 and eIF6. (A) HeLa cells were transiently transfected with constructs for GFP-BCCIPa or GFP- BCCIPb. 24 h after transfection, cells were fixed and analyzed for the localization of endogenous RPL23, eIF6 or MRTO4 by indirect immunofluorescence and of GFP-BCCIPa/b by direct fluorescence using confocal microscopy. Scale bar, 20 lm. (B) HASt-tagged full-length BCCIPb, BCCIPbDC3, or BCCIPbDC7 were expressed in HEK293 cells and used as TAP baits. TAP eluates were analyzed by SDS–PAGE followed by silver staining. (C) HASt-tagged BCCIPb wild-type or the indicated C-terminal truncation variants were transiently expressed in HeLa cells. Protein localization was determined by indirect immunofluorescence 24 h after transfection as in (A). Scale bar,20lm. 3688 E. Wyler et al. / FEBS Letters 588 (2014) 3685–3691

To improve expression and solubility of eIF6, we fused a zz-tag depletion of BCCIPb would affect the subcellular localization of to its N terminus and truncated the C-terminal 20 amino acids, as RPL23 and eIF6 in HeLa cells. Immunofluorescence analysis of these are likely unstructured [24]. IMAC experiments revealed that control cells showed that RPL23 localized predominantly to the zz-eIF6DC binds to the RPL23/BCCIPb dimer (Fig. 1C). We could cytoplasm as expected for a ribosomal protein, whereas the 60S thus show that the three proteins are sufficient to form the tri- subunit biogenesis factor eIF6 was enriched in nucleoli. meric complex, without any additional factors or modifications Overexpression of BCCIPb, but not of BCCIPa, led to an accumu- needed for complex formation. However, in the absence of lation of RPL23 and eIF6 in the nucleoplasm (Fig. 2A). Localization RPL23, binding of zz-eIF6DC to BCCIPb could not be reconstituted of MRTO4, another trans-acting factor involved in 60S subunit in these in vitro binding studies (data not shown), which might biogenesis [23], was not affected by BCCIPb overexpression indicate that eIF6 associates with BCCIPb indirectly via RPL23. A (Fig. 2A). Western blot analysis did not reveal any major changes direct interaction between RPL23 and eIF6 has been previously in the levels of RPL23 or eIF6 upon overexpression of BCCIPb (data documented for both their archaeal (L14; IF6) and yeast homologs not shown). (rpL23; Tif6) [25,26]. Currently, we cannot exclude that an interac- To analyze whether the observed effect of overexpression of tion between BCCIPb and eIF6 in the absence of RPL23 can exist in BCCIPb was dependent on the ability of BCCIPb to form a complex principle. For instance, BCCIPb-eIF6 interaction might require an with its two interaction partners, we set out to generate truncation intact C terminus of eIF6, certain posttranslational modifications mutants of BCCIPb deficient in RPL23 and eIF6 binding. Removal of or a specific configuration of these factors on pre-ribosomal the three C-terminal amino acids of BCCIPb led to a reduced asso- particles. ciation with both RPL23 and eIF6, as revealed by TAP of HEK293 cell extracts expressing HASt-BCCIPbDC3 (Fig. 2B). Deletion of 3.2. Overexpression of BCCIPb leads to nucleoplasmic accumulation of the seven C-terminal amino acids strongly decreased complex RPL23 formation. Notably, the truncated bait proteins could be retrieved in similar quantities as wild-type BCCIPb, indicating that the To investigate the functional importance of the observed overall stability of the bait protein was not compromised by the protein interactions, we next analyzed how overexpression and truncations.

Fig. 3. BCCIPb depletion results in a reduction of nucleolar eIF6. (A) HeLa cells were treated with the indicated siRNAs for 72 h. Cells were fixed and analyzed by immunofluorescence using the indicated antibodies. Scale bar, 20 lm. (B) Quantification of the experiment performed in (A). Nucleolar intensity of MRTO4 (black) or eIF6 (blue) signal was measured in 15 nucleoli per condition and normalized against control cells. Bars are depicted with standard deviation. (C) Western blot analysis of cells from (A) using the indicated antibodies. E. Wyler et al. / FEBS Letters 588 (2014) 3685–3691 3689

Fig. 4. BCCIPb depletion destabilizes extra-ribosomal RPL23. HeLa cells were left untreated or depleted of either BCCIPa or BCCIPb by RNA interference. Cell extracts (inputs) were fractionated on linear 10–45% sucrose gradients. Gradient fractions were precipitated and analyzed by SDS–PAGE followed by Western blotting using the indicated antibodies.

When HASt-BCCIPbDC3 or HASt-BCCIPbDC7 were expressed in in the ‘free’ protein fractions of the sucrose gradient (Fig. 4 and HeLa cells, they localized to the nucleus and were expressed at [20]). We therefore examined whether this extra-ribosomal pool similar levels as the wild-type protein. However, while overexpres- of RPL23 might be stabilized by presence of BCCIPb. Extracts of sion of HASt-BCCIPbDC3 caused a slight nucleoplasmic accumula- HeLa cells depleted of either BCCIPa or BCCIPb by RNA interference tion of RPL23, overexpression of HASt-BCCIPbDC7 did not (Fig. 2C). were fractionated by sucrose gradient centrifugation. The RPL23 This demonstrates that the effect of overexpression of BCCIPb on content in the upper part of the gradient was unchanged after the localization of RPL23 requires its ability to bind its partners. depletion of BCCIPa. In contrast, depletion of BCCIPb almost com- The effect of BCCIPb overexpression on RPL23 was not altered pletely eliminated RPL23 from the light part of the gradient, when eIF6 was co-overexpressed (data not shown), suggesting that whereas co-sedimentation of RPL23 with ribosomes was unaf- the phenotype is not due to limiting amounts of eIF6. fected (Fig. 4). Together, this suggests that BCCIPb might function Depletion of BCCIPb caused a decrease in the nucleolar signal of to stabilize a pool of RPL23 that is not incorporated into ribosomal eIF6, accompanied by a slight decrease in eIF6 protein levels subunits. (Fig. 3), further substantiating the link between BCCIPb and eIF6. If BCCIPb was an indispensable chaperoning factor for extra- ribosomal RPL23, then the cellular concentration of BCCIPb should 3.3. BCCIPb stabilizes a nuclear pool of extra-ribosomal RPL23 determine how much extra-ribosomal RPL23 cells can maintain. To test this model, we first examined whether cellular BCCIPb levels In our TAP experiments (Fig. 1), we had isolated HASt-BCCIPb in are sufficient to allow for stabilization of over-expressed, extra- a complex with eIF6 and RPL23 but had failed to detect any obvi- ribosomal RPL23. We used an N-terminally FLAG-tagged variant ous association with pre-ribosomal particles. To analyze whether of RPL23 that has been previously employed as tool to study endogenous BCCIPb might co-fractionate with precursors of 60S extra-ribosomal functions of RPL23 (see below and [17,27]). Nota- subunits, we used sucrose gradient centrifugation of HeLa cell bly, FLAG-RPL23 could not be efficiently expressed in the absence extracts. Unlike the 60S trans-acting factors eIF6 or NMD3, which of additional BCCIPb (Fig. 5A, inputs). Upon co-expression of partially co-sedimented with ribosomal proteins in the dense HASt-BCCIPb, however, the levels of FLAG-RPL23 were consider- fractions of the gradient (Fig. 4), both isoforms of BCCIP were only ably higher. After sucrose gradient centrifugation, FLAG-RPL23 detected in the lighter part of the gradient, where free proteins and migrated at the top part of the gradient, like BCCIPb (Fig. 5A). smaller protein complexes are found. We conclude that BCCIPb Analysis of FLAG-RPL23 at the singe cell level using immunoflu- might either be unable to associate with pre-60S particles or that orescence confirmed that the levels of BCCIPb limit FLAG-RPL23 such interaction is too transient or unstable to be detected by expression. When transfected alone, FLAG-RPL23 was expressed co-sedimentation or TAP. at low levels and localized to nucleoli (Fig. 5B, top left). Co-overex- Inspection of the sedimentation behavior of RPL23, however, pression of BCCIPb, but not of BCCIPa, allowed for higher expres- revealed that in contrast to other ribosomal proteins like RPL10/ sion as evidenced by the strong fluorescent signal obtained for uL16, RPS3/uS3, or RPL23A/uL23, an unusually large portion of FLAG-RPL23 by immunofluorescence. Under these conditions, both RPL23 was not associated with ribosomal subunits and migrated GFP-BCCIPb and FLAG-RPL23 were majorly nucleoplasmic. Taken 3690 E. Wyler et al. / FEBS Letters 588 (2014) 3685–3691

Fig. 5. BCCIPb stabilizes extra-ribosomal RPL23 in the nucleus. (A) HeLa cells were left untreated or transiently transfected with constructs for expression of either FLAG- RPL23 alone or together with HASt-BCCIPb. 24 h later, cell extracts were prepared (inputs), fractionated on a sucrose gradient and analyzed by SDS–PAGE followed by Western blotting using the indicated antibodies. (B) Constructs for FLAG-RPL23 and GFP-BCCIPa or GFP-BCCIPb were transiently transfected into HeLa cells. After 24 h, cells were fixed and analyzed by indirect immunofluorescence using an anti-FLAG antibody to detect exogenous FLAG-RPL23 and using direct fluorescence for GFP-BCCIP or GFP- BCCIPb. Pictures were taken by confocal microscopy at low and high exposure to account for the different expression levels. Scale bar, 20 lm. together, our data indicate that BCCIPb is necessary for the stabil- manifest in cells, such as primary cells, that do not overproduce ization of extra-ribosomal RPL23 in the nucleus. ribosomal constituents like many cancer cells do. As BCCIPb forms a complex with nuclear RPL23 and eIF6, one Alternatively, the function of BCCIPb as an RPL23 chaperone might speculate that BCCIPb functions as a chaperone for newly might be required to control the activity of BCCIPb as a cell cycle synthesized RPL23, likely by preventing degradation of free regulator, or to promote an extraribosomal function of RPL23 in RPL23. BCCIPb might even promote incorporation of RPL23, per- the nucleus. Based on the previous observations that BCCIP overex- haps in a binary complex with eIF6, into nascent 60S subunits. If pression leads to p21-mediated cell cycle arrest that is dependent so, then one would predict that BCCIPb is in turn required for on p53, and BCCIP depletion results in reduced p21 levels and a 60S biogenesis. Although we have repeatedly tested this assump- failure to appropriately respond to DNA damage [3–5], our results tion using 60S biogenesis reporters in different HeLa cell back- open the possibility that these effects involve a crosstalk with grounds, there was no effect in the majority of experiments. This RPL23 and/or eIF6. Clearly, the trimeric complex between BCCIPb, could indicate that either the RPL23 chaperoning function of RPL23 and eIF6 is ideally poised to constitute part of a relay mech- BCCIPb is dispensable for 60S production or that its role is redun- anism between ribosome biogenesis and cell cycle control. dant with other RPL23 chaperones such as nucleostemin [17] or eIF6 itself. In support for a role of BCCIPb in 60S biogenesis comes Acknowledgements the observation that yeast bcp1ts cells display a strong nuclear 60S subunit biogenesis defect [14]. It is currently unclear whether this We thank Martin Eilers (University of Würzburg) for the allele of BCP1 behaves as dominant-negative or loss-of-function. FLAG-RPL23 construct and valuable discussion, Jens Pfannstiel Perhaps, a function of human BCCIPb in ribosome biogenesis only and Berit Würtz (Serviceeinheit LSC, Universität Hohenheim, E. Wyler et al. / FEBS Letters 588 (2014) 3685–3691 3691

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