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Human Molecular Genetics Advance Access originally published online on June 1, 2005
Human Molecular Genetics 2005 14(14):2035-2043; doi:10.1093/hmg/ddi208
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© The Author 2005. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oupjournals.org

The Treacher Collins syndrome (TCOF1) gene product is involved in pre-rRNA methylation

Bianca Gonzales1, Dale Henning1, Rolando B. So1, Jill Dixon2, Michael J. Dixon2,3 and Benigno C. Valdez1,*

1Department of Pharmacology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA, 2Dental School and 3Faculty of Life Sciences, Michael Smith Building, University of Manchester, Oxford Road, Manchester M13 9PT, UK

* To whom correspondence should be addressed. Tel: +1 7137987908; Fax: +1 7137983145; Email: bvaldez{at}bcm.tmc.edu

Received April 20, 2005; Revised May 19, 2005; Accepted May 26, 2005


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Treacher Collins syndrome (TCS) is characterized by defects in craniofacial development, which results from mutations in the TCOF1 gene. TCOF1 encodes the nucleolar phosphoprotein treacle, which interacts with upstream binding factor (UBF) and affects transcription of the ribosomal DNA gene. The present study shows participation of treacle in the 2'-O-methylation of pre-rRNA. Antisense-mediated down-regulation of treacle expression in Xenopus laevis oocytes reduced 2'-O-methylation of pre-rRNA. Analysis of RNA isolated from wild-type and Tcof1+/– heterozygous mice embryos from strains that exhibit a lethal phenotype showed significant reduction in 2'-O-methylation at nucleotide C463 of 18S rRNA. The level of pseudouridylation of U1642 of 18S rRNA from the same RNA samples was not affected suggesting specificity. There is no significant difference in rRNA methylation between wild-type and heterozygous embryos of DBAxBALB/c mice, which have no obvious craniofacial phenotype. The function of treacle in pre-rRNA methylation is most likely mediated by its direct physical interaction with NOP56, a component of the ribonucleoprotein methylation complex. Although treacle co-localizes with UBF throughout mitosis, it co-localizes with NOP56 and fibrillarin, a putative methyl transferase, only during telophase when rDNA gene transcription and pre-rRNA methylation are known to commence. These observations suggest that treacle might link RNA polymerase I-catalyzed transcription and post-transcriptional modification of pre-rRNA. We hypothesize that haploinsufficiency of treacle in TCS patients results in inhibition of production of properly modified mature rRNA in addition to inhibition of rDNA gene transcription, which consequently affects proliferation and proper differentiation of specific embryonic cells during development.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Loss-of-function mutations in the TCOF1 gene have been demonstrated to underlie Treacher Collins syndrome (TCS), an autosomal dominant disorder of craniofacial development (1Go–7Go). The TCOF1 gene encodes treacle, a nucleolar phosphoprotein (8Go). Treacle displays homology to Nopp140, a trafficking nucleolar phosphoprotein (9Go), which interacts with box C/D snoRNP complexes and RNA polymerase I (10Go,11Go). Knock-out of the murine ortholog of TCOF1 produces embryos that die immediately postnatally as a result of severe craniofacial anomalies (12Go). However, the penetrance and severity of the craniofacial phenotypes observed in Tcof1+/– mice depend on the genetic background on which the mutation is placed (13Go) which might explain the lack of genotype–phenotype correlation observed in TCS patients.

Haploinsufficiency, rather than a dominant-negative effect, has been suggested to be the mechanism underlying the pathogenesis of TCS (14Go,15Go). Expression of treacle below its optimum level might result in inhibition of proper proliferation and/or differentiation of specific neural crest cells (16Go), which are precursors to the facial primordia and branchial arches in the development of the vertebrate head. In fact, treacle is highly expressed in the first and second branchial arches during early embryogenesis; these are branchial arches that give rise to structures affected during early development of TCS patients (17Go). Identification of the cellular functions of treacle will lead to a more detailed understanding of the molecular pathogenesis of TCS.

The localization of treacle to the nucleolus suggests a possible function in rRNA biogenesis. A recent report from our laboratories demonstrated a function of treacle in rDNA gene transcription through interaction with the upstream binding factor (UBF), a transcription factor for RNA polymerase I (18Go). Another group reported an association of treacle with the ribonucleoprotein complex responsible for the 2'-O-methylation of pre-rRNA, suggesting an additional function for treacle in the rRNA biogenesis pathway (19Go). Here, we present evidence that down-regulation of expression of treacle in frog oocytes and mouse embryos results in decreased methylation of 18S pre-rRNA and show that this function of treacle is probably mediated by its interaction with NOP56 protein, a component of the pre-rRNA methylation complex.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Down-regulation of Xenopus Treacle inhibits pre-rRNA methylation in frog oocytes
We cloned the cDNA that encodes the Xenopus laevis ortholog of TCOF1 (GenBank accession no. AY731504) and designed antisense oligodeoxynucleotides, which decreased the Xenopus Tcof1 expression to various levels when microinjected individually into Xenopus oocytes (Fig. 1A). The most effective oligodeoxynucleotide, BV1222, was used to study the function of Xenopus treacle (xtreacle) in pre-rRNA methylation because it most effectively caused a reduction in treacle.



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  		Figure 1. Down-regulation of treacle in Xenopus oocytes inhibits rRNA methylation. (A) Three antisense oligodeoxynucleotides were microinjected individually into oocytes and assayed for their efficacy to decrease the level of xTcof1 mRNA using RT–PCR. Expression of Xenopus B23 (xB23) was used as an internal control. (B–D) Site-specific RNase H cleavage directed by 2'-O-methyl RNA–DNA chimeric oligonucleotide. The top strand in (B) is an 18S rRNA sequence hybridized to a 2'-O-methyl RNA–DNA chimera (bottom strand). In the chimera, deoxynucleotides are indicated as bold letters and 2'-O-methyl ribonucleotides as plain letters. Methylation of nucleotide C427 is expected to inhibit RNase H cleavage (B) and absence of methylation results in RNase H cleavage (C). The bold arrow indicates the cleavage site. Total 32P-labeled RNAs isolated from microinjected oocytes were hybridized with the RNA–DNA chimera and treated with RNase H. Equal counts (c.p.m.) were loaded in each lane. The positions of 40S pre-rRNA and its RNase H cleavage products are indicated (D).

 
Joan Steitz's laboratory developed a sensitive method to determine methylations of pre-rRNA in X. laevis oocytes (20Go). The same procedure was used previously in the analysis of U14 snoRNA function in 2'-O-methylation of the pre-18S rRNA in Xenopus oocytes (21Go). The method utilizes RNase H cleavage directed by a 2'-O-methyl RNA–DNA chimeric oligonucleotide; the methylated target RNA (e.g. 40S precursor) that is hybridized with the chimeric oligonucleotide will not be cleaved by RNase H (Fig. 1B). Removal of the methyl group causes the target RNA to become more sensitive to RNase H cleavage (Fig. 1C). We used this method to determine the effect of xtreacle down-regulation on the 2'-O-methylation of nucleotide C427 of pre-18S rRNA from Xenopus oocytes. The chimeric oligonucleotide was annealed with 32P-metabolically labeled RNA from control and BV1222-treated Xenopus oocytes and digested with RNase H. Figure 1D shows that the control antisense oligonucleotide did not affect the methylation of nucleotide C427 as shown by the absence of RNase H cleavage products (lanes 1 and 2). The xTcof1 antisense oligonucleotide (BV1222) decreased the methylation of nucleotide C427 as indicated by the decrease in 40S pre-rRNA and the appearance of RNase H digestion products (lanes 3–5).

Inhibition of pre-rRNA methylation in Tcof1+/– heterozygous mice embryos
The generation and characterization of Tcof1+/– heterozygous mice embryos exhibiting craniofacial phenotypes were previously described (12Go). The penetrance and severity of these phenotypes in Tcof1 heterozygous mice depends on their genetic background. For example, DBAxBALB/c strain does not show craniofacial defects, whereas DBAxCBA and DBAxC57BL/6 strains exhibit a lethal phenotype (13Go). Mice embryos from these strains were analyzed for the level of pre-rRNA methylation using a modified method from Joan Steitz's laboratory (20Go) (see Materials and Methods). Figure 2A shows the assay for the level of methylation of mouse rRNA, which includes RT–PCR analysis of the RNase H-digests. However, this method does not discriminate between precursors and mature rRNAs. As both cytoplasmic and nuclear rRNAs were isolated, the samples contained a high level of mature rRNAs. RT–PCR amplification of nucleotides 213–575 of 18S rRNA, which included the chimeric oligonucleotide binding and RNase H cleavage sites, from wild-type and heterozygous DBAxBALB/c mice did not show any difference, suggesting similar levels of methylation of nucleotide C463 of 18S rRNA (Fig. 2B). The same analysis of 18S rRNA from DBAxCBA embryos, which exhibit a lethal phenotype (13Go), shows decreased levels of 18S rRNA methylation in the Tcof1 heterozygotes (compare lanes 5 and 7 in Fig. 2B). We pooled our data for strains DBAxCBA (three pairs of wild-type and heterozygotes) and DBAxC57BL6 (two pairs of wild-type and heterozygotes), and a quantitative analysis shows a statistically significant difference (P<0.01) in the level of methylation of C463 of 18S rRNA between wild-type and heterozygous mice (Fig. 2C). The results suggest that the decrease in the methylation of pre-rRNA might be a contributing factor to the observed differences in phenotypic penetrance.



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  		Figure 2. Assay for methylation of 18S rRNA from wild-type and Tcof1+/– heterozygous mice embryos. (A) Diagram showing an additional step to the method described in Fig. 1. RT–PCR was done using primers Pr1 and Pr2 to amplify the region containing the RNase H cleavage site and primers Pr3 and Pr4 to amplify a different region in 18S rRNA as an internal control. (B) Total RNA (1.0 µg) was mixed with 15 ng RNA–DNA chimeric oligonucleotide in 9 µl buffer, boiled for 3 min, incubated at 37°C for 10 min and mixed with RNase H as described under Materials and Methods. RT–PCR was performed and the reaction products were resolved on a 6% polyacrylamide-SDS gel and analyzed by phosphorous imaging. For quantitative analysis, the upper signals corresponding to nucleotides 213–575 were normalized relative to the internal control signals (nucleotides 1421–1688 of 18S rRNA). The obtained normalized values in the presence and absence of chimeric oligonucleotide for the same RNA samples were compared (e.g. lane 1 versus lane 2). The resulting numbers for the wild-type (+/+) and heterozygote (+/–) mice embryos were then compared (C). Data from DBAxCBA and DBAxC57BL/6, which showed similar phenotypes, were combined and analyzed as one group as shown in (C). Statistical analysis using Student's t-test shows a significant difference (P<0.01) in the level of methylation of rRNA in the wild-type (+/+) and heterozygous (+/–) DBAxCBA and DBAxC57BL/6 strains (indicated by asterisks).

 
Treacle is not involved in pseudouridylation of 18S rRNA
To determine whether down-regulation of treacle in mouse affects pseudouridylation of 18S rRNA, we compared the levels of pseudouridylation of U1642 of 18S using 1-cyclohexyl-3-(2-morpholinoethyl) carbodiimide metho-p-toluenesulfonate (CMCT) adduct formation, which blocks reverse transcription from a specific primer (22Go). An intense band appears, the length of which ends one base downstream of the pseudouridyaltion site ({psi}). Figure 3 shows an intense band in samples treated with CMCT but not in untreated RNA samples. This band corresponds to U1641, which is one base downstream from pseudouridylated U1642. Normalization of this band relative to the two dark bands near the top of each lane did not show differences in the pseudouridylation of U1642 of 18S rRNA in wild-type and heterozygous DBAxCBA mice embryos.



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  		Figure 3. Comparison of the levels of pseudouridylation of nucleotide U1642 of 18S rRNA in wild-type (Tcof1+/+) and heterozygous (Tcof1+/–) mice embryos. RNA samples were incubated at 37°C in the absence (–CMCT) or presence (+CMCT) of CMCT as described earlier (22Go). Reverse transcription was done using BV1253 oligodeoxynucleotide as primer. The dotted line shows the product of reverse transcription that ended one base downstream of the pseudouridylation ({psi}) site.

 
Direct physical interaction between treacle and NOP56
Hayano et al. (19Go) previously reported that treacle and NOP56 are components of a common ribonucleoprotein complex involved in pre-rRNA methylation and suggested physical interaction of the two proteins. We initially screened a human cDNA library in a yeast expression vector for proteins that interacted with human treacle. One of the positive clones encoded the C-terminal (amino acids 367–594) of NOP56. Additional deletion analyses show that amino acids 742–1488 of human treacle interact with full-length NOP56 (Fig. 4A). Interestingly, a similar region of treacle interacts with UBF (18Go). The direct physical interaction of treacle and NOP56 is supported by the results of the co-immunoprecipitation experiment (Fig. 4B).



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  		Figure 4. Direct physical interaction of treacle and NOP56. (A) The COOH-end of treacle interacts with NOP56. Full-length and three deletion mutants of human treacle were analyzed for their interaction with full-length NOP56 using the yeast two-hybrid system. Growth in the absence of Trp and Leu suggests the presence of treacle and NOP56 expression constructs. Growth in the absence of Trp, Leu and His indicates the interaction between the two proteins. Absence of growth in the 1–285 and 1–963 treacle mutants suggests lack of interaction of these domains with NOP56. (B) Immunoprecipitation of treacle–NOP56 complex. HeLa cells were transfected with constructs that expressed double-FLAG-tagged (1Go) treacle amino acids 933–1488 or (2Go) treacle amino acids 1228–1488. Nuclear extracts were mixed with anti-FLAG monoclonal antibody resin and the precipitates were analyzed with anti-FLAG, anti-NOP56, anti-Fib (fibrillarin) and anti-Gu{alpha} antibodies. Both anti-FLAG and anti-Fib are monoclonal antibodies, which recognize mouse IgG. NOP56 and fibrillarin, but not Gu{alpha}, precipitated with FLAG-treacle. Numbers on the left refer to molecular weight markers in kilodaltons.

 
Methylations of human pre-rRNA at specific ribose moieties are catalyzed by a ribonucleoprotein complex containing proteins and guide snoRNAs with the box C/D sequence motifs. The core proteins include NOP56, NOP58, a 15.5 kDa protein and fibrillarin, which is believed to contain the methyltransferase activity (23Go,24Go). To determine whether other components of this methylation complex interact with human treacle, the full-length cDNA that encodes fibrillarin was subcloned into pGBKT7 vector and assayed by the yeast two-hybrid system. No interaction between human treacle and fibrillarin was observed (data not shown). However, the immunoprecipitation experiment shows presence of fibrillarin in a complex brought down by FLAG-treacle (Fig. 4B), suggesting that treacle interacts with the methylation complex through NOP56.

Co-localization of treacle with components of 2'-O-methylation complex
To prove the association of treacle with the complex that 2'-O-methylates pre-rRNA, its co-localization with NOP56 and fibrillarin was determined during mitosis. As rRNA production is suppressed during early mitosis and commences during telophase, we hypothesized that treacle begins to associate with the methylation complex at the onset of rDNA gene transcription and pre-rRNA modification. Indeed, Figure 5A shows the co-localization of treacle and NOP56 in the condensed chromosomes during late telophase but not during anaphase. Although UBF and treacle co-localize to punctate structures throughout mitosis (18Go), NOP56 seems to co-localize with treacle in the condensed chromosomes only during the latter stage of mitosis when synthesis of pre-rRNA is known to begin. It is possible that treacle recruits the NOP56 complex as soon as pre-rRNA synthesis commences. This view is supported by a similar co-localization of treacle with fibrillarin in the condensed chromosomes only during telophase (Fig. 5B). Overall, the direct physical interaction of treacle with NOP56 and its association with the 2'-O-methyl transferase complex during the onset of pre-rRNA production support a function for treacle in the methylation of pre-rRNA.



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  		Figure 5. Co-localization of treacle and known components of the pre-rRNA methylation complex during late mitosis. (A) HeLa cells were transiently transfected with a construct expressing treacle GFP fusion protein (green) and analyzed by indirect immunofluorescence using affinity purified anti-NOP56 antibody (red). Nuclear DNA was visualized with Hoechst stain (blue). (B) Non-transfected HeLa cells were stained with anti-treacle (green) and anti-fibrillarin (red) antibodies. The field shows cells in different stages of cell division including interphase (I), anaphase (A) and telophase (T).

 
Binding of treacle to specific regions of the rDNA gene
Examination of the cDNA-derived amino acid sequence of the human treacle using the website http://motif.genome.ad.jp shows 13 HMG-1 and HMG-Y DNA binding domains (A+T hook), which are distributed throughout the molecule. To determine whether treacle directly binds to rDNA sequences in vivo, a chromatin immunoprecipitation (ChIP) assay was done. Figure 6 shows binding of treacle within nucleotides –240 to +370 of the rDNA gene. No immunoprecipitation of nucleotide –510 to –210 of the rDNA promoter and nucleotides 4377–5026 of the 28S gene suggested specificity of our ChIP assay. The results suggest that treacle binds to the proximal region of the promoter and the 5' end region of ETS1 of the rDNA gene. This DNA binding activity of treacle might be important in the regulation of RNA polymerase I activity and the methylation of the pre-rRNA transcript.



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  		Figure 6. ChIP assay. Fragmented genomic DNA (gDNA) from non-synchronized HeLa cells was immunoprecipitated using anti-treacle antibody or control immunoglobulins (IgG). The precipitated DNA was amplified by PCR using the indicated primer sets (below) as described under Materials and Methods. The asterisks indicate binding of treacle to –240 to +370 nt of the rDNA gene. The arrow at +1 position shows start of RNA pol I transcription. ETS1, external transcribed sequence 1.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
We recently reported that treacle stimulates rDNA gene transcription by interacting with transcription factor UBF. Down-regulation of treacle expression using RNA interference in HeLa cells or elimination of one Tcof1 allele in mouse both resulted in a decreased level of rDNA gene transcription (18Go). An additional function for treacle was suggested by another previous report on the association of treacle with the nucleolar ribonucleoprotein complex that methylates pre-rRNA (19Go). We investigated this possibility by examining the status of methylation of rRNA from Xenopus oocytes with down-regulated treacle and from wild-type and Tcof1+/– heterozygous mice littermates, and then studied a possible molecular mechanism of treacle function in pre-rRNA methylation. In both systems, low levels of Tcof1 expression result in a lower level of methylation of nucleotide C427 of 18S in Xenopus oocytes and its homologous nucleotide C463 of 18S rRNA in mice. It is interesting to note that pseudouridylation of U1642 of 18S rRNA in mice is not affected by down-regulation of treacle. It remains to be determined whether other methylation and pseudouridylation sites in 5.8S, 18S and 28S rRNAs are inhibited by down-regulation of treacle expression. Because there is only one model for 2'-O-ribose methylation machinery known to date and guided by different small nucleolar RNAs, it is possible that treacle is a part of the machinery that methylates >105 nt in mammalian rRNAs. Haploinsufficiency of treacle might lower the activity of the methylation complex, and together with decreased rDNA gene transcription (18Go) results in lower production of mature rRNAs. When compared with mice strains, which do not show craniofacial phenotypes, the Tcof1 heterozygotes of strains that exhibit a lethal phenotype have a significantly lower level of rRNA methylation (Fig. 2). The 15% difference obtained in the level of rRNA methylation between wild-type and heterozygous sensitive strains might be less than what we would expect. This might be due to a more rapid turnover of undermethylated rRNAs and/or inhibition of pre-rRNA processing (25Go–28Go).

We next determined a possible mechanism of this newly identified function of treacle. To prove unequivocally that this function of treacle in the methylation of pre-rRNA is mediated by a previously reported interaction of treacle with NOP56 (19Go), direct physical interaction of the two proteins was examined by the yeast two-hybrid analysis and by the co-immunoprecipitation assays. Results from both types of experiments support a direct physical interaction between treacle and NOP56 (Fig. 4). These results are further supported by co-localization of treacle with NOP56 during the late stage of mitosis when rDNA gene transcription commences (Fig. 5A). Although no physical interaction between treacle and fibrillarin was observed experimentally, these two proteins also co-localized during the late stage of mitosis (Fig. 5B), suggesting that the methylation complex containing NOP56 and fibrillarin co-localizes with treacle during late mitosis and this co-localization is most likely mediated by a direct physical interaction between treacle and NOP56.

Identification of this function of treacle in pre-rRNA methylation and our previous report on its function in rDNA transcription (18Go) emphasize the importance of treacle in the biogenesis of rRNA. We envisioned a model (Fig. 7) in which binding of treacle to the ETS1 region physically blocks RNA polymerase I during early mitosis when RNA pol I transcription is shutdown. Binding of treacle to the ETS1 region might prevent RNA pol I from advancing. Post-translational modifications and/or interaction of treacle with UBF during late telophase would let treacle dissociate from ETS1 and allow RNA polymerase I either to begin transcription or to elongate any short transcripts which were produced prior to onset of mitosis (28Go). It is possible that modified treacle binds to the promoter region of the rDNA gene and recruits more UBF to stimulate transcription. Such a scenario would be consistent with the binding of treacle to the ETS1 and promoter regions of the rDNA gene shown by our ChIP assay (Fig. 6). During activation of RNA pol I, treacle might simultaneously recruit the ribonucleoprotein 2'-O-methylation complex by interacting with NOP56. Methylation of pre-rRNA is an early event and a prerequisite for subsequent pre-rRNA processing making treacle's proximity to the site of rDNA transcription more relevant. It remains to be determined whether the same treacle molecule bound to UBF can also interact with NOP56. As the C-terminal of treacle interacts with both UBF and NOP56, it is likely that the treacle molecules associated with RNA pol I machinery are different from those associated with the methylation complex. The exact molecular mechanism of treacle function in the methylation reaction is not known at this time but we believe, it might provide a scaffold or framework for the methylation complex.



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  		Figure 7. A simplified model showing the functions of treacle in the biosynthesis of pre-rRNA. See Discussion for description.

 
The identification of a newer role for treacle in pre-rRNA methylation reinforces its relevance in the production of biologically active rRNA (i.e. correct conformation). Although the specific role of ribose methylation is not known, methylation of pre-rRNA precursor is necessary for the production of mature rRNAs in X. laevis oocytes (25Go–27Go). Post-transcriptional methylation of pre-rRNA is believed to channel localized folding and rigid or stable structural rearrangement within the rRNA during the assembly of ribosomes (28Go,29Go).

It is not uncommon for a nucleolar protein to have more than one function in the nucleolus. For example, dyskerin is a putative pseudouridine synthase involved in the post-transcriptional modification of pre-rRNA and it is also a component of the telomerase complex underlying the relevance of dyskerin in rRNA biogenesis and telomere maintenance. Mutation in the DKC1 gene that encodes dyskerin results in dyskeratosis congenita and increased susceptibility to cancer (30Go).

Our present results demonstrating a function for treacle in pre-rRNA methylation and our previously reported data on the function of treacle in rDNA gene transcription lead us to hypothesize that mutations in the TCOF1 gene in TCS patients can significantly inhibit the production of biologically active mature rRNAs. The pre-rRNA molecules that are transcribed might be undermethylated in any of the known 105–107 methylation sites in human 5.8S, 18S and 28S rRNAs (31Go). Insufficient production of mature rRNAs might be deleterious to the proliferation and proper differentiation of specific embryonic cells including the cephalic neural crest cells that eventually differentiate to craniofacial tissues, thus explaining the biochemical pathogenesis of TCS.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Microinjection of antisense oligodeoxynucleotides into oocytes and analysis of rRNA
Oocytes were surgically removed from a female X. laevis obtained from Xenopus Express and tumbled with 2 mg/ml type I collagenase (Sigma) in Ca2+-free modified Barth's saline medium (20 mM HEPES, pH 7.5, 88 mM NaCl, 1 mM KCl, 2.4 mM NaHCO3 and 0.82 mM MgSO4) at room temperature until most oocytes were free. Oocytes were washed thoroughly with this medium and stored overnight at 18°C in a modified Barth's saline medium (20 mM HEPES, pH 7.5, 88 mM NaCl, 1 mM KCl, 2.4 mM NaHCO3, 0.82 mM MgSO4, 0.33 mM Ca(NO3)2 and 0.41 mM CaCl2).

Stages V and VI oocytes were selected for cytoplasmic microinjection with 32 nl antisense oligonucleotide (1 ng/nl). After the indicated time of incubation at 18°C in modified Barth's saline medium, oocytes were microinjected with 23.1 nl [{alpha}-32P]GTP (3000 Ci/mmol and 10 uCi/µl). After further incubation for 1 h at 18°C, oocytes were homogenized in 50 µl homogenization buffer per oocyte (50 mM NaCl, 50 mM Tris–HCl, pH 7.5, 5 mM EDTA, 0.5% SDS and 200 µ/ml proteinase K) and incubated at 37°C for 1 h. Samples were extracted with phenol:chloroform:iso-amyl alcohol (25:24:1) twice and with chloroform once. Nucleic acids were precipitated with ethanol, dried and dissolved in water.

Isolation of total RNA from mouse embryos
Genotyped E11.5 mouse embryos were homogenized in TRIzol reagent (Invitrogen) using a tissue homogenizer. Total RNA was isolated according to the procedure provided by the manufacturer. The precipitated RNA was dissolved in water and its absorbance at 260 nm was determined.

Methylation assays
Methylation of pre-rRNA in Xenopus oocytes was analyzed as previously described (18Go). Briefly, the indicated amount of 32P-labeled total RNA from antisense-treated oocytes was incubated with 30 ng RNA–DNA chimeric oligonucleotide BV1240 (5'-UUdGdGdAdTGUGGUAGCCGUUU-3': all underlined ribonucleotides are 2'-O-methylated) in a buffer containing 20 mM Tris–HCl, pH 7.5, 10 mM MgCl2, 100 mM KCl, 0.1 mM DTT, 5% sucrose, 5 U RNAsin and 1 U RNase H (Invitrogen). The same chimeric oligonucleotide was previously used in the analysis of 2'-O-methylation of 18S pre-rRNA in Xenopus oocytes (21Go). The reaction mixture was incubated at 37°C for 30 min after which 0.5 µl of 0.5 M EDTA was added. A formamide-containing loading buffer was added and the mixture was incubated at 65°C for 15 min prior to loading onto a 1% agarose-formaldehyde gel. The RNA samples on the gel was transferred to Hybond-N nylon membrane and analyzed by phosphorous imaging.

This methylation procedure was modified for the analysis of rRNA from mice embryos. RNA (1 µg) was mixed with 15 ng RNA–DNA chimeric oligonucleotide BV1240 in a buffer containing 20 mM Tris–HCl, pH 7.5, 10 mM MgCl2, 100 mM KCl, 0.1 mM DTT and 5% sucrose. The sequence of BV1240 (mentioned earlier) is perfectly complementary to nucleotides 448–466 of mouse 18S rRNA sequence. The mixture was boiled for 3 min and incubated at 37°C for 10 min. RNase H (0.5 U, Epicenter) was added to make the final volume of the reaction to 10 µl. The reaction mixture was incubated at 37°C for 30 min, boiled for 3 min and placed on ice. The digest was diluted 2-fold with water and 0.5 µl (25 ng RNA) was used for RT–PCR.

Reverse transcription–polymerase chain reaction
A onestep RT–PCR kit (Qiagen, was used to analyze the RNAse H-digested RNA. A 20 µl reaction mixture contained 1x buffer, 0.4 mM dNTPs, 0.8 µl onestep RT–PCR enzyme mix, 0.15 µM 32P-end-labeled BV1250 (5'-GCGTGCATTTATCAGATCAAAACCA-3'), 0.15 µM 32P]-end-labeled BV1253 (5'-GGCAGGGACTTAATCAACGCAAGCT-3'), 0.15 µM BV1251 (5'-TAAAGTGGACTCATTCCAATTACAG-3'), 0.15 µM BV1252 (5'-AGCGGTCGGCGTCCCCCAACTTCTT-3') and 25 ng RNA. PTC-100 Programmable Thermal Controller (MJ Research Inc.) was used to do reverse transcription at 50°C for 30 min followed by 95°C heating for 15 min to denature reverse transcriptase and activate HotStarTaq DNA polymerase. The succeeding 12 cycles of PCR included denaturation at 94°C for 0.5 min, annealing at 58°C for 0.5 min and extension at 72°C for 40 s. A final extension reaction was done at 72°C for 10 min. The RT–PCR product was mixed with 2 µl Bluejuice (Invitrogen), and a 6 µl mixture was analyzed using a 6% polyacrylamide–SDS gel. After electrophoresis at 100 V, the gel was vacuum-dried and exposed to a phosphorous imaging screen. All radioactive signals were measured using the Molecular Dynamics Storm 860 phosphorimager equipped with ImageQuant software for quantitative analysis.

Pseudouridylation
RNA samples from wild-type and Tcof1+/– heterozygous mice embryos were analyzed for the level of pseudouridylation of nucleotide U1642 of 18S rRNA as described earlier (22Go). Treatment of RNA with CMCT was done at 37°C for 12 min. Oligodeoxynucleotide BV1253 (5'-GGCAGGGACTTAATCAACGCAAGCT-3'), which is complementary to nucleotides 1664–1688 of mouse 18S rRNA, was used as the primer for the reverse transcription reaction. Samples were analyzed using a 6% polyacrylamide sequencing gel.

Yeast two-hybrid analysis
Full-length human TCOF1 cDNA was subcloned into pGADT7 and pGBKT7 yeast expression vectors. The pGBKT7 clone was used to screen a human cDNA expression library in pACT vector using the MATCHMAKER two-hybrid system (BD Biosciences). Also, full-length human NOP56 and fibrillarin cDNAs were subcloned into pGBKT7 vector and analyzed for their interaction with treacle. Clones were grown in a triple drop-out medium without tryptophan, leucine and histidine to screen for protein–protein interactions. Further confirmation of the interaction was seen by the blue color of clones grown on triple drop-out medium containing 5-bromo-4-chloro-3-indolyl-{alpha}-D-galactopyranoside (BD Biosciences).

Immunoprecipitation assay
The cDNA fragment that coded for amino acids 717–1488 of human treacle was PCR-amplified and subcloned into the BglII/XhoI sites of pSG5–KF2M vector. HeLa cells were transfected with this plasmid for 48 h. Nuclei were isolated and resuspended in 10 mM Tris–HCl, pH 7.6, 1 mM EDTA, 400 mM NaCl, 10% glycerol, 0.5% NP-40, 5 mM NaF, 1 mM DTT and 0.5 mM Na3VO4 with complete protease inhibitor mix (Roche). Nuclei were lysed by sonication and centrifuged for 20 min at 18 000g, 4°C. Nuclear extract (1 mg) was diluted to 0.5 ml with lysis buffer and mixed with 0.5 ml of dilution buffer (10 mM Tris–HCl, pH 7.6, 1 mM EDTA, 20% glycerol, 0.5% NP-40 5 mM NaF, 1 mM DTT, 0.5 mM Na3VO4 and protease inhibitors). RNase A (0.2 µg/µl) and DNase I (0.5 U/µl) were added. The reaction mix was kept on ice for 10–15 min and then centrifuged at 10 000g for 10 min. The supernatant was tumbled with anti-FLAG M2-agarose overnight at 4°C. The resin was washed in three consecutive steps: NET-gel buffer (50 mM Tris–HCl, pH 7.5, 500 mM NaCl, 0.1% NP-40, 1 mM EDTA and 0.25% gelatin), NET-gel buffer with 0.1% SDS and a final wash buffer (10 mM Tris–HCl, pH 7.6 and 0.1% NP-40). The resin was boiled in Laemmli buffer and used for western blot analysis.

ChIP assay
A ChIP assay kit (Upstate) was used. Non-synchronized HeLa cells were treated with 1% formaldehyde for 10 min, washed and sonicated in lysis buffer to give 200–5000 bp genomic DNA fragments. The sonicate was tumbled with anti-treacle antibody including a rabbit IgG negative control. Complexes were immunoprecipitated with protein A agarose. After reversal of crosslinks at 65°C and RNase A/proteinase K treatments, precipitated DNA was PCR-amplified with primer sets specific to the promoter region of rDNA gene, 5' of the ETS1 and 28S. Genomic DNA (40 ng) was used as positive control template.


   ACKNOWLEDGEMENTS
 
This work was supported by Public Health Service Grant DK52341 from NIDDK, National Institutes of Health to B.C.V. and Medical Research Council Grant G81/535 to M.J.D. B.G. was supported by Public Health Service R25 GM56929, an Initiative for Minority Student Development grant. We thank the laboratory of Dr M. Jamrich for care of the Xenopus and Drs N.J. Watkins and R. Luhrmann for the anti-NOP56 antibody.

Conflict of Interest statement. None declared.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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