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BOR - Papers in Press, published online ahead of print July 19, 2006.
Biol Reprod 2006, 10.1095/biolreprod.106.054072
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BIOLOGY OF REPRODUCTION 75, 690–696 (2006)
DOI: 10.1095/biolreprod.106.054072
© 2006 by the Society for the Study of Reproduction, Inc.

Research Article

Implication of Nucleolar Protein SURF6 in Ribosome Biogenesis and Preimplantation Mouse Development1

Ludmila G. Romanova 34 , Martin Anger 3, Olga V. Zatsepina 4, and Richard M. Schultz 2 3

Department of Biology,3 University of Pennsylvania, Philadelphia, Pennsylvania 19104-6018 Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry,4 RAS, Moscow, 117997, Russia

ABSTRACT

The step-wise assembly of a functional nucleolus, which occurs over the first few cell cycles during preimplantation development, is poorly understood. In this study, we examined the function of the evolutionary conserved nucleolar protein SURF6 in preimplantation mouse embryo development. Immunocytochemical analyses revealed that the localization of SURF6 was similar but not identical to those of fibrillarin and B23/nucleophosmin 1, which are involved in rRNA processing and ribosome biogenesis in mammalian somatic cells. Surf6 mRNA, which is expressed in oocytes and maternally inherited in the zygote, reached a peak level of expression during the 8-cell stage of embryo development, at which time rDNA is highly transcribed. Knock-down of Surf6 mRNA by RNAi led to a decrease in both the mRNA and protein levels, and resulted in developmental arrest at the 8-cell/morula stage, as well as a decrease in the level of 18S rRNA. These results suggest that Surf6 is essential for mouse preimplantation development, presumably by regulating ribosome biogenesis.

developmental biology, early development, embryo, gametogenesis, gene regulation

INTRODUCTION

The nucleolus is the site for the transcription of ribosomal DNA (rDNA), which encodes the 18S, 5.8S, and 28S ribosomal RNA (rRNA), the processing of the 45–47S rRNA transcripts, and the assembly of preribosomal particles. The nucleolus, which is composed of 700 proteins, also plays a role in other cellular processes, including cell cycle regulation and apoptosis [1]. In contrast to somatic cells, the early development of mammalian embryos is characterized by a period of rDNA transcriptional quiescence and the presence of nucleolar precursors (called the nucleolar precursor bodies, NPBs) that nucleate the assembly of the mature nucleoli starting from the 2-cell embryo stage onwards [2].

The active reassembly of a functional nucleolus is triggered by the resumption of rDNA transcription, which occurs in the middle of the second cell cycle in mouse embryos or in the later stages of embryo development in other mammalian species [3–5]. The major proteins of the mature nucleolus, i.e., B23/nucleophosmin 1, which is an abundant preribosome assembly factor, nucleolin, which is involved in early stages of rRNA processing, NOLC1, which apparently functions as a chaperone for the snoRNP complexes, fibrillarin, which is an early pre-rRNA processing protein, as well as the RNA polymerase I (pol I) transcription machinery constituents RPO1–2 and UBTF, are anchored to the NPB surface [5–9] concomitant with the reactivation of rDNA transcription. In preimplantation mouse embryos, maturation of the nucleolus is apparently completed no earlier than the morulae stage, at which time characteristic nucleolar subdomains, which include the fibrillar center, dense fibrillar component, and granular component, can be unequivocally identified by electron microscopy [2, 10]. However, the molecular mechanisms that govern nucleolus assembly remain unclear.

To date, the dynamics of nucleolus reassembly have been described in terms of the behaviors of only a few major nucleolar proteins; little is known regarding their expression patterns. Moreover, despite the finding that certain proteins of the mature nucleolus, e.g., pescadillo, fibrillarin, and B23/nucleophosmin 1, are essential for early mouse embryo development [11–13], a particular role for nucleolar proteins in preimplantation development has not been confirmed.

Surf6 is a member of the Surfeit gene locus, which is the tightest known cluster of genes in the mammalian genome [14]. The product of the murine Surf6 gene is the highly basic 355-amino acid nucleolar protein SURF6, which localizes to the granular component of the nucleolus. SURF6 binds more strongly to RNA than DNA, and in somatic cells its distribution during the cell cycle is similar, but not identical, to those of fibrillarin and B23/nucleophosmin 1 [15]. The SURF6 amino acid sequence does not contain any recognizable functional domains, which makes it difficult to ascribe a particular function to this protein. Nevertheless, a highly evolutionary conserved domain is present in the C-terminus, and GFP-fusion SURF6 proteins from distinct taxonomic groups that containing this domain are targeted to the nucleolus [16]. Moreover, knockout of the Surf6 yeast homolog yk1082c gene, rrp14, the gene product of which (RRP14) interacts with proteins involved in ribosome biosynthesis [17, 18], results in a loss of cell viability [19]. These observations suggest that SURF6 may also be involved in ribosome biogenesis, although the precise role of this protein in mammalian cells, including embryos, remains unknown.

We report here that in mouse oocytes and preimplantation embryos, SURF6 displays a unique distribution profile and is present in the nuclei of transcriptionally competent cells. Moreover, RNAi-mediated knockdown of Surf6 mRNA and the SURF6 protein results in developmental arrest at the morula stage, with a concomitant reduction in the level of 18S rRNA. These results provide the first evidence that Surf6 is essential for early mammalian development, and provide direct evidence of Surf6 participation in ribosome biosynthesis in mouse embryos.

MATERIALS AND METHODS

Collection of Oocytes and Embryos

Fully-grown germinal vesicle (GV)-intact oocytes were obtained from eCG-primed, six-week-old CF-1 females (Harlan), as previously described [20]. To prevent resumption of meiosis during isolation, 2.5 µM milrinone was added to the MEM collection medium (Sigma-Aldrich, St. Louis, MO). Metaphase II (MII)-arrested eggs were obtained 14–16 h after hCG injection from females that were primed previously with eCG. Embryos of various developmental stages were obtained from CF-1 females that were stimulated with eCG and hCG and mated to B6D2F1/J males. Embryos were collected in the following intervals after hCG stimulation: 1-cell embryos, 18–20 h; 2-cell embryos, 40–42 h (early), 44–46 h (mid), and 48 h (late); 4-cell embryos, 52–54 h; 8-cell embryos, 64–66 h; and blastocysts, 92–94 h. All animal experiments were approved by the Institutional Animal Use and Care Committee and were consistent with National Institutes of Health guidelines.

Antibodies, Immunolabeling, and Confocal Microscopy

Oocytes and embryos were fixed with 2% paraformaldehyde in standard phosphate-buffered saline [PBS; 0.14 M NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, 1.5 mM KH2PO4 (p? 7.2–7.4)] for 1 h at room temperature, followed by permeabilization with 0.1% Triton X-100 in PBS [137 mM NaCl, 2.7 mM KCl, 6.7 mM Na2HPO4, 1.5 mM KH2PO4 (pH 7.3)] for 30 min at room temperature. The cells were then incubated in blocking solution (2% BSA in PBS) and processed for single or double immunolabelling. At least 20 oocytes or embryos at each developmental stage were used for immunostaining.

The following primary antibodies were used: rabbit polyclonal anti-SURF6 antibody (1:200 dilution) [15]; mouse monoclonal anti-B23/nucleophosmin 1 antibody (1:100 dilution) (Sigma); human autoimmune serum against fibrillarin (1:100 dilution) [21]; mouse monoclonal anti-p80-coilin antibody (1:200 dilution) (BD Biosciences Pharmingen, San Diego, CA).

Oocytes and embryos were incubated with the primary antibodies for 1 h at room temperature, washed in the blocking solution, and then incubated with secondary antibodies, while minimizing light exposure. The following secondary antibodies were used: Cy5-conjugated anti-rabbit IgG and Cy5-conjugated anti-human IgG (Jackson Immunoresearch Laboratories, West Grove, PA); and Alexa 488-conjugated anti-mouse IgG and Alexa 488-conjugated anti-rabbit IgG (Molecular Probes, Eugene, OR). All the secondary antibodies were used at a dilution of 1:500.

After incubation with secondary antibodies, the samples were washed and the DNA was stained with 1 µM SYTOX Green (Molecular Probes) for 15 min. The samples were mounted on microscopic slides in VectorShield (Molecular Probes). Specimens that were incubated in blocking solution without primary or secondary antibodies served as controls. Fluorescence microscopy was performed with the Leica TCS SP laser-scanning confocal microscope using an oil-immersion 40x objective.

Total RNA Isolation, Reverse Transcription, and Real-Time RT-PCR Analysis

Total RNA samples were isolated from groups of 5–50 oocytes/embryos using the Absolutely RNA Microprep Kit (Stratagene, La Jolla, CA) and subjected to reverse transcription, as described previously [22], with the modification that 2 ng of Egfp RNA was added per sample to the lysis buffer at the beginning of the RNA isolation.

All of the equipment, probes, chemicals and consumables for real-time RT-PCR were purchased from Applied Biosystems (Foster City, CA). The levels of the mRNAs were quantified using the Real-Time ABI Prism Detection System 7000, and the amount of cDNA used for each reaction was equivalent to the amount in a single oocyte/embryo. The ABI Assay On Demand probe and the primers Mm00486494_m1 were used for the analysis of Surf6 expression. Three independent sets of samples were collected and analyzed from each stage. All of the reactions were run in triplicate. Relative expression was calculated using {Delta}{Delta}Ct method; samples were normalized to the Egfp values and the amount of transcript present in GV-intact oocytes was arbitrarily set at 1. To quantify the 18S rRNA content in the RNAi experiments, the ABI Assay By Design 18Sexon-aF and probe/primers sets, which are specific for the 18S coding sequence, were used.

In Vitro Synthesis of Double-Stranded (ds) RNA

The 550-bp Surf6 and 750-bp Egfp dsRNAs were synthesized using the MEGAscript RNAi Kit (Ambion, Austin, TX). DNA templates were prepared by PCR with primers that contained the T7 polymerase promoter template, as described previously [22]. The following Surf6 primers were used: forward, 5'-AGCGGCTAATACGACT CACTATAGGGAGATATGGATCACAAGACTAAAGC-3'; and reverse, 5'-AGCGGCTAATACGACTCAC TATAGGGAGATGCATCCTGATCACGAAGCTC-3'. The Egfp primers were the same as those used previously [22]. The dsRNA was column-purified and diluted with elution buffer to the concentration of 0.5–1 µg/µl.

Microinjection

One-cell embryos were collected 18–20 h after hCG injection, and those embryos with apparent pronuclei were used for microinjection, as described previously [23]. Embryos were microinjected in bicarbonate-free Whitten's medium (pH 7.3) [24] that was supplemented with 10 mM Hepes (pH 7.2), and then cultured in KSOM medium [25] at 37°C in 5% CO2/95% O2 for the appropriate time intervals.

RESULTS

In order to study the dynamics of SURF6 in mouse oocytes and preimplantation embryos, we first examined SURF6 localization in 48 mouse oocytes and 150 preimplantation embryos by immunocytochemistry (Fig. 1a–j). We also examined SURF6 colocalization with the rRNA processing protein markers fibrillarin and B23/nucleophosmin 1 (Fig. 1k–t), the localizations of which are similar to that of SURF6 during the cell cycle [15].


Figure 1
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  		FIG. 1. Distribution of SURF6 in mouse oocytes and preimplantation embryos. In NSN oocytes, SURF6 forms discrete foci at the surface of the nucleolus (a) and is also seen within small patches attached to chromatin blocks (arrow); SURF6 partly colocalizes with B23 (k) and fibrillarin (p). In SN oocytes (b) and MII eggs (c), the intense immunolabeling of the cytoplasm with anti-SURF6 antibodies increases. SURF6 is not detected in 1-cell (d) and early 2-cell (38–40 h post-hCG) embryos (e), but in mid 2-cell embryos (42–44 h post-hCG) it becomes visible in the large patches attached to NPBs (f, arrow). In late 2-cell embryos (46–48 h post-hCG), SURF6 surrounds the NPB surface and partially colocalizes with B23/nucleophosmin 1 (l) and fibrillarin (q). At the 4-cell stage (g, m, r) and 8-cell stage (h, n, s), SURF6 is clearly seen at the NPB surface, where it forms a looser and more extended layer than in 2-cell embryos and partially colocalizes with B23 (m, n) and fibrillarin (r, s). In morulae (i), the distribution of SURF6 is similar to that in somatic nucleoli, i.e., SURF6 partially colocalizes with B23/nucleophosmin 1 (o) and fibrillarin (t). No specific immunolabeling of oocytes is observed when the anti-SURF6 antibody was omitted (j). However, there is a variable degree of non-specific cytoplasmic labeling in oocytes and early embryos. Note that the panels in black and white show a section of the entire embryo, whereas the nucleus is shown in the color panels. Bar = 10 µm

In all the GV oocytes that lacked a perinucleolar chromatin rim (the so-called ‘non-surrounded oocytes' or NSN, which are transcriptionally competent [26]) SURF was clearly visible at the periphery of the NLB (nucleolus-like body) (Fig. 1a), where it partially colocalized with B23/nucleophosmin 1 (Fig. 1k) and fibrillarin (Fig. 1p). In addition, the SURF6 signal was observed as small dots that were attached to chromatin blocks either within the nucleoplasm or associated with nucleoli (Fig. 1a). In contrast, SURF6 was undetectable in any nuclear compartment of the transcriptionally inert oocytes, which were characterized by the presence of a rim of condensed chromatin around the nucleolus (the so-called ‘surrounded-type oocytes' or SN [26]). We noted a variable degree of cytoplasmic staining in the oocytes and eggs, which precluded determination of whether SURF6 was present in MII eggs (Fig. 1c).

SURF6 was not detected in the nuclei of the 1- or 2-cell embryos (18–20 h and 38–40 h post-hCG, respectively; 40 embryos examined) (Fig. 1 d and e). SURF6 first became clearly visible in the nuclei of the mid 2-cell embryos (42–44 h post-hCG), where it accumulated in round structures that were associated with the NPB surface (Fig. 1f). These structures are probably Cajal (coiled) bodies, since p80-coilin, which is a Cajal body marker, showed colocalization (Fig. 2). In the late 2-cell embryos (46–48 h post-hCG), which are able to transcribe rDNA [4, 5], SURF6 was mainly distributed over the NPB surface, where it partially colocalized with B23/nucleophosmin 1 and fibrillarin (Fig. 1, l and q; 20 embryos examined). In 4-cell embryos (Fig. 1, g, m, and r; 30 embryos examined) and 8-cell embryos (Fig. 1, h, n, and s; 25 embryos examined), SURF6 remained at the NPB periphery, but its localization became more extended than in the 2-cell embryos (Fig. 1, f, l, and q, and Fig. 2b). In morulae (Fig. 1, o and t; 30 embryos examined) and blastocysts (30 embryos examined), SURF6 distribution was essentially similar to that in the nucleoli of mouse somatic cells. Therefore, SURF6 accumulates in oocyte and embryo nuclei when they are transcriptionally competent (NSN oocytes, late 2-cell embryos-blastocysts), and its localization is similar but not identical to that of B23/nucleophosmin 1 and fibrillarin. In addition, the downregulation of rRNA synthesis in SN oocytes results in failure to detect SURF6 in the nucleus.


Figure 2
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  		FIG. 2. Double immunolabelling of a mid 2-cell embryo (42–44 h post hCG) for p80-coilin (a) and SURF6 (b), and a merged image (c). The arrows point to the Cajal bodies (arrows) that are associated with NPBs. Bar = 10 µm

Temporal Patterns of Surf6 Transcripts in Oocytes and Preimplantation Embryos

Real-time RT-PCR revealed that Surf6 mRNA was present throughout preimplantation development and that its expression was highest in 8-cell embryos (Fig. 3). It is noteworthy that Surf6 transcripts were readily detected in MII eggs and 1-cell embryos, indicating that the maternal transcript is inherited by zygotes from oocytes. The absence of immunoreactive SURF6 in 1-cell and early 2-cell embryos when the transcript was clearly present suggests that the maternally-derived SURF6 protein is targeted for destruction within the period between the initiation of oocyte maturation and the early 2-cell stages. Zygotic expression of Surf6 is probably initiated by the 4-cell stage, as evidenced by a slight increase in transcript abundance between the 2-cell and 4-cell stage. Transient peak expression of Surf6 was observed during the 8-cell stage. Consistent with this finding, SURF6 protein was detected at these later stages by immunocytochemistry.


Figure 3
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  		FIG. 3. Temporal pattern of Surf6 expression during oocyte maturation and preimplantation development. Real-time RT-PCR analysis of the relative abundance of Surf6 transcripts in the mouse. One-cell, 2-cell, 4-cell, 8-cell, and blastocysts were harvested 8–20 h, 44–46 h, 52–54 h, 64–66 h, and 92–94 h post-hCG. The values are normalized relative to the relative amount of Surf6 mRNA in GV oocytes. The experiment was performed three times, and the data are presented as the mean ± SEM. Differences in the relative amount of Surf6 transcripts between each pair of developmental stages are statistically significant (t-test, P < 0.05)

Surf6 Knockdown Results in Embryo Arrest at the Morulae Stage and a Decrease in 18S rRNA

The role of SURF6 in preimplantation development was assessed by employing an RNAi approach using Surf6 dsRNA to target selectively Surf6 mRNA. Oocytes and preimplantation embryos are amenable to this approach because they lack an interferon response, which would result in cell death, and off-targeting does not appear to be a concern [27]. In addition, when this approach was used to target a member of either the Msy or Bnc family, a high degree of specificity was observed [28, 29].

One-cell embryos were injected with Surf6 dsRNA, while control embryos were injected with an equivalent amount of Egfp dsRNA (Table 1). We chose to inject 1-cell embryos because SURF6 protein is not readily detected in these embryos (Fig. 1d). The embryos were then cultured in vitro, and Surf6 mRNA and SURF6 protein expression were then analyzed in 4-cell embryos (Fig. 4). Surf6 dsRNA injection led to a marked reduction in the level of Surf6 mRNA (Fig. 4), and more importantly, a reduction in the level of SURF6 protein, which was not detected in the 4-cell embryos, as judged by immunolabeling with specific antibodies (Fig. 4).


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  		TABLE 1. Effect of Surf6 dsRNA on preimplantation development


Figure 4
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  		FIG. 4. Efficiency of Surf6 mRNA and protein targeting in 4-cell mouse embryos after injection of Surf6 dsRNA. The data are expressed as the mean ± SEM, where n = 3. A significant decrease in the level of Surf6 mRNA is observed in embryos injected with Surf6 dsRNA as compared to the control Egfp dsRNA-injected embryos (t-test, P < 0.05). Shown below are confocal images documenting a marked reduction in SURF6 protein (red). DNA (green) is counterstained with SYTOX Green. Bar = 10 µm

Monitoring the injected embryos for preimplantation development revealed that, in contrast to the Egfp dsRNA-injected embryos, the Surf6 dsRNA-injected embryos did not develop with high incidence to the blastocyst stage but became arrested at the morula stage (Table 1 and Fig. 5). Many of these embryos appeared to be fragmented and possessed a dark cytoplasm. Of note is that when the Surf6 dsRNA-injected embryos developed to the blastocyst stage, SURF6 protein was readily detected by immunocytochemistry (data not shown). The incomplete depletion of SURF6 in these embryos was most likely due to insufficient amounts of Surf6 dsRNA being injected, and this provides a tight correlation between SURF6 expression and development.


Figure 5
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  		FIG. 5. Phenotype of Surf6 knockdowns in mouse embryos developing in vitro. In the control group (A) injected with Egpf dsRNA, the majority of the embryos successfully form blastocyst (arrows), whereas the majority of the embryos injected with Surf6 dsRNA (B) fail to develop to the blastocyst stage, are arrested at the morula stage, and exhibit signs of degeneration (arrows). Bar = 100 µm

Given the potential function of SURF6 in ribosome biogenesis based on its localization in the nucleolus, we measured the levels of 18S rRNA in Surf6 dsRNA- and Egfp dsRNA-injected embryos that had reached the 4-cell stage. We elected to make the measurement at this time-point because these embryos transcribe rDNA [5, 10] and appear to be healthy. Observing a reduction in rRNA at the later stages could simply be a consequence of apparent embryo degeneration at these later times. Using real-time RT-PCR and primers and a probe set that detect specifically the overall 18S RNA pool, i.e., mature 18S rRNA and unprocessed pre-rRNA transcripts, a decrease of 40% was observed (Fig. 6). This observation is consistent with a function for SURF6 in ribosome biogenesis.


Figure 6
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  		FIG. 6. Evaluation of the 18S rRNA contents of the control and knockdown 4-cell mouse embryos. After dsRNA injections, the embryos were cultured in vitro and five 4-cell-stage embryos from each group were withdrawn for total RNA isolation and subsequent real-time RT-PCR analysis. The experiment was conducted three times on different groups of injected embryos. The observed decrease in 18S rRNA level is statistically significant (t-test, P < 0.05)

DISCUSSION

Although SURF6 is a nucleolar protein, there is a paucity of information regarding its function in somatic cells, let alone during mammalian development. The results described here suggest that SURF6 is essential for preimplantation development and implicate a role for SURF6 in ribosome biogenesis.

Colocalization of SURF6 with two major participants in rRNA processing (i.e., fibrillarin and B23/nucleophosmin 1), as well as its association with structures involved in nucleolar reassembly and rDNA transcription and processing (i.e., NPBs and Cajal bodies in embryos) provide highly suggestive evidence that SURF6 participates in nucleologenesis during preimplantation development. Of note is that, unlike other nucleolar proteins studied to date, SURF6 is present only when oocytes or embryos transcribe rDNA, i.e., SURF6 is present in the nuclei of NSN oocytes and late 2-cell embryos, and is absent from SN oocytes, 1-cell embryos, and early 2-cell embryos. This profile is dissimilar to those of other nucleolar proteins that have been studied to date, such as B23/nucleophosmin 1, fibrillarin, UBTF (UBF), and RPO1–2 (RPA116), which are detected in SN oocytes and in 1-cell embryos [5, 30]. Therefore, the presence of SURF6 in nuclei may provide a reliable marker for on-going rRNA synthesis.

The localization of SURF6 is to some extent similar to those of fibrillarin and B23/nucleoplasmin 1. Similar to fibrillarin, SURF6 is recruited to Cajal bodies in the early embryo, consistent with the suggestion that in early embryos, these proteins are recruited to Cajal bodies before being transported to nucleoli [31]. SURF6 also forms a continuous rim at the NBP surface, which is the site of rDNA transcription, with fibrillarin and B23/nucleophosmin 1 in transcriptionally competent embryos (2-cell to 8-cell stages). Taken together, these observations suggest that SURF6 is involved in rRNA synthesis in early mouse embryos, either in processes that differ from those mediated by fibrillarin and B23/nucleophosmin 1 or in collaboration with these proteins.

The increase in Surf6 transcript abundance in the 1-cell embryo may reflect the fact that Surf6 is one of the few genes transcribed at this time. It seems unlikely that this increase is due to the recruitment of maternal mRNA due to polyadenylation, as random primers were used for the reverse transcription reaction; the efficiency of the reverse transcription reaction using random primers should be independent of the length of the poly(A) tail that accompanies mRNA recruitment. In addition, neither a consensus CPE sequence nor a dodecauridine track is present in the Surf6 mRNA [32]. What is curious is that despite the presence of Surf6 mRNA in 1-cell embryos, SURF6 protein is not detected. Thus, the early embryo appears to need to remove this protein when it does not transcribe rDNA genes.

The transient increase in Surf6 expression in 8-cell embryos precedes the complete maturation of the nucleoli [2, 10]. Of note is that genes involved in ribosome biogenesis are transiently and preferentially expressed in 8-cell embryos [33]. The similar expression profile of Surf6 suggests that SURF6 could play such a role; in support of this is the finding that reducing the amount of SURF6 protein in 4-cell embryos using RNAi results in a reduced amount of 18S rRNA in 4-cell embryos that transcribe rDNA genes. This suggests that SURF6 participates in rRNA synthesis, and is in agreement with data obtained with yeast, which show that the SURF6 homolog yk1082c interacts with proteins required for ribosome formation [17]. Of particular note is that reducing the level of SURF6, which leads to a decrease in the level of 18S rRNA, results in developmental arrest at the 8-cell/morula stage, with apparent degeneration of the embryos; zygotic ribosomes are probably first generated at this time. A similar phenotype is observed in fibrillarin-null mouse embryos, which undergo massive apoptosis at the morula stage [12], and mouse embryos that lack the nucleolar protein pescadillo fail to undergo morulae-blastocyst transition due to the disruption of ribosome biosynthesis and proper nucleolar assembly [11]. Taken together, these data and our present data support the proposal that in preimplantation mouse embryos, protein synthesis is supported by maternal ribosomes until this stage. Thus, mouse embryos may differ from early embryos in other mammalian species, such as cattle and pigs, in which rDNA transcription and nucleolus maturation occur at later developmental stages [34]. In addition, our results provide for the first time direct evidence that Surf6 is involved in ribosome biogenesis in mammalian embryos.

It is generally accepted that the major role of the nucleolus is the synthesis of preribosomal particles. Nevertheless, recently it has become apparent that this organelle plays roles that are unrelated to ribosome production. For example, the nucleolar protein nucleostemin controls stem and cancer cell proliferation [35], and the nucleolar remodeling complex mediates de novo DNA methylation, histone acetylation, and heterochromatin protein binding [36]. The nucleolus and nucleolar proteins are also involved in cell cycle regulation and telomerase activity [37, 38]. In addition, recent proteomic analysis identified a panel of proteins that are not involved in ribosomal biosynthesis [39]. Recent studies show that ribosome biosynthesis is tightly linked to cell proliferation, and that the expression of major nucleolar proteins varies throughout the cell cycle transition [40, 41]. Thus, Surf6 may have functions other than those required for ribosome biogenesis.

ACKNOWLEDGMENTS

The authors thank Dr. M. Polzykov for his sustained interest in this project, Dr. Charalambos Magoulas for providing the anti-SURF6 antibody and plasmid, and Dr. Jason Knott for help with the RNAi microinjection experiments.

FOOTNOTES

2 Correspondence: Richard M. Schultz, Department of Biology, University of Pennsylvania, 415 South University Avenue, Philadelphia, PA 19104-6018. FAX: 215 898 8780; rschultz{at}sas.upenn.edu Back

1 Supported by a grant from the NIH (HD22681 to R.M.S.) and in part by the Russian Foundation for Fundamental Research (project 04-04-48391 to L.G.R.). Back

Received: 31 May 2006.

First decision: 24 June 2006.

Accepted: 12 July 2006.

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