The Treacher Collins syndrome (TCOF1) product is involved in ribosomal DNA gene transcription by interacting with upstream binding factor

Benigno C. Valdez*†, Dale Henning*, Rolando B. So*, Jill Dixon‡, and Michael J. Dixon‡

*Department of Pharmacology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030; and ‡School of Biological Sciences and Department of Dental Medicine and Surgery, University of Manchester, 3.239 Stopford Building, Oxford Road, Manchester M13 9PT, United Kingdom

Edited by Mark T. Groudine, Fred Hutchinson Cancer Research Center, Seattle, WA, and approved June 8, 2004 (received for review April 8, 2004) Treacher Collins syndrome (TCS) is an autosomal dominant disorder penetrance and severity of the craniofacial defects observed on characterized by an abnormality of craniofacial development that Tcof1ϩ/Ϫ mice (18). arises during early embryogenesis. TCS is caused by in Despite several genetic studies of TCOF1, the cellular function the gene TCOF1, which encodes the nucleolar phosphoprotein of treacle remains unknown. The localization of treacle to the treacle. Even though the genetic alterations causing TCS have been suggests a role in the production of rRNA. A recent uncovered, the mechanism underlying its pathogenesis and the report showed a physical interaction between treacle and function of treacle remain unknown. Here, we show that treacle is pNop56, a component of the ribonucleoprotein complex that involved in ribosomal DNA gene transcription by interacting with 2Ј-O-ribose methylates pre-rRNA (19). Here, we present evi- upstream binding factor (UBF). Immunofluorescence labeling dence that treacle is involved in mammalian ribosomal DNA shows treacle and UBF colocalize to specific nucleolar organizer (rDNA) gene transcription by interacting with upstream binding regions and cosegregate within nucleolar caps of actinomycin factor (UBF), a RNA polymerase I (RNA pol I) transcription D-treated HeLa cells. Biochemical analysis shows the association of factor (20–22). treacle and UBF with . Immunoprecipitation and the yeast two-hybrid system both suggest physical interaction of the Materials and Methods two nucleolar phosphoproteins. Down-regulation of treacle ex- Cells and Antibodies. HeLa cells were grown in DMEM with 10% pression using specific short interfering RNA results in inhibition of FBS, penicillin G, and streptomycin sulfate at 37°C, 5% CO2. ribosomal DNA transcription and cell growth. A similar correlation Rabbit anti-treacle antibody (Ab 014) against amino acids 1–55 ؉/؊ is observed in Tcof mouse embryos that exhibit craniofacial of human treacle has been characterized (12). Other anti-treacle defects and growth retardation. Thus, treacle haploinsufficiency in antibodies were produced in rabbits by using recombinant TCS patients might result in abnormal development caused by polypeptides (amino acids 214–290 and 1166–1339) expressed in inadequate ribosomal RNA production in the prefusion neural folds Escherichia coli. Other antibodies included anti-Gu␣ (23), anti- during the early stages of embryogenesis. The elucidation of a nucleolin (24), anti-nucleophosmin (25), anti-UBF (Santa Cruz physiological function of treacle provides important information of Biotechnology), and anti-BrdUrd (Sigma). relevance to the molecular dissection of the biochemical pathology of TCS. Indirect Immunofluorescence. Cells grown on slides were analyzed by indirect immunofluorescence staining as described (26). Cells reacher Collins syndrome (TCS) is an autosomal dominant were examined with a Nikon Eclipse TE2000-U inverted micro- Tdisorder of craniofacial development. The structures af- scope equipped with a Coolsnap digital color camera. fected in TCS patients arise from the first and second branchial MEDICAL SCIENCES arches during early embryogenesis (1). Early studies resulted in Western Blot Analysis. Samples were boiled in Laemmli buffer for mapping of the TCS locus to 5q31-q34 (2–4). Using 3 min. extracts were electrophoresed on 6% or 9% segregation analysis in affected families, the TCS gene, desig- polyacrylamide-SDS gels and blotted onto Immun-Blot poly(vi- nated TCOF1, was positionally cloned (5). Tcof1 heterozygous nylidene difluoride) membrane (Bio-Rad). Immunochemilumi- mice die perinatally as a result of severe craniofacial anomalies nescence was done by using the ECL-plus Western blotting arising from increased apoptosis in the prefusion neural folds, detection system (Amersham Pharmacia Biosciences). The where the highest level of Tcof1 expression is observed in WT membrane was incubated with one antibody followed by en- mice (6). hanced chemiluminescence to detect the antigen. The membrane Early reports showed that the TCOF1 gene is comprised of 26 was stripped and probed with another antibody. exons (7, 8). Recently, we discovered an additional exon between exons 6 and 7 (9). The encoded protein, treacle, has homology Analysis of Chromosome Spreads and Chromosome-Associated Pro- with Nopp140, a trafficking nucleolar phosphoprotein (10). teins. HeLa cells were blocked in mitosis with 0.1 ␮g͞ml colchi- Although treacle and Nopp140 share common characteristics, cine for 6 h. Chromosome spreads were prepared as described the inability of treacle to colocalize with Nopp140 to Cajal bodies (27). Chromosome-associated were separated from suggests different and distinct functions (11). cytoplasmic proteins, and fractions were analyzed by Western The C-terminal region of treacle is important for localization blot as described above. to the nucleolus (12, 13). It was hypothesized that mutations in the TCOF1 gene (7, 14–17) could result in mislocalization of truncated proteins and, consequently, loss of treacle functions, This paper was submitted directly (Track II) to the PNAS office. suggesting that TCS results from treacle haploinsufficiency. No Abbreviations: TCS, Treacher Collins syndrome; UBF, upstream binding factor; rDNA, genotype–phenotype correlation has been observed, which ribosomal DNA; siRNA, short interfering RNA; BrUTP, bromouridine; RNA pol I, RNA might explain the wide clinical variability observed among TCS polymerase I. patients. This variability may be explained by a recent study that †To whom correspondence should be addressed. E-mail: [email protected]. demonstrated that genetic background has a major effect on the © 2004 by The National Academy of Sciences of the USA

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0402492101 PNAS ͉ July 20, 2004 ͉ vol. 101 ͉ no. 29 ͉ 10709–10714 Downloaded by guest on October 2, 2021 Treatment with Actinomycin D. HeLa cells were grown overnight on RT-PCR product was cloned into the HindIII and BamHI sites slides, treated with 50 ng͞ml actinomycin D for2hat37°C, 5% of pBluescript vector (Stratagene). The construct was linearized 32 CO2, and analyzed by indirect immunofluorescence staining. with HindIII, and a P-labeled riboprobe was synthesized with a T7 Maxiscript kit (Ambion, Austin, TX). The labeled probe Preparation of GFP–Fusion Construct. The human treacle cDNA was was gel-purified and used for the RNase protection assay with an amplified by RT–PCR using BV1049 (5Ј-GGGGCGTGCA- RPA III kit (Ambion). GATCTCCGGCCGGCCGGGGGT-3Ј) and BV1050 (5Ј- A similar procedure was done to analyze the level of pre- GGTGCTGGTGGTACCGCTACAGTCTGCTCTGCTGT- rRNA in mouse embryos. BV1229 (5Ј-GGTACTGACAAGCT- CTTCTT-3Ј). The RT-PCR product was digested with BglII and TCCTTTCCCTATTAACACTAAAGGA-3Ј) and BV1230 (5Ј- KpnI and subcloned into the pEGFP-N1 vector (Clontech). The AGGCTGAAGGGATCCGAAATAAGGTGGCCCTCAAC- resulting clone was sequenced and used to transfect HeLa cells CACA-3Ј) were used to amplify nucleotides ϩ13 to ϩ320 of with Lipofectamine 2000 (Invitrogen) and analyzed 24–48 h mouse pre-rRNA. BV680 (5Ј-CATGCCATCAAGCTTCTGG- after transfection. ACCTGGCTGGC-3Ј) and BV1233 (5Ј-GGCTGGAAAG- GATCCTCAGGGCATCGGAACCGCTCG-3Ј) were used to Immunoprecipitation of Treacle Complex. The cDNA fragment that amplify nucleotides 558–778 of mouse ␤-actin. The amplified coded for amino acids 717-1488 of treacle was PCR-amplified products were cloned, and 32P-labeled probes were prepared as ͞ and subcloned into the BglII XhoI sites of pSG5-KF2M vector. described for the human pre-rRNA probe. HeLa cells were transfected for 48 h. Nuclei were isolated and resuspended in 10 mM Tris⅐HCl, pH 7.6͞1 mM EDTA͞400 mM 32P-Metabolic Labeling of Cells. HeLa cells were transfected with 40 NaCl͞10% glycerol͞0.5% Nonidet P-40͞5mMNaF͞1mM nM siRNA as described above. This transfection was repeated ͞ DTT 0.5 mM Na3VO4 with complete protease inhibitor mix after 24 h. Three days after the second transfection, cells were (Roche Applied Science). Nuclei were lysed by sonication and incubated in phosphate-free medium (Sigma) for 3.5 h. The centrifuged for 20 min at 18,000 ϫ g,4°C. Nuclear extract (1 mg) medium was replaced with fresh phosphate-free medium con- ␮ ͞ ϭ 32 was made to 0.5 ml with lysis buffer and mixed with 0.5 ml of taining 40 Ci ml (1 Ci 37 GBq) [ P] orthophosphate dilution buffer (10 mM Tris⅐HCl, pH 7.6͞1 mM EDTA͞20% (Amersham Pharmacia Biosciences), and 32P-labeled RNA was glycerol͞0.5% Nonidet P-40͞5mMNaF͞1mMDTT͞0.5 mM isolated and analyzed as described (28). ͞ ␮ ͞ Na3VO4 protease inhibitors). RNase A (0.2 g ul) and DNase I (0.5 units͞␮l) were added, kept on ice for 10–15 min, and then Bromouridine (BrUTP) Incorporation Assay. Transfected HeLa cells ͞ centrifuged at 10,000 ϫ g for 10 min. The supernatant was mixed were washed quickly with KHB solution (30 mM KCl 10 mM with anti-FLAG M2-agarose and tumbled overnight at 4°C. The Hepes, pH 7.4) at 37°C as described (29). The cells were then ␮ resin was washed in three consecutive steps: NET-gel buffer (50 overlaid with 150 l of KHB solution containing 10 mM BrUTP mM Tris⅐HCl, pH 7.5͞500 mM NaCl͞0.1% Nonidet P-40͞1mM and incubated at 37°C for 10 min. The BrUTP medium was EDTA͞0.25% gelatin), NET-gel buffer with 0.1% SDS, and a removed, and the cells were washed twice with growth medium ␮ final wash buffer (10 mM Tris⅐HCl, pH 7.6͞0.1% Nonidet P-40). and incubated in 300 l of growth medium at 37°C for 20 min. The slide was placed in 100% methanol at Ϫ20°C for 20 min and Yeast Two-Hybrid Analysis. The longest isoforms of treacle and transferred into acetone at room temperature for 30 sec. It was UBF cDNAs were subcloned into pGADT7 and pGBKT7 yeast washed with PBS for 10 min, dipped in water, and air-dried. expression vectors, respectively, and protein–protein interaction Antibody staining was done at 37°Cfor2hbyusing a mixture of anti-BrdUrd and anti-treacle antibodies (1:100 in PBS con- was determined in yeast by using the Matchmaker Two-Hybrid ͞ System (Becton Dickinson Biosciences). Clones were grown in a taining 0.05% Tween 20 and 0.05 units ml of RNase inhibitor). triple drop-out medium without tryptophan, leucine, and histi- Cells were washed three times with PBS for 20 min and incubated dine to screen for protein interaction. Further confirmation of with a mixture of rhodamine-conjugated anti-mouse IgG and the interaction was seen by the blue color of clones grown on FITC-conjugated anti-rabbit IgG at 37°C for 1 h followed by triple dropout medium containing 5-bromo-4-chloro-3-indolyl- washing. ␣ -D-galactopyranoside (Becton Dickinson Biosciences). Results Treatment with Short Interfering RNA (siRNA). An siRNA oligori- Treacle Colocalizes with UBF in Interphase and Mitotic Cells. We first bonucleotide, synthesized by Dharmacon, was used to down- hypothesized that treacle would colocalize with nucleolar pro- regulate the level of the TCOF1 mRNA. The sequence of teins known to be involved in pre-rRNA processing. HeLa cells siRNA1011 corresponds to the 5Ј end of exon 3 of TCOF1: in interphase and mitosis were doubly immunostained with antibodies against treacle and Gu␣, C23, or B23. Double staining 5ЈAACCUCAGAGCUUGGUCGGdTdT3Ј of interphase cells shows colocalization of treacle with Gu␣, C23, dTdTUUGGAGUCUCGAACCAGCC5Ј. and B23 (I in Fig. 1A). However, the magnification is not high enough to pinpoint their localization to different subnucleolar A control siRNA, si934Scr, was as described (28), and its compartments. More distinct differences in their localizations sequence corresponds to a scrambled sequence of the human are observed in mitotic cells (M in Fig. 1A). Whereas treacle ͞ ␣ nucleolar protein RNA helicase II Gu : localizes to punctate regions that overlap with the condensed 5ЈGUAACAAUGAGAGCACGGCdTdT3Ј , the other three nucleolar proteins disperse dTdTCAUUGUUACUCUCGUGCCG5Ј. throughout the dividing cells consistent with a previous report on the absence of association of pre-rRNA processing proteins with HeLa cells were transfected with siRNA oligoribonucleotides by the rDNA during mitosis (30); these processing proteins using Lipofectamine 2000. RT-PCR analysis of the total RNA form prenucleolar bodies during telophase or early G1 phase was done as described (9). (31), which subsequently fuse into nucleoli. This association of treacle with condensed chromosomes of RNase Protection Assay. BV1188 (5Ј-GTAGCTGACAAGCTT- mitotic cells suggests that it may colocalize with RNA pol I TCCTCTGGCGACCTGTCG-3Ј) and BV1189 (5Ј-GAGC- complex during mitosis. Previous reports have shown an asso- CGATCGGATCCGGCCACCCCCCACTCCGG-3Ј) were used ciation of UBF and other components of the RNA pol I complex to amplify by RT-PCR the first 360 nt of HeLa pre-rRNA. The with the condensed chromosomes of mitotic cells (27, 32, 33).

10710 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0402492101 Valdez et al. Downloaded by guest on October 2, 2021 Fig. 1. Cellular colocalization of treacle and UBF. (A) HeLa cells were indirectly stained with antibodies against treacle (Ab 014, red) and other antinucleolar proteins (green). Nuclear DNA was visualized by using Hoechst stain. Mitotic and interphase cells are marked M and I, respectively. (B) Treacle colocalizes with UBF at various stages of mitosis. HeLa cells were treated with 0.1 ␮g͞ml nocodazole for 4 h, incubated in fresh medium overnight, then analyzed by indirect immunofluorescence. (C) HeLa cells were blocked in mitosis with 0.1 ␮g͞ml colchicine for 6 h and analyzed by immunofluorescence labeling of chromosome spreads by using Hoechst stain (blue), anti-treacle (green), and anti-UBF (red) antibodies. (D) In a separate experiment HeLa cells were treated with 0.1 ␮g͞ml colchicine for 14 h, and mitotic cells were harvested by mechanical shock. Chromatin (Chr) and cytoplasmic (Cyt) extracts were prepared as described (27) and used for Western blot analysis. Cell extract (CE) refers to whole mitotic cells boiled in Laemmli buffer.

Double staining of HeLa cells in different stages of mitosis with structural change results in nucleolar proteins localizing to anti-treacle and anti-UBF antibodies shows colocalization of different subnuclear regions, including the , gran- treacle and UBF throughout mitosis (Fig. 1B). The colocaliza- ular region of the nucleolus, and nucleolar caps. To see where tion of treacle and UBF in specific spots within condensed treacle localizes in segregated nucleoli, HeLa cells were treated chromosomes of mitotic cells suggests an association of treacle with 50 ng͞ml actinomycin D for 2 h followed by indirect with the rDNA transcription machinery. Anti-treacle antibodies immunofluorescence analysis. Fig. 2A shows the localization of raised against three different regions of treacle all show the same treacle and RNA helicase II͞Gu␣ in the nucleoli of untreated result, suggesting that crossreaction of the anti-treacle antibody cells. Treatment with actinomycin D causes treacle to localize to with proteins other than treacle is probably not occurring (data nucleolar caps around the dense region of the nucleolus, whereas not shown). RNA helicase II͞Gu␣ translocates to the nucleoplasm. UBF, on the other hand, migrates to the same nucleolar caps that contain Treacle Colocalizes with UBF in Chromosome Spreads. Transcriptions treacle (Fig. 2B). Moreover, the C-terminal half of treacle of rDNA genes on chromosomes 13, 14, 15, 21, and 22 are active (amino acids 717-1488) is sufficient for its localization to these only during interphase. Although the production of rRNA is nucleolar caps (Fig. 2B). A higher magnification of these nu- suppressed during mitosis when the nucleoli are disassembled, cleolar caps, which are located at the periphery of a denser the rDNA transcription machinery remains associated with nucleolar region, is shown in Fig. 2C. The cosegregation of rDNA genes or nucleolar organizer regions (NORs). Chromo- treacle and UBF to the same nucleolar regions in cells treated some spread labeling using antibodies against anti-UBF and with actinomycin D suggests that they are components of the anti-RNA pol I antibodies has shown the association of the RNA same nucleolar complex. pol I machinery with NOR-bearing chromosomes (27). Immu- nofluorescence analysis of HeLa chromosome spreads using anti-treacle and anti-UBF antibodies shows the localization of the two antigens in the same chromosomal spots (Fig. 1C, MEDICAL SCIENCES superimposed) providing additional evidence that treacle is a part of the rDNA gene transcription machinery. These spots, which correspond to active NORs, differ in intensities within the same cell. Interestingly, the intensities of treacle and UBF vary in the same proportions, suggesting stoichiometric association of treacle and UBF with specific NORs. The same results were reported for UBF and RNA pol I (27).

Treacle Associates with Chromatin of Mitotic Cells. Sirri et al. (34) showed the association of UBF and TTF-1 with chromatin of mitotic cells by protein fractionation and Western blot analysis. A similar procedure was used to determine whether treacle associates with the condensed chromosomes. Immunoblot anal- yses of chromatin, cytoplasmic, and whole-cell extracts using anti-treacle, anti-UBF, and anti-Gu␣ antibodies show associa- tion of treacle and UBF, but not nucleolar RNA helicase Fig. 2. Effects of actinomycin D on the localization of nucleolar proteins. II͞Gu␣, with chromatin (Fig. 1D), consistent with the colocal- HeLa cells were treated with 50 ng͞ml actinomycin D for 2 h and stained by ization of treacle and UBF with the condensed chromosomes of indirect immunofluorescence. Nuclei with dark-phase nucleoli are shown. (A) Treacle (red) localization in untreated and treated cells is compared with RNA immunostained mitotic cells (Fig. 1 B and C). helicase II͞Gu␣ (green). (B) The localizations of full-length treacle and its C terminus both fused to GFP (green) are compared with endogenous UBF (red) Treacle and UBF Cosegregate in Cells Treated with Actinomycin D. in actinomycin D-treated cells. (C) Enlarged view of a superimposed phase and Actinomycin D inhibits rRNA production and causes segregation GFP fluorescence of actinomycin D-treated HeLa cell transfected with full- of the fibrillar and granular regions of the nucleolus. This length treacle–GFP construct.

Valdez et al. PNAS ͉ July 20, 2004 ͉ vol. 101 ͉ no. 29 ͉ 10711 Downloaded by guest on October 2, 2021 Fig. 3. Direct interaction between treacle and UBF. (A) HeLa cells were transfected with double-FLAG-tagged treacle (amino acids 717-1488) expres- sion construct and analyzed by immunofluorescence staining using anti-FLAG and anti-Gu␣ antibodies. Similar transfected cells were used for immunopre- cipitation analysis by tumbling nuclear extract with anti-FLAG antibody- agarose. Protein samples were analyzed by Western blot enhanced chemilu- minescence using anti-UBF antibody. The membrane was stripped and analyzed by using anti-Gu␣ antibody. (B) Analysis of the treacle–UBF interac- tion by the yeast two-hybrid system in a double drop-out medium (ϪTrp, ϪLeu) that permits growth of cells transfected with pGBKT7 and pGADT7 constructs, and in a triple drop-out medium (ϪTrp, ϪLeu, ϪHis) that screens for protein–protein interaction. The interaction of p53 and simian virus 40 large T antigen was used as a positive control.

Treacle Physically Interacts with UBF. The colocalization of treacle and UBF in all stages of mitosis, interphase cells, and actino- mycin D-segregated nucleoli suggests a physical interaction between the two nucleolar proteins. To test this hypothesis, the C-terminal half of treacle (amino acids 717-1488) was tagged with two copies in tandem of the FLAG epitope. The fusion protein localizes to the nucleoli (Fig. 3A). Immunoprecipitation using anti-FLAG resin in the presence of RNase A and DNase I shows that treacle can pull down UBF from the nuclear extract of FLAG-treacle-transfected cells (Fig. 3A). The slower migra- Fig. 4. Effects of siRNA-mediated down-regulation of treacle on rRNA tion of immunoprecipitated UBF on a polyacrylamide gel could production. (A) HeLa cells were treated with 40 nM siRNA or mock- be caused by differences in salt concentrations. Mixing the transfected. si1011 targets the TCOF1 mRNA and si934Scr is a control siRNA. nuclear extract and immunoprecipitate abolished this difference After 48 h of transfection, total RNA was isolated and analyzed by RT-PCR in the rate of migration (data not shown). The presence of two using TCOF1-specific primers and U1C primers as an internal control. In a UBF bands is consistent with its two isoforms (35, 36). separate 4-day transfection, cells were boiled in Laemmli buffer and analyzed by Western blot enhanced chemiluminescence using anti-treacle antibody. To further prove a physical interaction between treacle and ␣ UBF, the yeast two-hybrid system was used. The growth of yeast The blot was stripped and reprobed with anti-Gu antibody. Treacle comi- grates at Ϸ220 kDa near the top of the gel, whereas RNA helicase II͞Gu␣ cells, which harbor both treacle and UBF expression constructs, comigrates near the bottom of the gel at Ϸ90 kDa. (B) An RNase protection in a triple drop-out medium that lacks tryptophan, leucine, and assay shows a 47% decrease in the level of pre-rRNA in cells treated with si1011 histidine, indicates interaction of the two proteins (Fig. 3B). The compared to mock-transfected cells. Lane 1 contains 1͞10 of the riboprobe specificity of treacle–UBF interaction is shown by the inability of used in the other lanes. Lanes 2–5 contain 8 ␮g yeast RNA or total RNA isolated the yeast clones that harbor treacle and RNA helicase II͞Gu␣ from cells mock-treated or treated with the indicated siRNA. (C) Total RNAs expression constructs, or UBF and RNA helicase II͞Gu␣ ex- (1.0 ␮g) from 32P-metabolically labeled transfected cells were analyzed by gel pression constructs, to grow in a triple drop-out medium (Fig. electrophoresis. The chase times with nonradioactive phosphate are labeled 0, 0.5, and 1 h. The numbers below correspond to the amount of specific RNA 3B). Moreover, yeast cells that express treacle and UBF form band relative to mock-transfected samples (set at 100) obtained with IMAGE- blue colonies in triple drop-out medium that contains 5-bromo- ␣ QUANT software. (D) The same blot was stained with 0.2% methylene blue to 4-chloro-3-indolyl- -D-galactopyranoside, indicating protein– visualize total RNA. (E) BrUTP incorporation assay. HeLa cells grown on a protein interaction (data not shown). chamber slide were transfected twice with 40 nM si1011 (24 h apart) and analyzed for BrUTP incorporation 72 h after the last transfection. Mock- and siRNA-Mediated Down-Regulation of Treacle Expression Inhibits rRNA si1011-transfected cells were indirectly stained with anti-treacle (green) Production. An siRNA that targets the 5Ј end of exon 3 mRNA and anti-BrdUrd (red) antibodies. All pictures were taken at a preset bright- was used to decrease the level of treacle mRNA. Treatment of ness, contrast, and gamma level of 50, 50, and 1.0, respectively, and image scale range of 100–3,000. (F) Fluorescence intensity profiles of the images HeLa cells with 40 nM si1011 resulted in a significant reduction shown in E. of the TCOF1 mRNA and treacle protein, whereas the unrelated siRNA (si934Scr) had an insignificant effect (Fig. 4A). An RNase protection assay was done to determine the effects an insignificant effect (Fig. 4B), suggesting that down-regulation of siRNA-mediated down-regulation of treacle on rDNA gene of treacle expression inhibited rDNA transcription. transcription. A 32P-labeled antisense riboprobe that binds to the To further determine the effects of treacle down-regulation on 5Ј end of pre-rRNA was hybridized with 8 ␮g of total RNA from pre-rRNA production and processing, newly synthesized pre- siRNA-treated HeLa cells. Relative to mock-transfected cells, rRNA was labeled in vivo with 32P and chased with unlabeled the amount of protected pre-rRNA fragments decreased by 47% inorganic phosphate for 0, 0.5, and 1 h. Metabolic labeling (Fig. in cells treated with si1011, whereas an unrelated si934Scr had 4C, lane 4) shows a 48% decrease in the level of 47S pre-rRNA,

10712 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0402492101 Valdez et al. Downloaded by guest on October 2, 2021 possible biochemical explanation for the craniofacial defects in the Tcof1ϩ/Ϫ mice. Discussion Transcription of the Ͼ100 tandemly repeated rDNA genes in a mammalian cell is catalyzed by the RNA pol I machinery. The basal components of this machinery are known (38–41), and their activities are regulated by reversible acetylation and phos- phorylation (42–47). The effects of interaction of the RNA pol Fig. 5. Analysis of RNA from mouse embryos. RT-PCR was used to determine I complex with other nucleolar proteins are regulated transcrip- the levels of expression of Tcof1 in embryonic day 10.5 heterozygous (ϩ͞Ϫ) ϩ͞ϩ tion, elongation, and termination of pre-rRNA production (47). embryo relative to its WT ( ) littermate. (Left) Tcof1 signals were normal- The present study demonstrates the importance of the nucleolar ized by using U1C as an internal control. (Right) The level of pre-rRNA was determined by RNase protection assay using a 5Ј pre-rRNA probe and ␤-actin protein treacle to rDNA gene transcription. Several lines of as an internal control. Similar results were obtained with another set of evidence suggest that this function of treacle could be mediated embryos. by interaction with UBF. First, the colocalization of treacle and UBF in all stages of mitosis as shown by indirect immunofluorescence suggests that a transcript that is immediately processed to 45S, at0hofchase they are components of a ribonucleoprotein complex that asso- relative to mock-transfected cells and consistent with the results ciates with the condensed chromosomes (Fig. 1). The same of the RNase protection assay (Fig. 4B). The levels of 32S results were obtained when chromosome spreads were analyzed pre-rRNA, 28S, and 18S rRNA at the 0-h chase sample de- by immunofluorescence labeling (Fig. 1C). Indeed, isolation and creased at a similar proportion (Fig. 4C, lane 4), suggesting that analysis of the chromatin-associated proteins in mitotic cells down-regulation of treacle has more effect on rDNA gene show the presence of treacle and UBF in the same extract (Fig. transcription rather than on processing of the pre-rRNA tran- 1D). UBF binds to DNA, but it is not known whether treacle has script. An unrelated siRNA (si934Scr) did not affect the pro- a DNA-binding activity. Second, treatment of HeLa cells with duction of 47S͞45S pre-rRNA or its processing pattern (Fig. 4C, actinomycin D, a known inhibitor of RNA pol I that causes lanes 7–9). Fig. 4D shows the same blot stained with methylene nucleolar components to segregate, results in colocalization of blue to show equal loading of RNA. treacle and UBF to nucleolar caps around the periphery of a denser region of the nucleolus (Fig. 2). Third, an immunopre- The results of this 32P-metabolic labeling are supported by the cipitation assay pulled down treacle and UBF from nuclear BrUTP incorporation assay. HeLa cells treated with siRNA extracts treated with RNase A and DNase I, suggesting direct were allowed to incorporate BrUTP into newly synthesized physical interaction and negating the possibility of an RNA- or RNA. The cells were then analyzed by double staining with Ϸ DNA-mediated linkage (Fig. 3A). It remains to be determined anti-treacle and anti-BrdUrd antibodies. Because 70% or more whether phosphorylation or acetylation of UBF mediates its of the total RNA in a cell corresponds to rRNA (37), a greater interaction with treacle; both posttranslational modifications degree of BrUTP incorporation would reflect increased pre- activate UBF as far as RNA pol I activity is concerned (45, 48, rRNA and its processed products. Fig. 4E shows the si1011- 49). Lastly, the results of the yeast two-hybrid analysis further mediated decrease in the level of treacle (green); the level of support the interaction of treacle and UBF (Fig. 3B). BrUTP incorporation (red) proportionally decreased, suggest- UBF is known to interact with other nucleolar proteins. RNA ing that down-regulation of treacle resulted in a decrease in pol I transcription is initiated by an interaction between UBF rRNA production. and promoter selectivity factor SL1. Specifically, UBF interacts This decrease in rDNA transcription when treacle was down- with TATA binding protein (48) and TAF1 (50), which are regulated led us to examine the effects of overexpression of components of the SL1 complex, and PAF53 (51). UBF also

full-length and deletion mutant forms of treacle. There was no interacts with pRb, which inhibits RNA pol I activity (52). Our MEDICAL SCIENCES significant change in the level of 47S pre-rRNA (Fig. 6, which is data suggest that treacle is another activator of UBF because published as supporting information on the PNAS web site). down-regulation of treacle inhibits rDNA gene transcription. Expression of truncated forms of treacle (amino acids 1–1130 The interactions of UBF with a number of proteins including and 1–1245), similar to those expected in TCS patients, did not treacle demonstrate the complexity of regulation of the mam- result in inhibition of 47S production, suggesting absence of malian rDNA gene transcription. Three different types of experiments including RNase pro- dominant negative effect. Overall, our results are consistent with 32 a model that treacle haploinsufficiency decreases rRNA pro- tection assay, P-metabolic labeling, and BrUTP incorporation duction and, perhaps, contributes to the pathogenesis of TCS. assay prove the relevance of treacle–UBF interaction in rDNA gene transcription. When using any one of the three methods, -؉ ؊ siRNA-mediated down-regulation of expression of treacle re Decreased Pre-rRNA Production in Tcof1 / Mouse Embryos. Next, we sulted in an almost 50% inhibition of rDNA gene transcription determined whether or not down-regulation of Tcof1 expression (Fig. 4). The exact effect of this interaction of treacle with UBF decreases rDNA transcription in heterozygous mouse embryos. as it relates to rDNA gene transcription remains to be elucidated. Recent reports show that heterozygotes derived from a mixed ͞ Treacle might also play a role in of rRNA. Treacle 129-C57BL 6 background exhibit a severe TCS phenotype, interacts with pNop56 (19), a component of the ribonucleopro- whereas heterozygotes from a mixed 129-DBA background show tein complex that methylates pre-rRNA at the 2Ј-O-ribose a minimal phenotype (18). We genotyped embryos from moiety (53). Although the external and internal transcribed ϫ ͞ DBA C57BL 6 cross and determined the level of Tcof1 ex- sequences of pre-rRNA are not methylated (54), it is known that pression by RT-PCR. Fig. 5 shows 51% reduction in the level of methylation immediately occurs as pre-rRNA is transcribed or Tcof1 in heterozygous embryo relative to its WT littermate; just before its processing (55). These earlier observations and analysis of the same RNA samples showed a comparable reduc- our present data suggest that treacle might link transcription of tion in the level of pre-rRNA in the heterozygote embryo. rDNA genes to pre-rRNA modifications and processing remi- Overall, these results are consistent with the siRNA-mediated niscent of the linkage between RNA pol II-mediated transcrip- inhibition of pre-rRNA production we observed and provide a tions and splicing of pre-mRNA (56). As others have reported,

Valdez et al. PNAS ͉ July 20, 2004 ͉ vol. 101 ͉ no. 29 ͉ 10713 Downloaded by guest on October 2, 2021 this link between rDNA transcription and pre-rRNA processing also encodes a nucleolar phosphoprotein. of this gene is most likely controlled by cyclin-dependent kinases (57). resulted in smaller flies because of developmental delay (58). The observed inhibition of rRNA production in cells having The mfl gene is homologous to the human DKC1 gene, which is down-regulated treacle expression was accompanied by a de- mutated in patients with dyskeratosis congenita characterized by creased cell growth (data not shown), probably caused by a premature aging and increased susceptibility to cancer (59). ϩ Ϫ slowing of cellular metabolisms. This could explain why Tcof1 / Dyskerin, the DKC1-encoded protein, is involved in rRNA mouse embryos are smaller and developmentally delayed when pseudouridylation and its mutation causes reduced rRNA pro- compared with their WT littermates (6). duction (60, 61). The data from the present study suggest that haploinsuffi- In conclusion, identification of the functions of dyskerin and ciency of treacle in TCS patients might cause insufficient rRNA treacle clearly demonstrates the relevance of rRNA biogenesis in production in the prefusion neural folds, resulting in abnormal organism development and human diseases. Specifically, our craniofacial development. The cephalic cells prob- discovery of the function of treacle in rDNA gene transcription ably require a higher threshold concentration of rRNA for their should lead to a better understanding of the biochemical patho- survival and proper differentiation during early embryogenesis. genesis of TCS. Interestingly, no patient has been identified with both copies of the TCOF1 gene mutated, suggesting embryonic lethality and This work was supported by Public Health Service Grant DK52341 from underscoring the importance of treacle in development. the National Institute of Diabetes and Digestive and Kidney Diseases, Our study is not the first report on the relationship of rRNA National Institutes of Health (to B.C.V.) and Medical Research Council production and development. The Drosophila minifly (mfl) gene Grant G81͞535 (to M.J.D.).

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10714 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0402492101 Valdez et al. Downloaded by guest on October 2, 2021