RBD-1, a nucleolar RNA-binding protein, is essential for Caenorhabditi
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《核酸研究医学期刊》
Department of Biology, Graduate School of Science and Technology, Kobe University, 1-1 Rokkodaicho, Nadaku, Kobe 657-8501, Japan
*To whom correspondence should be addressed. Tel: +81 78 803 5796; Fax: +81 78 803 5720; Email: hsaka@kobe-u.ac.jp
ABSTRACT
RBD-1 is the Caenorhabditis elegans homolog of Mrd1p, which was recently shown to be required for 18S ribosomal RNA (rRNA) processing in yeast. To gain insights into the relationship between ribosome biogenesis and the development of multicellular organisms, we examined the expression and function of RBD-1. Maternal RBD-1 in the fertilized egg disappears immediately after cleavage starts, whereas zygotic RBD-1 first appears in late embryos and is localized in the nucleolus in most cells, although zygotic transcription of pre-rRNA is known to be initiated as early as the one-cell stage. RNA interference of the rbd-1 gene severely inhibits the processing of 18S rRNA in association with various developmental abnormalities, indicating its essential role in pre-rRNA processing and development in C.elegans. These results provide evidence for the linkage between ribosome biogenesis and the control of development and imply unexpected uncoupling of transcription and processing of pre-rRNA in early C.elegans embryos.
INTRODUCTION
In all eukaryotic cells, ribosome biogenesis is a very integrated process that occurs in the nucleolus (1). The nucleolus of the eukaryotic cell is densely packed with pre-ribosomal RNAs (pre-rRNAs) and a number of small nucleolar RNAs (snoRNAs) that are essential components involved in pre-rRNA processing . Maturation of rRNAs is achieved by post-transcriptional events including methylation, pseudouridylation and multiple cleavages, resulting in the generation of mature 18S, 5.8S and 25–28S rRNA species in all eukaryotic cells (4,5). These processes are accomplished via various cis-acting elements within pre-rRNAs (6) and a number of non-ribosomal trans-acting factors (7). Although the outline of the pre-rRNA processing pathway is roughly understood and the structures of two ribosomal subunits have recently been solved by X-ray crystallography and cryoelectron microscopy (8,9), little is understood about how ribosome biogenesis is controlled in eukaryotic cells and whether it is linked to more complex biological events, such as developmental processes in multicellular organisms.
Mrd1p was initially given this name because it contains multiple copies of an RNA-binding domain called RBD, also known as RRM (RNA recognition motif), and was recently shown to be a member of the group of non-ribosomal proteins that are involved in pre-rRNA processing in the yeast Saccharomyces cerevisiae (10). In yeast, Mrd1p is essential for viability and its depletion leads to a decrease in the levels of mature 18S rRNA and 40S ribosome and concomitant accumulation of 18S rRNA precursors, whereas 25S rRNA processing is not affected. Since Mrd1p can associate with pre-rRNA and two components of U3 small nucleolar ribonucleoprotein complex (snoRNP), Mrd1p is also likely to be a component of U3 snoRNP, which is known to be required for 18S rRNA processing (11). Since Mrd1p homologs are found in a wide range of metazoans, the homologs may also be involved in pre-rRNA processing. Indeed, in the dipteran Chironomus tentans, a homologous protein (Ct-RBD-1) is localized mainly in the nucleus and is likely to be involved in 18S rRNA processing (12). Interestingly, RNA interference (RNAi)-mediated depletion of the Caenorhabditis elegans homolog RBD-1 (according to its gene name in the database) and a truncation mutation in the zebrafish homolog Npo causes various developmental abnormalities (12,13). These observations imply that there may be the linkage between ribosome biogenesis and developmental events in multicellular organisms. However, the question as to whether the developmental abnormalities are correlated with defects in ribosome biogenesis still remains to be examined, since there is no direct evidence for the involvement of RBD-1 or Npo in pre-rRNA processing.
To address the question of this possible correlation, we examined in detail the role of RBD-1 in both ribosome biogenesis and development in C.elegans. We found that RBD-1 depletion by RNAi inhibits processing of 18S rRNA and subsequent formation of the 40S ribosomal subunit, and causes various developmental abnormalities simultaneously, indicating its essential role in pre-rRNA processing and development in C.elegans. We also found that RBD-1 is localized in the nucleolus, like Mrd1p and Ct-RBD-1 and that its zygotic expression starts in late embryos, although transcription of pre-rRNA by RNA polymerase I is known to start as early as the one-cell stage. We observed a similar expression pattern during embryogenesis for a component of U3 snoRNP, FIB-1 (14), a C.elegans homolog of the yeast Nop1p, which is an essential factor for 18S rRNA processing (15,16). These results provide evidence for the linkage between ribosome biogenesis and developmental events in multicellular organisms and imply that transcription and processing of pre-rRNA may be regulated differentially during early embryogenesis in C.elegans.
MATERIALS AND METHODS
Construction of GFP reporter gene fusion
The reporter construct expressing green fluorescent protein (GFP) under the control of the rbd-1 promoter was made as follows. The promoter region was PCR-amplified using C.elegans genomic DNA with the forward primer (–1931): TTG CAT GCT AAT GGT GAG TAG CTT TAT CCT GAA ATA AGA ACA C, and the reverse primer (+30): GGT CTA GAG CTT GTT TTT GAC AAT TAA TCG AGT TGT CAT G (the numbers in parentheses correspond to the nucleotide position relative to the first nucleotide of the rbd-1 open-reading frame). This genomic fragment was fused in-frame to a promoterless GFP vector, pPD95.77 (provided by Dr A. Fire). Microinjection of the resulting plasmid into C.elegans worms (Bristol type N2) was performed as described (17). Worm breeding and handling were conducted as described (18).
RNA interference
Sense and antisense RNAs were synthesized in vitro from yk417f6 cDNA which encodes RBD-1 (provided by Dr Y. Kohara). Both RNAs were annealed to form a double-stranded RNA (dsRNA). For RNAi, L4 hermaphrodites were soaked in 4 μl of dsRNA solution (2 μg/μl) for 16–24 h or dsRNA (1 μg/μl) was injected into the gonad arms of young adult hermaphrodites.
Northern blot analysis
Total RNA from wild-type and rbd-1(RNAi) animals were extracted with an RNA extraction kit (Micro-to-Midi Total RNA Purification System; Invitrogen). Approximately 4 μg of total RNA per lane were resolved on a 1.2% formaldehyde-containing agarose gel, transferred onto a nylon membrane (Roche Diagnostics), and hybridized with DIG-labeled antisense RNA probes. The antisense probes 1–9 and 18S probe correspond to the positions of nucleotides 511–609, 846–933, 2736–2791, 2969–3036, 3050–3157, 3342–3427, 1–210, 311–410, 411–510 and 1261–1677, respectively, of the C.elegans rDNA repeat (19).
Antibody preparation, western blot analysis and immunostaining
An rbd-1 cDNA fragment of 972 bp was subcloned into pGEX-4T3 (Amersham Biosciences) and the fusion protein GST-RBD-1 was over-expressed in the XL-2 blue Escherichia coli strain. GST-RBD-1 was affinity-purified using glutathione–Sepharose (Amersham Biosciences) and used to raise rabbit polyclonal antibodies. The anti-RBD-1 antibody was affinity-purified and used for western blot analyses (1:1000–1:2000 dilutions) as described previously (20). Isolation of worms at specific developmental stages was performed as described (21). Wild-type worms were immunostained using affinity-purified anti-RBD-1 antibodies (1:100 dilution) or anti-FIB-1 antibodies (22) (1:100 dilution) as described (23). Embryos and larvae were permeated for staining by the freeze-crack method and fixed with methanol/acetone according to standard procedures (24).
Sucrose gradient centrifugation
Wild type and rbd-1(RNAi) worms, respectively, were mixed with Lysing Matrix D (Bio101) and homogenized in buffer A by using a FastPrep homogenizator (Bio101). The lysates were loaded on a linear sucrose gradient (10–30%) in buffer B and centrifuged at 4°C in a Beckman MLS50 rotor at 40 000 r.p.m. for 120 min, followed by fractionation and monitoring at 260 nm.
Scanning electron microscopy
Worms were washed in M9 buffer before fixation in Parducz fixative (25). After extensive washes, samples were dehydrated in ethanol followed by amylacetate. The samples were processed in a critical-point drier, mounted and observed using a Hitachi S-2150N scanning electron microscope.
RESULTS
Expression and subcellular localization of RBD-1
We examined the expression pattern of RBD-1 at various developmental stages. To measure the levels of RBD-1 expression, we used synchronized populations at each developmental stage. Western blot analyses with anti-RBD-1 antibody reveal that RBD-1 is expressed constantly during the four larval stages and the adult stage, whereas the RBD-1 expression level in embryos is significantly lower than that at other stages (Fig. 1A). This suggests that RBD-1 expression is downregulated during embryogenesis. Consistently, immunostaining of embryos shows that RBD-1 expression is limited to late embryonic stages (Fig. 1B). Interestingly, a small amount of maternally supplied RBD-1 is also detectable in the fertilized egg (Fig. 1B, a and b: arrowhead). In contrast to zygotic RBD-1, maternal RBD-1 is seen as a number of granules in the cytoplasm and disappears shortly after cleavage begin, suggesting that maternal RBD-1 may be degraded rapidly in early embryos. To confirm the downregulation of RBD-1 in early embryos, we utilized a transgenic strain carrying the GFP gene under the control of the native promoter for the rbd-1 gene and monitored GFP fluorescence from a single embryo (Fig. 1C). As expected, prominent GFP fluorescence from the rbd-1 reporter gene is seen at the beginning of the morphogenesis stage, but not at the gastrulation stage, and persists with a gradual increase throughout subsequent embryogenesis. Interestingly, FIB-1, a C.elegans homolog of the yeast Nop1p, which is an essential component of U3 snoRNP (14–16), shows a very similar expression pattern to RBD-1 during C.elegans embryogenesis (Fig. 1D).
Figure 1. Expression of RBD-1 in C.elegans during development. (A) Western blot analysis of RBD-1 was performed with affinity-purified anti-RBD-1 antibodies. RBD-1 is expressed at a low level during embryogenesis and at a high level during post-embryonic development. Cell extracts were prepared from synchronized populations of wild-type animals. Equal amounts of total protein (10 μg) were electrophoresed on a 10% SDS–polyacrylamide gel and analyzed by western blotting with anti-RBD-1 antibody. Em, embryo; L1–L4, larval stages 1–4; Ad, adult. (B) Immunostaining of C.elegans embryos with anti-RBD-1 antibody. Embryos were immunostained with anti-RBD-1 antibody (a and c). The same embryos stained with DAPI are also shown to identify the stages of embryos (b and d). Arrowheads indicate the one-cell embryo. (C) Expression of the rbd-1::gfp transgene during embryogenesis. Temporal changes of GFP fluorescence in a single embryo at the developmental time indicated above (top) and Nomarski views of the same embryo are shown (bottom). (D) Expression of RBD-1 and FIB-1 in C.elegans embryos. Embryos were double-immunostained with anti-RBD-1 (a and c) and anti-FIB-1 antibodies (b and d). Embryos at the one-cell stage (a and b: indicated by arrowheads), at the six- to 30-cell stage (a and b) and at the morphogenesis stage (c and d) are shown.
During post-embryonic development, RBD-1 is expressed ubiquitously in L1 larvae and continues to be expressed until adulthood in both somatic and germline cells (Fig. 2A, a–f). To determine the subcellular localization of RBD-1, we closely inspected intestine cells since they have relatively large nuclei, making it possible to easily discriminate the cell compartments (Fig. 2B). As evidenced by its colocalization with a nucleolar marker protein FIB-1, RBD-1 is localized in the nucleolus. These results show that RBD-1 is a nucleolar protein, like its counterparts in yeast and C.tentans, and is expressed ubiquitously after the late embryonic stage.
Figure 2. Expression of RBD-1 during post-embryonic development. (A) Immunostaining views with anti-RBD-1 antibody of an L1 larva (a) and the gonad (c) and posterior region (e) of an adult hermaphrodite. The same samples were stained with DAPI to show nuclei (b, d and f). (B) Nucleolar localization of RBD-1 in adult intestine cells. Intestine cells were immunostained with anti-RBD-1 (a) and anti-FIB-1 (b) antibodies. The same sample was stained with DAPI to show nuclei (c). The nuclear edges are outlined with a dashed line. Germline cells are also seen at the left bottom part of each panel.
RBD-1 is essential for C.elegans development
To determine the possible function of RBD-1 during development of C.elegans, RNAi by injection was performed using adult hermaphrodites, and the phenotypes of the F1 progeny from the injected hermaphrodites were analyzed. RNAi of rbd-1 has no visible effects on embryogenesis, but causes various abnormalities during post-embryonic development (Fig. 3). After hatching, all F1 progeny exhibit growth retardation (Gro: 100%, n = 870). Approximately half of them show severe larval phenotypes such as larval lethality or arrest (Lvl or Lva: 24.8%) and defective molting (Mlt: 20.7%, Fig. 3B), resulting in larval death. The remaining progeny grow to adulthood but show abnormal gonad formation (Gon: 24.9%, Fig. 3C) and protruded vulva (Pvl: 12.9%, Fig. 3D).
Figure 3. Typical phenotypes observed in rbd-1(RNAi) animals. Nomarski (A–D) and scanning electron microscopic (E–H) views of rbd-1(RNAi) and wild-type animals. RNAi was performed by injection. Defective molting (B, Mlt), abnormal gonad formation (C, Gon) and protruded vulva (D, Pvl) are seen in rbd-1(RNAi) animals. Wild-type vulva is shown (A, arrowhead). Various abnormal phenotypes in the alae structure are seen on the body surface of rbd-1(RNAi) animals (F–H). Wild-type alae are shown (E).
In addition, most of the rbd-1(RNAi) adult hermaphrodites show malformation of the surface cuticle structure. In particular, the alae, which are protruding ridges formed over each lateral row of hypodermal seam cells, are abnormal in most rbd-1(RNAi) hermaphrodites. In wild-type animals, three lines of alae are seen along each lateral side of the animal (Fig. 3E). In contrast, in rbd-1(RNAi) hermaphrodites, the alae were disconnected at many points, increase in number or are severely deformed (Fig. 3F–H). These observations show that RBD-1 is required for various developmental processes in C.elegans.
Inhibition of 40S ribosomal subunit formation by RBD-1 depletion
It has been reported that depletion of Mrd1p reduces 18S rRNA synthesis and the formation of 40S ribosomal subunits in yeast (10). To test whether RBD-1 is also involved in ribosomal biogenesis, we performed RNAi of rbd-1 by soaking to deplete RBD-1 in C.elegans and analyzed the ribosome profile in rbd-1(RNAi) animals. The level of RBD-1 decreases significantly and concomitantly the level of 18S rRNA decreases to 60% as compared with the wild-type level, whereas 26S rRNA is not affected (Fig. 4A–C). Simultaneously, depletion of RBD-1 causes a prominent change in the ribosome profile in a sucrose density gradient (Fig. 4D). As expected, the formation of 40S subunits is severely inhibited in rbd-1(RNAi) animals. Accordingly, the level of 80S ribosome (a complex of 40S and 60S ribosomal subunits) decreases significantly in such animals, accompanied by a relative increase of the level of 60S ribosomal subunits. Thus, we concluded that RBD-1 is essential for ribosome biogenesis through 18S rRNA synthesis in C.elegans, like Mrd1p in yeast.
Figure 4. Inhibition of 18S rRNA synthesis by RNAi of rbd-1. (A) RBD-1 was significantly reduced in rbd-1(RNAi) animals. RNAi was performed by soaking. Equal amounts of total protein (10 μg) from mixed-stage wild-type (WT) and rbd-1(RNAi) animals were electrophoresed on a 10% SDS–polyacrylamide gel and analyzed by western blotting with anti-RBD-1 antibody. The Coomassie staining pattern of the same samples is shown below. (B) Synthesis of 18S rRNA, but not 26S rRNA, is reduced by RNAi of rbd-1. Equal amounts of total RNA (1 μg) from wild-type (WT) and rbd-1(RNAi) animals were electrophoresed on a 1% denaturing agarose gel and stained with ethidium bromide. (C) The relative ratio of the amounts of 18S rRNA to 26S rRNA was calculated by quantification of the rRNA bands in the 4% denaturing acrylamide gel stained with toluidine blue using the NIH image program. (D) Reduction of 40S ribosomal subunit and 80S ribosome and increase of 60S ribosomal subunit in an rbd-1(RNAi) animal. Ribosome profiles of the extracts from wild-type (WT) and rbd-1(RNAi) animals were analyzed by 10–30% sucrose density gradient centrifugation.
Pre-rRNA processing in C.elegans
In many organisms, mature 18S, 5.8S and 28S rRNAs are generated from a single pre-rRNA by multiple processing events that remove the external transcribed sequence (ETS) and internal transcribed sequence (ITS). However, there was no information about C.elegans pre-rRNA processing so far. Therefore, we decided to examine the outline of the pre-rRNA processing pathway in C.elegans by northern blot analysis using RNA probes which are specific to distinct regions of the primary rRNA transcript (Fig. 5). Probes 1 and 2 correspond to the positions within the putative 5' ETS region. The probe 1 region includes a predicted TATA-like sequence for transcription by RNA polymerase I (Fig. 5C, boxed sequence). Since RNA polymerase I transcription initiates just downstream of TATA-like sequences (26), it is expected that probe 1 hybridizes to the 5' end of the primary pre-rRNA. On the other hand, probe 2 hybridizes with the region just before the 5' end of 18S rRNA (Fig. 5A, site I). These two probes detect the same three kinds of rRNA intermediates a, b and d (Fig. 5B, lanes 1 and 2). Judging from the size, the largest band a corresponds to the pre-rRNA containing 18S, 5.8S and 26S rRNAs. Since both bands b and d are detected with a probe for the 18S rRNA coding region (data not shown), these RNA species are intermediates for 18S rRNA. Considering that there is no difference between the bands detected with probes 1 and 2, it is likely that there is no major processing site within the 5' ETS region of C.elegans pre-rRNA, although we could not exclude the possibility that the probe 1 region contains an additional processing site.
Figure 5. Examination of the pre-rRNA processing pathway in C.elegans. (A) Schematic representation of the C.elegans genomic region encoding rRNA genes, the primary pre-rRNA, its processing intermediates and mature rRNAs. Closed boxes indicate 18S, 5.8S and 26S rRNA regions. The cleavage sites (I–VIII) are shown on the primary pre-rRNA. Positions of the specific probes 1–9 used for northern blot analysis are shown below the rRNA genomic region. (B) Identification of pre-rRNA and its processing intermediates in wild-type C.elegans. Equal amounts of total RNA (4 μg) were electrophoresed on a 1.2% denaturing agarose gel and were analyzed by northern blotting using the specific probes indicated above. (C) Nucleotide sequence of the putative boundary of the 3' and 5' ETS regions. The probe 9 and 1 regions are indicated below. Two TATA-like sequences (boxed) and a T-rich sequence (underlined) are shown.
To examine the processing sites within the ITS region, we used four probes (Fig. 5B, lanes 3–6). Probes 3–5 correspond to the positions between the 3' end of 18S rRNA (site II) and the 5' end of 5.8S (site V). Probe 6 corresponds to the position immediately downstream of the 3' end of 5.8S rRNA (site VI). Probe 3 detects bands a, b and d, as do probes 1 and 2, confirming that there is no major processing site in the 5' ETS region. Probe 4 detects bands a, d and also a new c', but not band b. Probe 5 detects bands a and c', but not bands b and d. Probe 6 detects bands a and c' and a new band c. Considering the sizes of these bands, bands b and d correspond to the intermediates for 18S rRNA with different 3' ends, band c' corresponds to the intermediate for 5.8S and 26S rRNAs, and band c corresponds to the intermediate for 26S rRNA. These results indicate that there are at least two processing sites in the ITS1 region: one (site III) lies between site II and the 5' end of the probe 4 region, and the other (site IV) within the probe 4 region. It should be noted that the probe 4 region encompasses the site IV.
Finally, we examined the processing sites in the putative 3' ETS region using probes 7–9 which are complementary to the positions between the 3' end of 26S rRNA (site VIII) and the probe 1 region (Fig. 5B, lanes 7–9). Only band a is detected with these three probes, although the signal with probe 9 is very faint. In addition, we noticed that the hybridization signals for band a are relatively weak with probes 7–9 as compared with those with probes 1–6. This is possibly because band a includes at least two pre-rRNAs: the major one lacks the 3' ETS region, and the minor one contains the 3' ETS region, and these two pre-rRNAs cannot be resolved under our gel electrophoresis conditions because of their relatively large sizes. Although we could not identify any processing site in the 3' ETS region in this study, the results suggest that transcription termination of the primary pre-rRNA transcript occurs near the probe 9 region. We do not know the precise sites for transcription initiation and termination of the primary pre-rRNA transcript, but it is likely that both sites exist within the region encompassing the probes 9 and 1, since there are two putative TATA-like sequences and a T-rich sequence within the region which may function for initiation and termination of RNA polymerase I, respectively (Fig. 5C). The results also suggest that the elimination of the 3' ETS region is very rapid since the amount of the 3' ETS-containing pre-rRNA(s) is very low at the steady state, consistent with the previous finding that rRNA processing is initiated by rapid cleavage within the 3' ETS region of the primary transcript (27).
Taken together, the outline of the pre-rRNA processing pathway in C.elegans is depicted in Figure 5A. There are at least eight processing sites in the primary pre-rRNA including both ends of mature rRNAs. We do not exclude the possibility that there are more processing sites for very short-lived rRNA intermediates, especially in the 3' ETS region.
Inhibition of 18S rRNA processing by RBD-1 depletion
Since we have clarified the outline of the pre-rRNA processing pathway in C.elegans, we then compared the processing patterns of pre-rRNAs between the wild-type and rbd-1(RNAi) animals to examine in which steps of pre-rRNA processing RBD-1 is involved (Fig. 6). The most prominent differences are the levels of mature 18S rRNA and the band d intermediate. In rbd-1(RNAi) animals, the amount of mature 18S rRNA significantly decreases, whereas the amount of the band d intermediate significantly increases (Fig. 6B, see probes 1, 3, 4, 18S). This indicates that cleavage at the site III in the band d intermediate is inhibited by RBD-1 depletion and that the site III processing is a rate-limiting step for 18S rRNA synthesis. In contrast, the levels of the band c and c' intermediates are not affected in rbd-1(RNAi) animals, indicating that cleavages at site IV and its downstream sites do not depend upon RBD-1. In addition, the amount of the band a intermediates slightly increases in rbd-1(RNAi) animals. As discussed later, the accumulation of the band a intermediates suggests an additional processing site in the 5' ETS region. Taken together, these findings indicate that RBD-1 is involved at least in the site III processing during C.elegans 18S rRNA synthesis.
Figure 6. Inhibition of the site III cleavage of pre-rRNA by RNAi of rbd-1. (A) Schematic representation of the C.elegans pre-rRNA and its processing intermediates is shown as in Figure 5A. Positions of the specific probes (1, 3, 4, 6 and 18S rRNA) used for northern blot analysis are shown below the pre-rRNA. (B) Comparison of pre-rRNA, its processing intermediates and 18S rRNA between wild-type and rbd-1(RNAi) animals. Equal amounts of total RNA (4 μg) were electrophoresed on a 1.2% denaturing agarose gel and were analyzed by northern blotting using the specific probes indicated below.
DISCUSSION
In this study, we have clarified for the first time the outline of the pre-rRNA processing pathway in C.elegans and shown that a nucleolar RNA-binding protein, RBD-1, is required for 18S rRNA processing in C.elegans. The requirement of RBD-1 for 18S rRNA processing is essentially the same conclusion as that reported by Jin et al. (10) for the yeast counterpart Mrd1p, but we extend the functional conservation of RBD-1 to a multicellular organism. We have also shown that zygotic expression of both RBD-1 and FIB-1 only starts in late embryos and that RBD-1 depletion affects various developmental processes in C.elegans.
Previous studies on yeast pre-rRNA processing have shown that processing of 18S rRNA includes multiple cleavages at the sites A0, A1 and A2, and requires the U3 snoRNP function (27). Interaction of the hinge region of U3 snoRNA with a short segment upstream of the A0 site is required for the cleavages at the sites A0, A1 and A2 (28). In addition, interaction of the Box A sequence of U3 snoRNA with two internal segments of 18S rRNA forms a pseudoknot structure and is required for the cleavages at the sites A1 and A2 (29). These previous findings clearly show that U3 snoRNP binds the segments and promotes the 18S rRNA processing. The sites I, II and III in C.elegans correspond to the A1, D and A2 in yeast, respectively, and the cleavage of these sites apparently depends upon the RBD-1 function, since the site III processing is inhibited in association with the reduction of the level of mature 18S rRNA in rbd-1(RNAi) animals. In this study, we could not identify the cleavage site in C.elegans which corresponds to the yeast A0 site. Such an A0-like site may exist in the 5' ETS region in C.elegans pre-rRNA, especially in the probe 1 region, although we could not examine the possibility in this study. Cleavage of the putative A0-like site may also depend upon the RBD-1 function, on the analogy of the dependence of A0 site cleavage upon the function U3 snoRNP in yeast. If this is the case, the band a accumulation in rbd-1(RNAi) animals (Fig. 6) can be explained as a result from the defect of the A0-like site cleavage. In addition, we have identified two cleavage sites, III and IV, in the C.elegans ITS1 region, which correspond to the yeast A2 and A3 sites, respectively. In contrast, there is only a single A3-like site in the ITS1 region in Xenopus (30), suggesting some diversity in the pre-rRNA cleavage sites even in the higher eukaryotes.
The detailed mechanism of how U3 snoRNP functions in 18S rRNA processing still remains unclear. Recently, more than dozens of core components of U3 snoRNP have been identified in yeast using mass spectrographic analysis (31). Surprisingly, Mrd1p is not a member of the core components of U3 snoRNP, although it is also required for 18S rRNA processing in yeast, as does RBD-1 in C.elegans. Thus, it is conceivable that many extrinsic factors may be required for proper 18S rRNA processing other than U3 snoRNP. The mode of action of Mrd1p and its homologs including RBD-1 is not known at present, but one could speculate that they may transiently associate with pre-rRNA and/or U3 snoRNP, and help the function of U3 snoRNP for 18S rRNA processing, since they contain multiple RNA-binding domains and are required for all U3 snoRNP-involved cleavages.
An important outcome of this study is the clear demonstration of the linkage of ribosome biogenesis with several developmental events. Bj?rk et al. (12) described briefly that RNAi of rbd-1 causes various developmental phenotypes in C.elegans, as we observed in this study. However, they did not show any evidence for the RBD-1 function in 18S rRNA processing in C.elegans. In this respect, our results provide direct evidence for the first time that ribosome biogenesis is involved in developmental regulation in multicellular organisms. Previous studies in bacteria and yeast have demonstrated that cell growth (increase in cell size and number) is correlated closely with an increase in both protein synthesis and ribosome biogenesis . In higher eukaryotes, this correlation is seen in the diminished transcription of rRNAs during quiescence and the subsequent increase after stimulation with growth factors (33). Studies of hypertrophy in vertebrate cardiomyocytes have also linked rRNA and 5S RNA synthesis with control of cell size (34). These studies suggest that regulators of ribosome biogenesis may play an important role in cellular growth control. From this point of view, some of the phenotypes that we observed in rbd-1(RNAi) animals, such as the molting defect and cuticle malformation, seem to be closely related to protein synthesis. During the molting and subsequent growth processes, drastic and rapid changes occur in the nematode surface structures, and thus rapid massive production of proteins should be required for such processes. Accordingly, it appears that an insufficient quantity of 40S ribosomal subunits caused by RBD-1 depletion disturbs such rapid protein synthesis, resulting in severe defects in molting and cuticle formation. Since it is well known that RNAi efficiency varies with the dosage of dsRNA delivered in individual animals, target genes and cell types (35,36), it is not surprising that various phenotypes emerge upon RNAi of rbd-1. More complete inhibition of 18S rRNA synthesis by efficient RNAi of rbd-1 seems to lead to fatal effects, such as larval arrest and lethality that we observed in this study. Consistent with this, RNAi of fib-1 showed only such severe phenotypes (our unpublished data). An alternative explanation for the phenotypes caused by RNAi of rbd-1 is that RBD-1 may have a specific role for cuticle formation in addition to the role for 18S rRNA processing, like yeast Nop7p and Nop15p that are not only required for 28S rRNA processing but also have critical functions in DNA replication and cytokinesis, respectively (37,38).
Another important finding of this study is that RBD-1 is not present in early embryos, although 18S rRNA synthesis should require RBD-1. We have also shown similar limitation of the expression to late embryogenesis and thereafter for a component of U3 snoRNP, FIB-1, which is thought to be required for 18S rRNA synthesis in C.elegans. These observations imply that processing of 18S rRNA may be repressed in early embryos. It is likely that only the maternal stock of ribosomes is utilized for protein synthesis in early embryos and that such maternal ribosomes would have been fully consumed in late embryos. Thus, zygotic RBD-1 should start to be expressed in late embryos to newly synthesize 18S rRNA. Interestingly, it was reported that zygotic transcription of pre-rRNA starts as early as the one-cell stage and persists thereafter in C.elegans (39). Thus, the apparent absence of RBD-1 and FIB-1 in early embryos raises the possibility for the first time that transcription and processing of pre-rRNA are uncoupled during early embryogenesis.
ACKNOWLEDGEMENTS
We thank Dr Andrew Fire for the GFP expression vector, Dr Yuji Kohara for C.elegans EST clones, Dr John Aris for anti-FIB-1 antibody, and Dr Elizabeth Nakajima for reading the manuscript. This work was supported in part by grants from MEXT to H.S.
REFERENCES
Scheer,U. and Hock,R. (1999) Structure and function of the nucleolus. Curr. Opin. Cell Biol., 11, 385–390.
Maxwell,E.S. and Fournier,M.J. (1995) The small nucleolar RNAs. Annu. Rev. Biochem., 64, 897–934.
Smith,C.M. and Steitz,J.A. (1997) Sno storm in the nucleolus: new roles for myriad small RNPs. Cell, 89, 669–672.
Maden,B.E. and Hughes,J.M. (1997) Eukaryotic ribosomal RNA: the recent excitement in the nucleotide modification problem. Chromosoma, 105, 391–400.
Decatur,W.A. and Fournier,M.J. (2003) RNA-guided nucleotide modification of ribosomal and other RNAs. J. Biol. Chem., 278, 695–698.
van Nues,R.W., Venema,J., Rientjes,J.M., Dirks-Mulder,A. and Raue,H.A. (1995) Processing of eukaryotic pre-rRNA: the role of the transcribed spacers. Biochem. Cell Biol., 73, 789–801.
Fromont-Racine,M., Senger,B., Saveanu,C. and Fasiolo,F. (2003) Ribosome assembly in eukaryotes. Gene, 313, 17–42.
Ban,N., Nissen,P., Hansen,J., Moore,P.B. and Steitz,T.A. (2000) The complete atomic structure of the large ribosomal subunit at 2.4 ? resolution. Science, 289, 905–920.
Spahn,C.M., Beckmann,R., Eswar,N., Penczek,P.A., Sali,A., Blobel,G. and Frank,J. (2001) Structure of the 80S ribosome from Saccharomyces cerevisiae–tRNA-ribosome and subunit–subunit interactions. Cell, 107, 373–386.
Jin,S.B., Zhao,J., Bj?rk,P., Schmekel,K., Ljungdahl,P.O. and Wieslander,L. (2002) Mrd1p is required for processing of pre-rRNA and for maintenance of steady-state levels of 40 S ribosomal subunits in yeast. J. Biol. Chem., 277, 18431–18439.
Gavin,A.C., Bosche,M., Krause,R., Grandi,P., Marzioch,M., Bauer,A., Schultz,J., Rick,J.M., Michon,A.M., Cruciat,C.M. et al. (2002) Functional organization of the yeast proteome by systematic analysis of protein complexes. Nature, 415, 141–147.
Bj?rk,P., Bauren,G., Jin,S., Tong,Y.G., Burglin,T.R., Hellman,U. and Wieslander,L. (2002) A novel conserved RNA-binding domain protein, RBD-1, is essential for ribosome biogenesis. Mol. Biol. Cell, 13, 3683–3695.
Mayer,A.N. and Fishman,M.C. (2003) Nil per os encodes a conserved RNA recognition motif protein required for morphogenesis and cytodifferentiation of digestive organs in zebrafish. Development, 130, 3917–3928.
MacQueen,A.J. and Villeneuve,A.M. (2001) Nuclear reorganization and homologous chromosome pairing during meiotic prophase require C. elegans chk-2. Genes Dev., 15, 1674–1687.
Tollervey,D., Lehtonen,H., Carmo-Fonseca,M. and Hurt,E.C. (1991) The small nucleolar RNP protein NOP1 (fibrillarin) is required for pre-rRNA processing in yeast. EMBO J., 10, 573–583.
Tollervey,D., Lehtonen,H., Jansen,R., Kern,H. and Hurt,E.C. (1993) Temperature-sensitive mutations demonstrate roles for yeast fibrillarin in pre-rRNA processing, pre-rRNA methylation and ribosome assembly. Cell, 72, 443–457.
Epstein,H.F. and Shakes,D.C. (eds) (1995) Caenorhabditis elegans: Modern Biological Analysis of an Organism. Academic Press, San Diego, CA.
Brenner,S. (1974) The genetics of Caenorhabditis elegans. Genetics, 77, 71–94.
Ellis,R.E., Sulston,J.E. and Coulson,A.R. (1986) The rDNA of C. elegans: sequence and structure. Nucleic Acids Res., 14, 2345–2364.
Fujita,M., Takasaki,T., Nakajima,N., Kawano,T., Shimura,Y. and Sakamoto,H. (2002) MRG-1, a mortality factor-related chromodomain protein, is required maternally for primordial germ cells to initiate mitotic proliferation in C. elegans. Mech. Dev., 114, 61–69.
Hope,I. (1999) C. elegans: A Practical Approach. Oxford University Press, Oxford.
Aris,J.P. and Blobel,G. (1988) Identification and characterization of a yeast nucleolar protein that is similar to a rat liver nucleolar protein. J. Cell Biol., 107, 17–31.
Ahnn,J. and Fire,A. (1994) A screen for genetic loci required for body-wall muscle development during embryogenesis in Caenorhabditis elegans. Genetics, 137, 483–498.
Epstein,H.F., Berliner,G.C., Casey,D.L. and Ortiz,I. (1988) Purified thick filaments from the nematode Caenorhabditis elegans: evidence for multiple proteins associated with core structures. J. Cell Biol., 106, 1985–1995.
Small,E.B. and Marszalek,D.S. (1969) Scanning electron microscopy of fixed, frozen and dried protozoa. Science, 163, 1064–1065.
Paule,M.R. and White,R.J. (2000) Survey and summary: transcription by RNA polymerases I and III. Nucleic Acids Res., 28, 1283–1298.
Venema,J. and Tollervey,D. (1999) Ribosome synthesis in Saccharomyces cerevisiae. Annu. Rev. Genet., 33, 261–311.
Beltrame,M. and Tollervey,D. (1995) Base pairing between U3 and the pre-ribosomal RNA is required for 18S rRNA synthesis. EMBO J., 14, 4350–4356.
Sharma,K. and Tollervey,D. (1999) Base pairing between U3 small nucleolar RNA and the 5' end of 18S rRNA is required for pre-rRNA processing. Mol. Cell. Biol., 19, 6012–6019.
Borovjagin,A.V. and Gerbi,S.A. (2001) Xenopus U3 snoRNA GAC-Box A' and Box A sequences play distinct functional roles in rRNA processing. Mol. Cell. Biol., 21, 6210–6221.
Dragon,F., Gallagher,J.E., Compagnone-Post,P.A., Mitchell,B.M., Porwancher,K.A., Wehner,K.A., Wormsley,S., Settlage,R.E., Shabanowitz,J., Osheim,Y. et al. (2002) A large nucleolar U3 ribonucleoprotein required for 18S ribosomal RNA biogenesis. Nature, 417, 967–970.
Nomura,M., Gourse,R. and Baughman,G. (1984) Regulation of the synthesis of ribosomes and ribosomal components. Annu. Rev. Biochem., 53, 75–117.
Johnson,L.F., Williams,J.G., Abelson,H.T., Green,H. and Penman,S. (1975) Changes in RNA in relation to growth of the fibroblast. III. Posttranscriptional regulation of mRNA formation in resting and growing cells. Cell, 4, 69–75.
McDermott,P.J., Rothblum,L.I., Smith,S.D. and Morgan,H.E. (1989) Accelerated rates of ribosomal RNA synthesis during growth of contracting heart cells in culture. J. Biol. Chem., 264, 18220–18227.
Fraser,A.G., Kamath,R.S., Zipperlen,P., Martinez-Campos,M., Sohrmann,M. and Ahringer,J. (2000) Functional genomic analysis of C. elegans chromosome I by systematic RNA interference. Nature, 408, 325–330.
Gonczy,P., Echeverri,C., Oegema,K., Coulson,A., Jones,S.J., Copley,R.R., Duperon,J., Oegema,J., Brehm,M., Cassin,E. et al. (2000) Functional genomic analysis of cell division in C. elegans using RNAi of genes on chromosome III. Nature, 408, 331–336.
Du,Y.C. and Stillman,B. (2002) Yph1p, an ORC-interacting protein: potential links between cell proliferation control, DNA replication and ribosome biogenesis. Cell, 109, 835–848.
Oeffinger,M. and Tollervey,D. (2003) Yeast Nop15p is an RNA-binding protein required for pre-rRNA processing and cytokinesis. EMBO J., 22, 6573–6583.
Seydoux,G. and Dunn,M.A. (1997) Transcriptionally repressed germ cells lack a subpopulation of phosphorylated RNA polymerase II in early embryos of Caenorhabditis elegans and Drosophila melanogaster. Development, 124, 2191–2201.(Eiko Saijou, Toshinobu Fujiwara, Toshino)
*To whom correspondence should be addressed. Tel: +81 78 803 5796; Fax: +81 78 803 5720; Email: hsaka@kobe-u.ac.jp
ABSTRACT
RBD-1 is the Caenorhabditis elegans homolog of Mrd1p, which was recently shown to be required for 18S ribosomal RNA (rRNA) processing in yeast. To gain insights into the relationship between ribosome biogenesis and the development of multicellular organisms, we examined the expression and function of RBD-1. Maternal RBD-1 in the fertilized egg disappears immediately after cleavage starts, whereas zygotic RBD-1 first appears in late embryos and is localized in the nucleolus in most cells, although zygotic transcription of pre-rRNA is known to be initiated as early as the one-cell stage. RNA interference of the rbd-1 gene severely inhibits the processing of 18S rRNA in association with various developmental abnormalities, indicating its essential role in pre-rRNA processing and development in C.elegans. These results provide evidence for the linkage between ribosome biogenesis and the control of development and imply unexpected uncoupling of transcription and processing of pre-rRNA in early C.elegans embryos.
INTRODUCTION
In all eukaryotic cells, ribosome biogenesis is a very integrated process that occurs in the nucleolus (1). The nucleolus of the eukaryotic cell is densely packed with pre-ribosomal RNAs (pre-rRNAs) and a number of small nucleolar RNAs (snoRNAs) that are essential components involved in pre-rRNA processing . Maturation of rRNAs is achieved by post-transcriptional events including methylation, pseudouridylation and multiple cleavages, resulting in the generation of mature 18S, 5.8S and 25–28S rRNA species in all eukaryotic cells (4,5). These processes are accomplished via various cis-acting elements within pre-rRNAs (6) and a number of non-ribosomal trans-acting factors (7). Although the outline of the pre-rRNA processing pathway is roughly understood and the structures of two ribosomal subunits have recently been solved by X-ray crystallography and cryoelectron microscopy (8,9), little is understood about how ribosome biogenesis is controlled in eukaryotic cells and whether it is linked to more complex biological events, such as developmental processes in multicellular organisms.
Mrd1p was initially given this name because it contains multiple copies of an RNA-binding domain called RBD, also known as RRM (RNA recognition motif), and was recently shown to be a member of the group of non-ribosomal proteins that are involved in pre-rRNA processing in the yeast Saccharomyces cerevisiae (10). In yeast, Mrd1p is essential for viability and its depletion leads to a decrease in the levels of mature 18S rRNA and 40S ribosome and concomitant accumulation of 18S rRNA precursors, whereas 25S rRNA processing is not affected. Since Mrd1p can associate with pre-rRNA and two components of U3 small nucleolar ribonucleoprotein complex (snoRNP), Mrd1p is also likely to be a component of U3 snoRNP, which is known to be required for 18S rRNA processing (11). Since Mrd1p homologs are found in a wide range of metazoans, the homologs may also be involved in pre-rRNA processing. Indeed, in the dipteran Chironomus tentans, a homologous protein (Ct-RBD-1) is localized mainly in the nucleus and is likely to be involved in 18S rRNA processing (12). Interestingly, RNA interference (RNAi)-mediated depletion of the Caenorhabditis elegans homolog RBD-1 (according to its gene name in the database) and a truncation mutation in the zebrafish homolog Npo causes various developmental abnormalities (12,13). These observations imply that there may be the linkage between ribosome biogenesis and developmental events in multicellular organisms. However, the question as to whether the developmental abnormalities are correlated with defects in ribosome biogenesis still remains to be examined, since there is no direct evidence for the involvement of RBD-1 or Npo in pre-rRNA processing.
To address the question of this possible correlation, we examined in detail the role of RBD-1 in both ribosome biogenesis and development in C.elegans. We found that RBD-1 depletion by RNAi inhibits processing of 18S rRNA and subsequent formation of the 40S ribosomal subunit, and causes various developmental abnormalities simultaneously, indicating its essential role in pre-rRNA processing and development in C.elegans. We also found that RBD-1 is localized in the nucleolus, like Mrd1p and Ct-RBD-1 and that its zygotic expression starts in late embryos, although transcription of pre-rRNA by RNA polymerase I is known to start as early as the one-cell stage. We observed a similar expression pattern during embryogenesis for a component of U3 snoRNP, FIB-1 (14), a C.elegans homolog of the yeast Nop1p, which is an essential factor for 18S rRNA processing (15,16). These results provide evidence for the linkage between ribosome biogenesis and developmental events in multicellular organisms and imply that transcription and processing of pre-rRNA may be regulated differentially during early embryogenesis in C.elegans.
MATERIALS AND METHODS
Construction of GFP reporter gene fusion
The reporter construct expressing green fluorescent protein (GFP) under the control of the rbd-1 promoter was made as follows. The promoter region was PCR-amplified using C.elegans genomic DNA with the forward primer (–1931): TTG CAT GCT AAT GGT GAG TAG CTT TAT CCT GAA ATA AGA ACA C, and the reverse primer (+30): GGT CTA GAG CTT GTT TTT GAC AAT TAA TCG AGT TGT CAT G (the numbers in parentheses correspond to the nucleotide position relative to the first nucleotide of the rbd-1 open-reading frame). This genomic fragment was fused in-frame to a promoterless GFP vector, pPD95.77 (provided by Dr A. Fire). Microinjection of the resulting plasmid into C.elegans worms (Bristol type N2) was performed as described (17). Worm breeding and handling were conducted as described (18).
RNA interference
Sense and antisense RNAs were synthesized in vitro from yk417f6 cDNA which encodes RBD-1 (provided by Dr Y. Kohara). Both RNAs were annealed to form a double-stranded RNA (dsRNA). For RNAi, L4 hermaphrodites were soaked in 4 μl of dsRNA solution (2 μg/μl) for 16–24 h or dsRNA (1 μg/μl) was injected into the gonad arms of young adult hermaphrodites.
Northern blot analysis
Total RNA from wild-type and rbd-1(RNAi) animals were extracted with an RNA extraction kit (Micro-to-Midi Total RNA Purification System; Invitrogen). Approximately 4 μg of total RNA per lane were resolved on a 1.2% formaldehyde-containing agarose gel, transferred onto a nylon membrane (Roche Diagnostics), and hybridized with DIG-labeled antisense RNA probes. The antisense probes 1–9 and 18S probe correspond to the positions of nucleotides 511–609, 846–933, 2736–2791, 2969–3036, 3050–3157, 3342–3427, 1–210, 311–410, 411–510 and 1261–1677, respectively, of the C.elegans rDNA repeat (19).
Antibody preparation, western blot analysis and immunostaining
An rbd-1 cDNA fragment of 972 bp was subcloned into pGEX-4T3 (Amersham Biosciences) and the fusion protein GST-RBD-1 was over-expressed in the XL-2 blue Escherichia coli strain. GST-RBD-1 was affinity-purified using glutathione–Sepharose (Amersham Biosciences) and used to raise rabbit polyclonal antibodies. The anti-RBD-1 antibody was affinity-purified and used for western blot analyses (1:1000–1:2000 dilutions) as described previously (20). Isolation of worms at specific developmental stages was performed as described (21). Wild-type worms were immunostained using affinity-purified anti-RBD-1 antibodies (1:100 dilution) or anti-FIB-1 antibodies (22) (1:100 dilution) as described (23). Embryos and larvae were permeated for staining by the freeze-crack method and fixed with methanol/acetone according to standard procedures (24).
Sucrose gradient centrifugation
Wild type and rbd-1(RNAi) worms, respectively, were mixed with Lysing Matrix D (Bio101) and homogenized in buffer A by using a FastPrep homogenizator (Bio101). The lysates were loaded on a linear sucrose gradient (10–30%) in buffer B and centrifuged at 4°C in a Beckman MLS50 rotor at 40 000 r.p.m. for 120 min, followed by fractionation and monitoring at 260 nm.
Scanning electron microscopy
Worms were washed in M9 buffer before fixation in Parducz fixative (25). After extensive washes, samples were dehydrated in ethanol followed by amylacetate. The samples were processed in a critical-point drier, mounted and observed using a Hitachi S-2150N scanning electron microscope.
RESULTS
Expression and subcellular localization of RBD-1
We examined the expression pattern of RBD-1 at various developmental stages. To measure the levels of RBD-1 expression, we used synchronized populations at each developmental stage. Western blot analyses with anti-RBD-1 antibody reveal that RBD-1 is expressed constantly during the four larval stages and the adult stage, whereas the RBD-1 expression level in embryos is significantly lower than that at other stages (Fig. 1A). This suggests that RBD-1 expression is downregulated during embryogenesis. Consistently, immunostaining of embryos shows that RBD-1 expression is limited to late embryonic stages (Fig. 1B). Interestingly, a small amount of maternally supplied RBD-1 is also detectable in the fertilized egg (Fig. 1B, a and b: arrowhead). In contrast to zygotic RBD-1, maternal RBD-1 is seen as a number of granules in the cytoplasm and disappears shortly after cleavage begin, suggesting that maternal RBD-1 may be degraded rapidly in early embryos. To confirm the downregulation of RBD-1 in early embryos, we utilized a transgenic strain carrying the GFP gene under the control of the native promoter for the rbd-1 gene and monitored GFP fluorescence from a single embryo (Fig. 1C). As expected, prominent GFP fluorescence from the rbd-1 reporter gene is seen at the beginning of the morphogenesis stage, but not at the gastrulation stage, and persists with a gradual increase throughout subsequent embryogenesis. Interestingly, FIB-1, a C.elegans homolog of the yeast Nop1p, which is an essential component of U3 snoRNP (14–16), shows a very similar expression pattern to RBD-1 during C.elegans embryogenesis (Fig. 1D).
Figure 1. Expression of RBD-1 in C.elegans during development. (A) Western blot analysis of RBD-1 was performed with affinity-purified anti-RBD-1 antibodies. RBD-1 is expressed at a low level during embryogenesis and at a high level during post-embryonic development. Cell extracts were prepared from synchronized populations of wild-type animals. Equal amounts of total protein (10 μg) were electrophoresed on a 10% SDS–polyacrylamide gel and analyzed by western blotting with anti-RBD-1 antibody. Em, embryo; L1–L4, larval stages 1–4; Ad, adult. (B) Immunostaining of C.elegans embryos with anti-RBD-1 antibody. Embryos were immunostained with anti-RBD-1 antibody (a and c). The same embryos stained with DAPI are also shown to identify the stages of embryos (b and d). Arrowheads indicate the one-cell embryo. (C) Expression of the rbd-1::gfp transgene during embryogenesis. Temporal changes of GFP fluorescence in a single embryo at the developmental time indicated above (top) and Nomarski views of the same embryo are shown (bottom). (D) Expression of RBD-1 and FIB-1 in C.elegans embryos. Embryos were double-immunostained with anti-RBD-1 (a and c) and anti-FIB-1 antibodies (b and d). Embryos at the one-cell stage (a and b: indicated by arrowheads), at the six- to 30-cell stage (a and b) and at the morphogenesis stage (c and d) are shown.
During post-embryonic development, RBD-1 is expressed ubiquitously in L1 larvae and continues to be expressed until adulthood in both somatic and germline cells (Fig. 2A, a–f). To determine the subcellular localization of RBD-1, we closely inspected intestine cells since they have relatively large nuclei, making it possible to easily discriminate the cell compartments (Fig. 2B). As evidenced by its colocalization with a nucleolar marker protein FIB-1, RBD-1 is localized in the nucleolus. These results show that RBD-1 is a nucleolar protein, like its counterparts in yeast and C.tentans, and is expressed ubiquitously after the late embryonic stage.
Figure 2. Expression of RBD-1 during post-embryonic development. (A) Immunostaining views with anti-RBD-1 antibody of an L1 larva (a) and the gonad (c) and posterior region (e) of an adult hermaphrodite. The same samples were stained with DAPI to show nuclei (b, d and f). (B) Nucleolar localization of RBD-1 in adult intestine cells. Intestine cells were immunostained with anti-RBD-1 (a) and anti-FIB-1 (b) antibodies. The same sample was stained with DAPI to show nuclei (c). The nuclear edges are outlined with a dashed line. Germline cells are also seen at the left bottom part of each panel.
RBD-1 is essential for C.elegans development
To determine the possible function of RBD-1 during development of C.elegans, RNAi by injection was performed using adult hermaphrodites, and the phenotypes of the F1 progeny from the injected hermaphrodites were analyzed. RNAi of rbd-1 has no visible effects on embryogenesis, but causes various abnormalities during post-embryonic development (Fig. 3). After hatching, all F1 progeny exhibit growth retardation (Gro: 100%, n = 870). Approximately half of them show severe larval phenotypes such as larval lethality or arrest (Lvl or Lva: 24.8%) and defective molting (Mlt: 20.7%, Fig. 3B), resulting in larval death. The remaining progeny grow to adulthood but show abnormal gonad formation (Gon: 24.9%, Fig. 3C) and protruded vulva (Pvl: 12.9%, Fig. 3D).
Figure 3. Typical phenotypes observed in rbd-1(RNAi) animals. Nomarski (A–D) and scanning electron microscopic (E–H) views of rbd-1(RNAi) and wild-type animals. RNAi was performed by injection. Defective molting (B, Mlt), abnormal gonad formation (C, Gon) and protruded vulva (D, Pvl) are seen in rbd-1(RNAi) animals. Wild-type vulva is shown (A, arrowhead). Various abnormal phenotypes in the alae structure are seen on the body surface of rbd-1(RNAi) animals (F–H). Wild-type alae are shown (E).
In addition, most of the rbd-1(RNAi) adult hermaphrodites show malformation of the surface cuticle structure. In particular, the alae, which are protruding ridges formed over each lateral row of hypodermal seam cells, are abnormal in most rbd-1(RNAi) hermaphrodites. In wild-type animals, three lines of alae are seen along each lateral side of the animal (Fig. 3E). In contrast, in rbd-1(RNAi) hermaphrodites, the alae were disconnected at many points, increase in number or are severely deformed (Fig. 3F–H). These observations show that RBD-1 is required for various developmental processes in C.elegans.
Inhibition of 40S ribosomal subunit formation by RBD-1 depletion
It has been reported that depletion of Mrd1p reduces 18S rRNA synthesis and the formation of 40S ribosomal subunits in yeast (10). To test whether RBD-1 is also involved in ribosomal biogenesis, we performed RNAi of rbd-1 by soaking to deplete RBD-1 in C.elegans and analyzed the ribosome profile in rbd-1(RNAi) animals. The level of RBD-1 decreases significantly and concomitantly the level of 18S rRNA decreases to 60% as compared with the wild-type level, whereas 26S rRNA is not affected (Fig. 4A–C). Simultaneously, depletion of RBD-1 causes a prominent change in the ribosome profile in a sucrose density gradient (Fig. 4D). As expected, the formation of 40S subunits is severely inhibited in rbd-1(RNAi) animals. Accordingly, the level of 80S ribosome (a complex of 40S and 60S ribosomal subunits) decreases significantly in such animals, accompanied by a relative increase of the level of 60S ribosomal subunits. Thus, we concluded that RBD-1 is essential for ribosome biogenesis through 18S rRNA synthesis in C.elegans, like Mrd1p in yeast.
Figure 4. Inhibition of 18S rRNA synthesis by RNAi of rbd-1. (A) RBD-1 was significantly reduced in rbd-1(RNAi) animals. RNAi was performed by soaking. Equal amounts of total protein (10 μg) from mixed-stage wild-type (WT) and rbd-1(RNAi) animals were electrophoresed on a 10% SDS–polyacrylamide gel and analyzed by western blotting with anti-RBD-1 antibody. The Coomassie staining pattern of the same samples is shown below. (B) Synthesis of 18S rRNA, but not 26S rRNA, is reduced by RNAi of rbd-1. Equal amounts of total RNA (1 μg) from wild-type (WT) and rbd-1(RNAi) animals were electrophoresed on a 1% denaturing agarose gel and stained with ethidium bromide. (C) The relative ratio of the amounts of 18S rRNA to 26S rRNA was calculated by quantification of the rRNA bands in the 4% denaturing acrylamide gel stained with toluidine blue using the NIH image program. (D) Reduction of 40S ribosomal subunit and 80S ribosome and increase of 60S ribosomal subunit in an rbd-1(RNAi) animal. Ribosome profiles of the extracts from wild-type (WT) and rbd-1(RNAi) animals were analyzed by 10–30% sucrose density gradient centrifugation.
Pre-rRNA processing in C.elegans
In many organisms, mature 18S, 5.8S and 28S rRNAs are generated from a single pre-rRNA by multiple processing events that remove the external transcribed sequence (ETS) and internal transcribed sequence (ITS). However, there was no information about C.elegans pre-rRNA processing so far. Therefore, we decided to examine the outline of the pre-rRNA processing pathway in C.elegans by northern blot analysis using RNA probes which are specific to distinct regions of the primary rRNA transcript (Fig. 5). Probes 1 and 2 correspond to the positions within the putative 5' ETS region. The probe 1 region includes a predicted TATA-like sequence for transcription by RNA polymerase I (Fig. 5C, boxed sequence). Since RNA polymerase I transcription initiates just downstream of TATA-like sequences (26), it is expected that probe 1 hybridizes to the 5' end of the primary pre-rRNA. On the other hand, probe 2 hybridizes with the region just before the 5' end of 18S rRNA (Fig. 5A, site I). These two probes detect the same three kinds of rRNA intermediates a, b and d (Fig. 5B, lanes 1 and 2). Judging from the size, the largest band a corresponds to the pre-rRNA containing 18S, 5.8S and 26S rRNAs. Since both bands b and d are detected with a probe for the 18S rRNA coding region (data not shown), these RNA species are intermediates for 18S rRNA. Considering that there is no difference between the bands detected with probes 1 and 2, it is likely that there is no major processing site within the 5' ETS region of C.elegans pre-rRNA, although we could not exclude the possibility that the probe 1 region contains an additional processing site.
Figure 5. Examination of the pre-rRNA processing pathway in C.elegans. (A) Schematic representation of the C.elegans genomic region encoding rRNA genes, the primary pre-rRNA, its processing intermediates and mature rRNAs. Closed boxes indicate 18S, 5.8S and 26S rRNA regions. The cleavage sites (I–VIII) are shown on the primary pre-rRNA. Positions of the specific probes 1–9 used for northern blot analysis are shown below the rRNA genomic region. (B) Identification of pre-rRNA and its processing intermediates in wild-type C.elegans. Equal amounts of total RNA (4 μg) were electrophoresed on a 1.2% denaturing agarose gel and were analyzed by northern blotting using the specific probes indicated above. (C) Nucleotide sequence of the putative boundary of the 3' and 5' ETS regions. The probe 9 and 1 regions are indicated below. Two TATA-like sequences (boxed) and a T-rich sequence (underlined) are shown.
To examine the processing sites within the ITS region, we used four probes (Fig. 5B, lanes 3–6). Probes 3–5 correspond to the positions between the 3' end of 18S rRNA (site II) and the 5' end of 5.8S (site V). Probe 6 corresponds to the position immediately downstream of the 3' end of 5.8S rRNA (site VI). Probe 3 detects bands a, b and d, as do probes 1 and 2, confirming that there is no major processing site in the 5' ETS region. Probe 4 detects bands a, d and also a new c', but not band b. Probe 5 detects bands a and c', but not bands b and d. Probe 6 detects bands a and c' and a new band c. Considering the sizes of these bands, bands b and d correspond to the intermediates for 18S rRNA with different 3' ends, band c' corresponds to the intermediate for 5.8S and 26S rRNAs, and band c corresponds to the intermediate for 26S rRNA. These results indicate that there are at least two processing sites in the ITS1 region: one (site III) lies between site II and the 5' end of the probe 4 region, and the other (site IV) within the probe 4 region. It should be noted that the probe 4 region encompasses the site IV.
Finally, we examined the processing sites in the putative 3' ETS region using probes 7–9 which are complementary to the positions between the 3' end of 26S rRNA (site VIII) and the probe 1 region (Fig. 5B, lanes 7–9). Only band a is detected with these three probes, although the signal with probe 9 is very faint. In addition, we noticed that the hybridization signals for band a are relatively weak with probes 7–9 as compared with those with probes 1–6. This is possibly because band a includes at least two pre-rRNAs: the major one lacks the 3' ETS region, and the minor one contains the 3' ETS region, and these two pre-rRNAs cannot be resolved under our gel electrophoresis conditions because of their relatively large sizes. Although we could not identify any processing site in the 3' ETS region in this study, the results suggest that transcription termination of the primary pre-rRNA transcript occurs near the probe 9 region. We do not know the precise sites for transcription initiation and termination of the primary pre-rRNA transcript, but it is likely that both sites exist within the region encompassing the probes 9 and 1, since there are two putative TATA-like sequences and a T-rich sequence within the region which may function for initiation and termination of RNA polymerase I, respectively (Fig. 5C). The results also suggest that the elimination of the 3' ETS region is very rapid since the amount of the 3' ETS-containing pre-rRNA(s) is very low at the steady state, consistent with the previous finding that rRNA processing is initiated by rapid cleavage within the 3' ETS region of the primary transcript (27).
Taken together, the outline of the pre-rRNA processing pathway in C.elegans is depicted in Figure 5A. There are at least eight processing sites in the primary pre-rRNA including both ends of mature rRNAs. We do not exclude the possibility that there are more processing sites for very short-lived rRNA intermediates, especially in the 3' ETS region.
Inhibition of 18S rRNA processing by RBD-1 depletion
Since we have clarified the outline of the pre-rRNA processing pathway in C.elegans, we then compared the processing patterns of pre-rRNAs between the wild-type and rbd-1(RNAi) animals to examine in which steps of pre-rRNA processing RBD-1 is involved (Fig. 6). The most prominent differences are the levels of mature 18S rRNA and the band d intermediate. In rbd-1(RNAi) animals, the amount of mature 18S rRNA significantly decreases, whereas the amount of the band d intermediate significantly increases (Fig. 6B, see probes 1, 3, 4, 18S). This indicates that cleavage at the site III in the band d intermediate is inhibited by RBD-1 depletion and that the site III processing is a rate-limiting step for 18S rRNA synthesis. In contrast, the levels of the band c and c' intermediates are not affected in rbd-1(RNAi) animals, indicating that cleavages at site IV and its downstream sites do not depend upon RBD-1. In addition, the amount of the band a intermediates slightly increases in rbd-1(RNAi) animals. As discussed later, the accumulation of the band a intermediates suggests an additional processing site in the 5' ETS region. Taken together, these findings indicate that RBD-1 is involved at least in the site III processing during C.elegans 18S rRNA synthesis.
Figure 6. Inhibition of the site III cleavage of pre-rRNA by RNAi of rbd-1. (A) Schematic representation of the C.elegans pre-rRNA and its processing intermediates is shown as in Figure 5A. Positions of the specific probes (1, 3, 4, 6 and 18S rRNA) used for northern blot analysis are shown below the pre-rRNA. (B) Comparison of pre-rRNA, its processing intermediates and 18S rRNA between wild-type and rbd-1(RNAi) animals. Equal amounts of total RNA (4 μg) were electrophoresed on a 1.2% denaturing agarose gel and were analyzed by northern blotting using the specific probes indicated below.
DISCUSSION
In this study, we have clarified for the first time the outline of the pre-rRNA processing pathway in C.elegans and shown that a nucleolar RNA-binding protein, RBD-1, is required for 18S rRNA processing in C.elegans. The requirement of RBD-1 for 18S rRNA processing is essentially the same conclusion as that reported by Jin et al. (10) for the yeast counterpart Mrd1p, but we extend the functional conservation of RBD-1 to a multicellular organism. We have also shown that zygotic expression of both RBD-1 and FIB-1 only starts in late embryos and that RBD-1 depletion affects various developmental processes in C.elegans.
Previous studies on yeast pre-rRNA processing have shown that processing of 18S rRNA includes multiple cleavages at the sites A0, A1 and A2, and requires the U3 snoRNP function (27). Interaction of the hinge region of U3 snoRNA with a short segment upstream of the A0 site is required for the cleavages at the sites A0, A1 and A2 (28). In addition, interaction of the Box A sequence of U3 snoRNA with two internal segments of 18S rRNA forms a pseudoknot structure and is required for the cleavages at the sites A1 and A2 (29). These previous findings clearly show that U3 snoRNP binds the segments and promotes the 18S rRNA processing. The sites I, II and III in C.elegans correspond to the A1, D and A2 in yeast, respectively, and the cleavage of these sites apparently depends upon the RBD-1 function, since the site III processing is inhibited in association with the reduction of the level of mature 18S rRNA in rbd-1(RNAi) animals. In this study, we could not identify the cleavage site in C.elegans which corresponds to the yeast A0 site. Such an A0-like site may exist in the 5' ETS region in C.elegans pre-rRNA, especially in the probe 1 region, although we could not examine the possibility in this study. Cleavage of the putative A0-like site may also depend upon the RBD-1 function, on the analogy of the dependence of A0 site cleavage upon the function U3 snoRNP in yeast. If this is the case, the band a accumulation in rbd-1(RNAi) animals (Fig. 6) can be explained as a result from the defect of the A0-like site cleavage. In addition, we have identified two cleavage sites, III and IV, in the C.elegans ITS1 region, which correspond to the yeast A2 and A3 sites, respectively. In contrast, there is only a single A3-like site in the ITS1 region in Xenopus (30), suggesting some diversity in the pre-rRNA cleavage sites even in the higher eukaryotes.
The detailed mechanism of how U3 snoRNP functions in 18S rRNA processing still remains unclear. Recently, more than dozens of core components of U3 snoRNP have been identified in yeast using mass spectrographic analysis (31). Surprisingly, Mrd1p is not a member of the core components of U3 snoRNP, although it is also required for 18S rRNA processing in yeast, as does RBD-1 in C.elegans. Thus, it is conceivable that many extrinsic factors may be required for proper 18S rRNA processing other than U3 snoRNP. The mode of action of Mrd1p and its homologs including RBD-1 is not known at present, but one could speculate that they may transiently associate with pre-rRNA and/or U3 snoRNP, and help the function of U3 snoRNP for 18S rRNA processing, since they contain multiple RNA-binding domains and are required for all U3 snoRNP-involved cleavages.
An important outcome of this study is the clear demonstration of the linkage of ribosome biogenesis with several developmental events. Bj?rk et al. (12) described briefly that RNAi of rbd-1 causes various developmental phenotypes in C.elegans, as we observed in this study. However, they did not show any evidence for the RBD-1 function in 18S rRNA processing in C.elegans. In this respect, our results provide direct evidence for the first time that ribosome biogenesis is involved in developmental regulation in multicellular organisms. Previous studies in bacteria and yeast have demonstrated that cell growth (increase in cell size and number) is correlated closely with an increase in both protein synthesis and ribosome biogenesis . In higher eukaryotes, this correlation is seen in the diminished transcription of rRNAs during quiescence and the subsequent increase after stimulation with growth factors (33). Studies of hypertrophy in vertebrate cardiomyocytes have also linked rRNA and 5S RNA synthesis with control of cell size (34). These studies suggest that regulators of ribosome biogenesis may play an important role in cellular growth control. From this point of view, some of the phenotypes that we observed in rbd-1(RNAi) animals, such as the molting defect and cuticle malformation, seem to be closely related to protein synthesis. During the molting and subsequent growth processes, drastic and rapid changes occur in the nematode surface structures, and thus rapid massive production of proteins should be required for such processes. Accordingly, it appears that an insufficient quantity of 40S ribosomal subunits caused by RBD-1 depletion disturbs such rapid protein synthesis, resulting in severe defects in molting and cuticle formation. Since it is well known that RNAi efficiency varies with the dosage of dsRNA delivered in individual animals, target genes and cell types (35,36), it is not surprising that various phenotypes emerge upon RNAi of rbd-1. More complete inhibition of 18S rRNA synthesis by efficient RNAi of rbd-1 seems to lead to fatal effects, such as larval arrest and lethality that we observed in this study. Consistent with this, RNAi of fib-1 showed only such severe phenotypes (our unpublished data). An alternative explanation for the phenotypes caused by RNAi of rbd-1 is that RBD-1 may have a specific role for cuticle formation in addition to the role for 18S rRNA processing, like yeast Nop7p and Nop15p that are not only required for 28S rRNA processing but also have critical functions in DNA replication and cytokinesis, respectively (37,38).
Another important finding of this study is that RBD-1 is not present in early embryos, although 18S rRNA synthesis should require RBD-1. We have also shown similar limitation of the expression to late embryogenesis and thereafter for a component of U3 snoRNP, FIB-1, which is thought to be required for 18S rRNA synthesis in C.elegans. These observations imply that processing of 18S rRNA may be repressed in early embryos. It is likely that only the maternal stock of ribosomes is utilized for protein synthesis in early embryos and that such maternal ribosomes would have been fully consumed in late embryos. Thus, zygotic RBD-1 should start to be expressed in late embryos to newly synthesize 18S rRNA. Interestingly, it was reported that zygotic transcription of pre-rRNA starts as early as the one-cell stage and persists thereafter in C.elegans (39). Thus, the apparent absence of RBD-1 and FIB-1 in early embryos raises the possibility for the first time that transcription and processing of pre-rRNA are uncoupled during early embryogenesis.
ACKNOWLEDGEMENTS
We thank Dr Andrew Fire for the GFP expression vector, Dr Yuji Kohara for C.elegans EST clones, Dr John Aris for anti-FIB-1 antibody, and Dr Elizabeth Nakajima for reading the manuscript. This work was supported in part by grants from MEXT to H.S.
REFERENCES
Scheer,U. and Hock,R. (1999) Structure and function of the nucleolus. Curr. Opin. Cell Biol., 11, 385–390.
Maxwell,E.S. and Fournier,M.J. (1995) The small nucleolar RNAs. Annu. Rev. Biochem., 64, 897–934.
Smith,C.M. and Steitz,J.A. (1997) Sno storm in the nucleolus: new roles for myriad small RNPs. Cell, 89, 669–672.
Maden,B.E. and Hughes,J.M. (1997) Eukaryotic ribosomal RNA: the recent excitement in the nucleotide modification problem. Chromosoma, 105, 391–400.
Decatur,W.A. and Fournier,M.J. (2003) RNA-guided nucleotide modification of ribosomal and other RNAs. J. Biol. Chem., 278, 695–698.
van Nues,R.W., Venema,J., Rientjes,J.M., Dirks-Mulder,A. and Raue,H.A. (1995) Processing of eukaryotic pre-rRNA: the role of the transcribed spacers. Biochem. Cell Biol., 73, 789–801.
Fromont-Racine,M., Senger,B., Saveanu,C. and Fasiolo,F. (2003) Ribosome assembly in eukaryotes. Gene, 313, 17–42.
Ban,N., Nissen,P., Hansen,J., Moore,P.B. and Steitz,T.A. (2000) The complete atomic structure of the large ribosomal subunit at 2.4 ? resolution. Science, 289, 905–920.
Spahn,C.M., Beckmann,R., Eswar,N., Penczek,P.A., Sali,A., Blobel,G. and Frank,J. (2001) Structure of the 80S ribosome from Saccharomyces cerevisiae–tRNA-ribosome and subunit–subunit interactions. Cell, 107, 373–386.
Jin,S.B., Zhao,J., Bj?rk,P., Schmekel,K., Ljungdahl,P.O. and Wieslander,L. (2002) Mrd1p is required for processing of pre-rRNA and for maintenance of steady-state levels of 40 S ribosomal subunits in yeast. J. Biol. Chem., 277, 18431–18439.
Gavin,A.C., Bosche,M., Krause,R., Grandi,P., Marzioch,M., Bauer,A., Schultz,J., Rick,J.M., Michon,A.M., Cruciat,C.M. et al. (2002) Functional organization of the yeast proteome by systematic analysis of protein complexes. Nature, 415, 141–147.
Bj?rk,P., Bauren,G., Jin,S., Tong,Y.G., Burglin,T.R., Hellman,U. and Wieslander,L. (2002) A novel conserved RNA-binding domain protein, RBD-1, is essential for ribosome biogenesis. Mol. Biol. Cell, 13, 3683–3695.
Mayer,A.N. and Fishman,M.C. (2003) Nil per os encodes a conserved RNA recognition motif protein required for morphogenesis and cytodifferentiation of digestive organs in zebrafish. Development, 130, 3917–3928.
MacQueen,A.J. and Villeneuve,A.M. (2001) Nuclear reorganization and homologous chromosome pairing during meiotic prophase require C. elegans chk-2. Genes Dev., 15, 1674–1687.
Tollervey,D., Lehtonen,H., Carmo-Fonseca,M. and Hurt,E.C. (1991) The small nucleolar RNP protein NOP1 (fibrillarin) is required for pre-rRNA processing in yeast. EMBO J., 10, 573–583.
Tollervey,D., Lehtonen,H., Jansen,R., Kern,H. and Hurt,E.C. (1993) Temperature-sensitive mutations demonstrate roles for yeast fibrillarin in pre-rRNA processing, pre-rRNA methylation and ribosome assembly. Cell, 72, 443–457.
Epstein,H.F. and Shakes,D.C. (eds) (1995) Caenorhabditis elegans: Modern Biological Analysis of an Organism. Academic Press, San Diego, CA.
Brenner,S. (1974) The genetics of Caenorhabditis elegans. Genetics, 77, 71–94.
Ellis,R.E., Sulston,J.E. and Coulson,A.R. (1986) The rDNA of C. elegans: sequence and structure. Nucleic Acids Res., 14, 2345–2364.
Fujita,M., Takasaki,T., Nakajima,N., Kawano,T., Shimura,Y. and Sakamoto,H. (2002) MRG-1, a mortality factor-related chromodomain protein, is required maternally for primordial germ cells to initiate mitotic proliferation in C. elegans. Mech. Dev., 114, 61–69.
Hope,I. (1999) C. elegans: A Practical Approach. Oxford University Press, Oxford.
Aris,J.P. and Blobel,G. (1988) Identification and characterization of a yeast nucleolar protein that is similar to a rat liver nucleolar protein. J. Cell Biol., 107, 17–31.
Ahnn,J. and Fire,A. (1994) A screen for genetic loci required for body-wall muscle development during embryogenesis in Caenorhabditis elegans. Genetics, 137, 483–498.
Epstein,H.F., Berliner,G.C., Casey,D.L. and Ortiz,I. (1988) Purified thick filaments from the nematode Caenorhabditis elegans: evidence for multiple proteins associated with core structures. J. Cell Biol., 106, 1985–1995.
Small,E.B. and Marszalek,D.S. (1969) Scanning electron microscopy of fixed, frozen and dried protozoa. Science, 163, 1064–1065.
Paule,M.R. and White,R.J. (2000) Survey and summary: transcription by RNA polymerases I and III. Nucleic Acids Res., 28, 1283–1298.
Venema,J. and Tollervey,D. (1999) Ribosome synthesis in Saccharomyces cerevisiae. Annu. Rev. Genet., 33, 261–311.
Beltrame,M. and Tollervey,D. (1995) Base pairing between U3 and the pre-ribosomal RNA is required for 18S rRNA synthesis. EMBO J., 14, 4350–4356.
Sharma,K. and Tollervey,D. (1999) Base pairing between U3 small nucleolar RNA and the 5' end of 18S rRNA is required for pre-rRNA processing. Mol. Cell. Biol., 19, 6012–6019.
Borovjagin,A.V. and Gerbi,S.A. (2001) Xenopus U3 snoRNA GAC-Box A' and Box A sequences play distinct functional roles in rRNA processing. Mol. Cell. Biol., 21, 6210–6221.
Dragon,F., Gallagher,J.E., Compagnone-Post,P.A., Mitchell,B.M., Porwancher,K.A., Wehner,K.A., Wormsley,S., Settlage,R.E., Shabanowitz,J., Osheim,Y. et al. (2002) A large nucleolar U3 ribonucleoprotein required for 18S ribosomal RNA biogenesis. Nature, 417, 967–970.
Nomura,M., Gourse,R. and Baughman,G. (1984) Regulation of the synthesis of ribosomes and ribosomal components. Annu. Rev. Biochem., 53, 75–117.
Johnson,L.F., Williams,J.G., Abelson,H.T., Green,H. and Penman,S. (1975) Changes in RNA in relation to growth of the fibroblast. III. Posttranscriptional regulation of mRNA formation in resting and growing cells. Cell, 4, 69–75.
McDermott,P.J., Rothblum,L.I., Smith,S.D. and Morgan,H.E. (1989) Accelerated rates of ribosomal RNA synthesis during growth of contracting heart cells in culture. J. Biol. Chem., 264, 18220–18227.
Fraser,A.G., Kamath,R.S., Zipperlen,P., Martinez-Campos,M., Sohrmann,M. and Ahringer,J. (2000) Functional genomic analysis of C. elegans chromosome I by systematic RNA interference. Nature, 408, 325–330.
Gonczy,P., Echeverri,C., Oegema,K., Coulson,A., Jones,S.J., Copley,R.R., Duperon,J., Oegema,J., Brehm,M., Cassin,E. et al. (2000) Functional genomic analysis of cell division in C. elegans using RNAi of genes on chromosome III. Nature, 408, 331–336.
Du,Y.C. and Stillman,B. (2002) Yph1p, an ORC-interacting protein: potential links between cell proliferation control, DNA replication and ribosome biogenesis. Cell, 109, 835–848.
Oeffinger,M. and Tollervey,D. (2003) Yeast Nop15p is an RNA-binding protein required for pre-rRNA processing and cytokinesis. EMBO J., 22, 6573–6583.
Seydoux,G. and Dunn,M.A. (1997) Transcriptionally repressed germ cells lack a subpopulation of phosphorylated RNA polymerase II in early embryos of Caenorhabditis elegans and Drosophila melanogaster. Development, 124, 2191–2201.(Eiko Saijou, Toshinobu Fujiwara, Toshino)