RBD-1, a nucleolar RNA-binding protein, is essential for Caenorhabditis elegans early development through 18S ribosomal RNA processing

 
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RBD-1, a nucleolar RNA-binding protein, is essential for Caenorhabditis elegans early development through 18S ribosomal RNA processing
Published online February 10, 2004

1028±1036 Nucleic Acids Research, 2004, Vol. 32, No. 3
DOI: 10.1093/nar/gkh264

RBD-1, a nucleolar RNA-binding protein, is essential
for Caenorhabditis elegans early development
through 18S ribosomal RNA processing
Eiko Saijou, Toshinobu Fujiwara, Toshinobu Suzaki, Kunio Inoue and Hiroshi Sakamoto*

Department of Biology, Graduate School of Science and Technology, Kobe University, 1-1 Rokkodaicho, Nadaku,
Kobe 657-8501, Japan

Received December 8, 2003; Revised and Accepted January 14, 2004

ABSTRACT                                                                   cryoelectron microscopy (8,9), little is understood about how
                                                                           ribosome biogenesis is controlled in eukaryotic cells and
RBD-1 is the Caenorhabditis elegans homolog of                             whether it is linked to more complex biological events, such as
Mrd1p, which was recently shown to be required for                         developmental processes in multicellular organisms.

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18S ribosomal RNA (rRNA) processing in yeast. To                              Mrd1p was initially given this name because it contains
gain insights into the relationship between                                multiple copies of an RNA-binding domain called RBD, also
ribosome biogenesis and the development of multi-                          known as RRM (RNA recognition motif), and was recently
cellular organisms, we examined the expression                             shown to be a member of the group of non-ribosomal proteins
and function of RBD-1. Maternal RBD-1 in the ferti-                        that are involved in pre-rRNA processing in the yeast
lized egg disappears immediately after cleavage                            Saccharomyces cerevisiae (10). In yeast, Mrd1p is essential
starts, whereas zygotic RBD-1 ®rst appears in late                         for viability and its depletion leads to a decrease in the levels
embryos and is localized in the nucleolus in most                          of mature 18S rRNA and 40S ribosome and concomitant
cells, although zygotic transcription of pre-rRNA is                       accumulation of 18S rRNA precursors, whereas 25S rRNA
                                                                           processing is not affected. Since Mrd1p can associate with
known to be initiated as early as the one-cell stage.
                                                                           pre-rRNA and two components of U3 small nucleolar
RNA interference of the rbd-1 gene severely inhibits                       ribonucleoprotein complex (snoRNP), Mrd1p is also likely
the processing of 18S rRNA in association with                             to be a component of U3 snoRNP, which is known to be
various developmental abnormalities, indicating its                        required for 18S rRNA processing (11). Since Mrd1p
essential role in pre-rRNA processing and develop-                         homologs are found in a wide range of metazoans, the
ment in C.elegans. These results provide evidence                          homologs may also be involved in pre-rRNA processing.
for the linkage between ribosome biogenesis and                            Indeed, in the dipteran Chironomus tentans, a homologous
the control of development and imply unexpected                            protein (Ct-RBD-1) is localized mainly in the nucleus and is
uncoupling of transcription and processing of pre-                         likely to be involved in 18S rRNA processing (12).
rRNA in early C.elegans embryos.                                           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
                                                                           zebra®sh homolog Npo causes various developmental abnor-
INTRODUCTION                                                               malities (12,13). These observations imply that there may be
In all eukaryotic cells, ribosome biogenesis is a very integrated          the linkage between ribosome biogenesis and developmental
process that occurs in the nucleolus (1). The nucleolus of the             events in multicellular organisms. However, the question as to
eukaryotic cell is densely packed with pre-ribosomal RNAs                  whether the developmental abnormalities are correlated with
(pre-rRNAs) and a number of small nucleolar RNAs                           defects in ribosome biogenesis still remains to be examined,
(snoRNAs) that are essential components involved in pre-                   since there is no direct evidence for the involvement of RBD-1
rRNA processing [reviewed by Maxwell and Fournier (2) and                  or Npo in pre-rRNA processing.
Smith and Steitz (3)]. Maturation of rRNAs is achieved by                     To address the question of this possible correlation, we
post-transcriptional events including methylation, pseudo-                 examined in detail the role of RBD-1 in both ribosome
uridylation and multiple cleavages, resulting in the generation            biogenesis and development in C.elegans. We found that
of mature 18S, 5.8S and 25±28S rRNA species in all                         RBD-1 depletion by RNAi inhibits processing of 18S rRNA
eukaryotic cells (4,5). These processes are accomplished via               and subsequent formation of the 40S ribosomal subunit, and
various cis-acting elements within pre-rRNAs (6) and a                     causes various developmental abnormalities simultaneously,
number of non-ribosomal trans-acting factors (7). Although                 indicating its essential role in pre-rRNA processing and
the outline of the pre-rRNA processing pathway is roughly                  development in C.elegans. We also found that RBD-1 is
understood and the structures of two ribosomal subunits                    localized in the nucleolus, like Mrd1p and Ct-RBD-1 and that
have recently been solved by X-ray crystallography and                     its zygotic expression starts in late embryos, although

*To whom correspondence should be addressed. Tel: +81 78 803 5796; Fax: +81 78 803 5720; Email: hsaka@kobe-u.ac.jp

Nucleic Acids Research, Vol. 32 No. 3 ã Oxford University Press 2004; all rights reserved
RBD-1, a nucleolar RNA-binding protein, is essential for Caenorhabditis elegans early development through 18S ribosomal RNA processing
Nucleic Acids Research, 2004, Vol. 32, No. 3     1029

transcription of pre-rRNA by RNA polymerase I is known to       was af®nity-puri®ed and used for western blot analyses
start as early as the one-cell stage. We observed a similar     (1:1000±1:2000 dilutions) as described previously (20).
expression pattern during embryogenesis for a component of      Isolation of worms at speci®c developmental stages was
U3 snoRNP, FIB-1 (14), a C.elegans homolog of the yeast         performed as described (21). Wild-type worms were immuno-
Nop1p, which is an essential factor for 18S rRNA processing     stained using af®nity-puri®ed anti-RBD-1 antibodies (1:100
(15,16). These results provide evidence for the linkage         dilution) or anti-FIB-1 antibodies (22) (1:100 dilution) as
between ribosome biogenesis and developmental events in         described (23). Embryos and larvae were permeated for
multicellular organisms and imply that transcription and        staining by the freeze-crack method and ®xed with methanol/
processing of pre-rRNA may be regulated differentially during   acetone according to standard procedures (24).
early embryogenesis in C.elegans.
                                                                Sucrose gradient centrifugation
                                                                Wild type and rbd-1(RNAi) worms, respectively, were mixed
MATERIALS AND METHODS                                           with Lysing Matrix D (Bio101) and homogenized in buffer A
                                                                [50 mM Tris±HCl (pH 7.5), 25 mM KCl, 5 mM MgCl2, 0.5%
Construction of GFP reporter gene fusion                        Triton X-100, 250 mM sucrose] by using a FastPrep
The reporter construct expressing green ¯uorescent protein      homogenizator (Bio101). The lysates were loaded on a linear
(GFP) under the control of the rbd-1 promoter was made as       sucrose gradient (10±30%) in buffer B [10 mM Tris±HCl
follows. The promoter region was PCR-ampli®ed using             (pH 7.5), 10 mM MgCl2, 100 mM NH4Cl] and centrifuged at
C.elegans genomic DNA with the forward primer (±1931):          4°C in a Beckman MLS50 rotor at 40 000 r.p.m. for 120 min,

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TTG CAT GCT AAT GGT GAG TAG CTT TAT CCT GAA                     followed by fractionation and monitoring at 260 nm.
ATA AGA ACA C, and the reverse primer (+30): GGT CTA            Scanning electron microscopy
GAG CTT GTT TTT GAC AAT TAA TCG AGT TGT CAT
G (the numbers in parentheses correspond to the nucleotide      Worms were washed in M9 buffer before ®xation in Parducz
position relative to the ®rst nucleotide of the rbd-1 open-     ®xative (25). After extensive washes, samples were
reading frame). This genomic fragment was fused in-frame to     dehydrated in ethanol followed by amylacetate. The samples
a promoterless GFP vector, pPD95.77 (provided by Dr A.          were processed in a critical-point drier, mounted and observed
Fire). Microinjection of the resulting plasmid into C.elegans   using a Hitachi S-2150N scanning electron microscope.
worms (Bristol type N2) was performed as described (17).
Worm breeding and handling were conducted as described
(18).                                                           RESULTS
RNA interference                                                Expression and subcellular localization of RBD-1
Sense and antisense RNAs were synthesized in vitro              We examined the expression pattern of RBD-1 at various
from yk417f6 cDNA which encodes RBD-1 (provided by              developmental stages. To measure the levels of RBD-1
Dr Y. Kohara). Both RNAs were annealed to form a double-        expression, we used synchronized populations at each devel-
stranded RNA (dsRNA). For RNAi, L4 hermaphrodites were          opmental stage. Western blot analyses with anti-RBD-1
soaked in 4 ml of dsRNA solution (~2 mg/ml) for 16±24 h or      antibody reveal that RBD-1 is expressed constantly during
dsRNA (1 mg/ml) was injected into the gonad arms of young       the four larval stages and the adult stage, whereas the RBD-1
adult hermaphrodites.                                           expression level in embryos is signi®cantly lower than that at
                                                                other stages (Fig. 1A). This suggests that RBD-1 expression is
Northern blot analysis                                          downregulated during embryogenesis. Consistently, immuno-
Total RNA from wild-type and rbd-1(RNAi) animals were           staining of embryos shows that RBD-1 expression is limited to
extracted with an RNA extraction kit (Micro-to-Midi Total       late embryonic stages (Fig. 1B). Interestingly, a small amount
RNA Puri®cation System; Invitrogen). Approximately 4 mg of      of maternally supplied RBD-1 is also detectable in the
total RNA per lane were resolved on a 1.2% formaldehyde-        fertilized egg (Fig. 1B, a and b: arrowhead). In contrast to
containing agarose gel, transferred onto a nylon membrane       zygotic RBD-1, maternal RBD-1 is seen as a number of
(Roche Diagnostics), and hybridized with DIG-labeled            granules in the cytoplasm and disappears shortly after
antisense RNA probes. The antisense probes 1±9 and 18S          cleavage begin, suggesting that maternal RBD-1 may be
probe correspond to the positions of nucleotides 511±609,       degraded rapidly in early embryos. To con®rm the down-
846±933, 2736±2791, 2969±3036, 3050±3157, 3342±3427,            regulation of RBD-1 in early embryos, we utilized a transgenic
1±210, 311±410, 411±510 and 1261±1677, respectively, of the     strain carrying the GFP gene under the control of the native
C.elegans rDNA repeat (19).                                     promoter for the rbd-1 gene and monitored GFP ¯uorescence
                                                                from a single embryo (Fig. 1C). As expected, prominent GFP
Antibody preparation, western blot analysis and                 ¯uorescence from the rbd-1 reporter gene is seen at the
immunostaining                                                  beginning of the morphogenesis stage, but not at the
An rbd-1 cDNA fragment of 972 bp was subcloned into             gastrulation stage, and persists with a gradual increase
pGEX-4T3 (Amersham Biosciences) and the fusion protein          throughout subsequent embryogenesis. Interestingly, FIB-1,
GST-RBD-1 was over-expressed in the XL-2 blue Escherichia       a C.elegans homolog of the yeast Nop1p, which is an essential
coli strain. GST-RBD-1 was af®nity-puri®ed using                component of U3 snoRNP (14±16), shows a very similar
glutathione±Sepharose (Amersham Biosciences) and used to        expression pattern to RBD-1 during C.elegans embryogenesis
raise rabbit polyclonal antibodies. The anti-RBD-1 antibody     (Fig. 1D).
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Figure 1. Expression of RBD-1 in C.elegans during development. (A) Western blot analysis of RBD-1 was performed with af®nity-puri®ed 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 mg) 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
¯uorescence 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                       (Fig. 3E). In contrast, in rbd-1(RNAi) hermaphrodites, the alae
ubiquitously in L1 larvae and continues to be expressed until                  were disconnected at many points, increase in number or are
adulthood in both somatic and germline cells (Fig. 2A, a±f).                   severely deformed (Fig. 3F±H). These observations show that
To determine the subcellular localization of RBD-1, we                         RBD-1 is required for various developmental processes in
closely inspected intestine cells since they have relatively                   C.elegans.
large nuclei, making it possible to easily discriminate the cell
compartments (Fig. 2B). As evidenced by its colocalization                     Inhibition of 40S ribosomal subunit formation by RBD-1
with a nucleolar marker protein FIB-1, RBD-1 is localized in                   depletion
the nucleolus. These results show that RBD-1 is a nucleolar                    It has been reported that depletion of Mrd1p reduces 18S
protein, like its counterparts in yeast and C.tentans, and is                  rRNA synthesis and the formation of 40S ribosomal subunits
expressed ubiquitously after the late embryonic stage.                         in yeast (10). To test whether RBD-1 is also involved in
                                                                               ribosomal biogenesis, we performed RNAi of rbd-1 by
RBD-1 is essential for C.elegans development                                   soaking to deplete RBD-1 in C.elegans and analyzed the
To determine the possible function of RBD-1 during devel-                      ribosome pro®le in rbd-1(RNAi) animals. The level of RBD-1
opment of C.elegans, RNAi by injection was performed using                     decreases signi®cantly and concomitantly the level of 18S
adult hermaphrodites, and the phenotypes of the F1 progeny                     rRNA decreases to ~60% as compared with the wild-type
from the injected hermaphrodites were analyzed. RNAi of                        level, whereas 26S rRNA is not affected (Fig. 4A±C).
rbd-1 has no visible effects on embryogenesis, but causes                      Simultaneously, depletion of RBD-1 causes a prominent
various abnormalities during post-embryonic development                        change in the ribosome pro®le in a sucrose density gradient
(Fig. 3). After hatching, all F1 progeny exhibit growth                        (Fig. 4D). As expected, the formation of 40S subunits is
retardation (Gro: 100%, n = 870). Approximately half of                        severely inhibited in rbd-1(RNAi) animals. Accordingly, the
them show severe larval phenotypes such as larval lethality or                 level of 80S ribosome (a complex of 40S and 60S ribosomal
arrest (Lvl or Lva: 24.8%) and defective molting (Mlt: 20.7%,                  subunits) decreases signi®cantly in such animals, accompan-
Fig. 3B), resulting in larval death. The remaining progeny                     ied by a relative increase of the level of 60S ribosomal
grow to adulthood but show abnormal gonad formation (Gon:                      subunits. Thus, we concluded that RBD-1 is essential for
24.9%, Fig. 3C) and protruded vulva (Pvl: 12.9%, Fig. 3D).                     ribosome biogenesis through 18S rRNA synthesis in
   In addition, most of the rbd-1(RNAi) adult hermaphrodites                   C.elegans, like Mrd1p in yeast.
show malformation of the surface cuticle structure. In
particular, the alae, which are protruding ridges formed over                  Pre-rRNA processing in C.elegans
each lateral row of hypodermal seam cells, are abnormal in                     In many organisms, mature 18S, 5.8S and 28S rRNAs are
most rbd-1(RNAi) hermaphrodites. In wild-type animals, three                   generated from a single pre-rRNA by multiple processing
lines of alae are seen along each lateral side of the animal                   events that remove the external transcribed sequence (ETS)
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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 immuno-
                                                                               Figure 3. Typical phenotypes observed in rbd-1(RNAi) animals. Nomarski
stained with anti-RBD-1 (a) and anti-FIB-1 (b) antibodies. The same sample
                                                                               (A±D) and scanning electron microscopic (E±H) views of rbd-1(RNAi) and
was stained with DAPI to show nuclei (c). The nuclear edges are outlined
                                                                               wild-type animals. RNAi was performed by injection. Defective molting
with a dashed line. Germline cells are also seen at the left bottom part of
                                                                               (B, Mlt), abnormal gonad formation (C, Gon) and protruded vulva (D, Pvl)
each panel.
                                                                               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).
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                      immediately downstream of the 3¢ end of 5.8S rRNA (site VI).
using RNA probes which are speci®c to distinct regions of the                  Probe 3 detects bands a, b and d, as do probes 1 and 2,
primary rRNA transcript (Fig. 5). Probes 1 and 2 correspond to                 con®rming that there is no major processing site in the 5¢ ETS
the positions within the putative 5¢ ETS region. The probe 1                   region. Probe 4 detects bands a, d and also a new c¢, but not
region includes a predicted TATA-like sequence for tran-                       band b. Probe 5 detects bands a and c¢, but not bands b and d.
scription by RNA polymerase I (Fig. 5C, boxed sequence).                       Probe 6 detects bands a and c¢ and a new band c. Considering
Since RNA polymerase I transcription initiates just down-                      the sizes of these bands, bands b and d correspond to the
stream of TATA-like sequences (26), it is expected that probe                  intermediates for 18S rRNA with different 3¢ ends, band c¢
1 hybridizes to the 5¢ end of the primary pre-rRNA. On the                     corresponds to the intermediate for 5.8S and 26S rRNAs, and
other hand, probe 2 hybridizes with the region just before the                 band c corresponds to the intermediate for 26S rRNA. These
5¢ end of 18S rRNA (Fig. 5A, site I). These two probes detect                  results indicate that there are at least two processing sites in
the same three kinds of rRNA intermediates a, b and d                          the ITS1 region: one (site III) lies between site II and the 5¢
(Fig. 5B, lanes 1 and 2). Judging from the size, the largest band              end of the probe 4 region, and the other (site IV) within the
a corresponds to the pre-rRNA containing 18S, 5.8S and 26S                     probe 4 region. It should be noted that the probe 4 region
rRNAs. Since both bands b and d are detected with a probe for                  encompasses the site IV.
the 18S rRNA coding region (data not shown), these RNA                            Finally, we examined the processing sites in the putative 3¢
species are intermediates for 18S rRNA. Considering that                       ETS region using probes 7±9 which are complementary to the
there is no difference between the bands detected with probes                  positions between the 3¢ end of 26S rRNA (site VIII) and the
1 and 2, it is likely that there is no major processing site within            probe 1 region (Fig. 5B, lanes 7±9). Only band a is detected
the 5¢ ETS region of C.elegans pre-rRNA, although we could                     with these three probes, although the signal with probe 9 is
not exclude the possibility that the probe 1 region contains an                very faint. In addition, we noticed that the hybridization
additional processing site.                                                    signals for band a are relatively weak with probes 7±9 as
   To examine the processing sites within the ITS region, we                   compared with those with probes 1±6. This is possibly because
used four probes (Fig. 5B, lanes 3±6). Probes 3±5 correspond                   band a includes at least two pre-rRNAs: the major one lacks
to the positions between the 3¢ end of 18S rRNA (site II) and                  the 3¢ ETS region, and the minor one contains the 3¢ ETS
the 5¢ end of 5.8S (site V). Probe 6 corresponds to the position               region, and these two pre-rRNAs cannot be resolved under our
1032     Nucleic Acids Research, 2004, Vol. 32, No. 3

                                                                              Inhibition of 18S rRNA processing by RBD-1 depletion
                                                                              Since we have clari®ed 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 signi®cantly decreases, whereas the amount of the
                                                                              band d intermediate signi®cantly 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

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                                                                              intermediates suggests an additional processing site in the 5¢
Figure 4. Inhibition of 18S rRNA synthesis by RNAi of rbd-1. (A) RBD-1        ETS region. Taken together, these ®ndings indicate that
was signi®cantly reduced in rbd-1(RNAi) animals. RNAi was performed by
soaking. Equal amounts of total protein (10 mg) from mixed-stage wild-type    RBD-1 is involved at least in the site III processing during
(WT) and rbd-1(RNAi) animals were electrophoresed on a 10% SDS±polya-         C.elegans 18S rRNA synthesis.
crylamide 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 mg) from wild-type (WT) and
rbd-1(RNAi) animals were electrophoresed on a 1% denaturing agarose gel       DISCUSSION
and stained with ethidium bromide. (C) The relative ratio of the amounts of
18S rRNA to 26S rRNA was calculated by quanti®cation of the rRNA              In this study, we have clari®ed for the ®rst time the outline of
bands in the 4% denaturing acrylamide gel stained with toluidine blue using   the pre-rRNA processing pathway in C.elegans and shown
the NIH image program. (D) Reduction of 40S ribosomal subunit and             that a nucleolar RNA-binding protein, RBD-1, is required for
80S ribosome and increase of 60S ribosomal subunit in an rbd-1(RNAi)          18S rRNA processing in C.elegans. The requirement of
animal. Ribosome pro®les of the extracts from wild-type (WT) and
rbd-1(RNAi) animals were analyzed by 10±30% sucrose density gradient          RBD-1 for 18S rRNA processing is essentially the same
centrifugation.                                                               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
gel electrophoresis conditions because of their relatively large
                                                                              developmental processes in C.elegans.
sizes. Although we could not identify any processing site in                     Previous studies on yeast pre-rRNA processing have shown
the 3¢ ETS region in this study, the results suggest that                     that processing of 18S rRNA includes multiple cleavages at
transcription termination of the primary pre-rRNA transcript                  the sites A0, A1 and A2, and requires the U3 snoRNP function
occurs near the probe 9 region. We do not know the precise                    (27). Interaction of the hinge region of U3 snoRNA with a
sites for transcription initiation and termination of the primary             short segment upstream of the A0 site is required for the
pre-rRNA transcript, but it is likely that both sites exist within            cleavages at the sites A0, A1 and A2 (28). In addition,
the region encompassing the probes 9 and 1, since there are                   interaction of the Box A sequence of U3 snoRNA with two
two putative TATA-like sequences and a T-rich sequence                        internal segments of 18S rRNA forms a pseudoknot structure
within the region which may function for initiation and                       and is required for the cleavages at the sites A1 and A2 (29).
termination of RNA polymerase I, respectively (Fig. 5C). The                  These previous ®ndings clearly show that U3 snoRNP binds
results also suggest that the elimination of the 3¢ ETS region is             the segments and promotes the 18S rRNA processing. The
very rapid since the amount of the 3¢ ETS-containing pre-                     sites I, II and III in C.elegans correspond to the A1, D and A2
rRNA(s) is very low at the steady state, consistent with the                  in yeast, respectively, and the cleavage of these sites
previous ®nding that rRNA processing is initiated by rapid                    apparently depends upon the RBD-1 function, since the site
cleavage within the 3¢ ETS region of the primary transcript                   III processing is inhibited in association with the reduction of
(27).                                                                         the level of mature 18S rRNA in rbd-1(RNAi) animals. In this
   Taken together, the outline of the pre-rRNA processing                     study, we could not identify the cleavage site in C.elegans
pathway in C.elegans is depicted in Figure 5A. There are at                   which corresponds to the yeast A0 site. Such an A0-like site
least eight processing sites in the primary pre-rRNA including                may exist in the 5¢ ETS region in C.elegans pre-rRNA,
both ends of mature rRNAs. We do not exclude the possibility                  especially in the probe 1 region, although we could not
that there are more processing sites for very short-lived rRNA                examine the possibility in this study. Cleavage of the putative
intermediates, especially in the 3¢ ETS region.                               A0-like site may also depend upon the RBD-1 function, on the
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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 speci®c probes 1±9 used for northern blot analysis are shown below the rRNA genomic region.
(B) Identi®cation of pre-rRNA and its processing intermediates in wild-type C.elegans. Equal amounts of total RNA (4 mg) were electrophoresed on a 1.2%
denaturing agarose gel and were analyzed by northern blotting using the speci®c 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.

analogy of the dependence of A0 site cleavage upon the                          suggesting some diversity in the pre-rRNA cleavage sites even
function U3 snoRNP in yeast. If this is the case, the band a                    in the higher eukaryotes.
accumulation in rbd-1(RNAi) animals (Fig. 6) can be                                The detailed mechanism of how U3 snoRNP functions in
explained as a result from the defect of the A0-like site                       18S rRNA processing still remains unclear. Recently, more
cleavage. In addition, we have identi®ed two cleavage sites, III                than dozens of core components of U3 snoRNP have been
and IV, in the C.elegans ITS1 region, which correspond to the                   identi®ed in yeast using mass spectrographic analysis (31).
yeast A2 and A3 sites, respectively. In contrast, there is only a               Surprisingly, Mrd1p is not a member of the core components
single A3-like site in the ITS1 region in Xenopus (30),                         of U3 snoRNP, although it is also required for 18S rRNA
1034    Nucleic Acids Research, 2004, Vol. 32, No. 3

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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 speci®c 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 mg) were electrophoresed on a 1.2% denaturing agarose gel and were analyzed by northern blotting using the speci®c probes indicated below.

processing in yeast, as does RBD-1 in C.elegans. Thus, it is                 ribosome biogenesis [reviewed in Nomura et al. (32)]. In
conceivable that many extrinsic factors may be required for                  higher eukaryotes, this correlation is seen in the diminished
proper 18S rRNA processing other than U3 snoRNP. The                         transcription of rRNAs during quiescence and the subsequent
mode of action of Mrd1p and its homologs including RBD-1 is                  increase after stimulation with growth factors (33). Studies of
not known at present, but one could speculate that they may                  hypertrophy in vertebrate cardiomyocytes have also linked
transiently associate with pre-rRNA and/or U3 snoRNP, and                    rRNA and 5S RNA synthesis with control of cell size (34).
help the function of U3 snoRNP for 18S rRNA processing,                      These studies suggest that regulators of ribosome biogenesis
since they contain multiple RNA-binding domains and are                      may play an important role in cellular growth control. From
required for all U3 snoRNP-involved cleavages.                               this point of view, some of the phenotypes that we observed in
   An important outcome of this study is the clear demonstra-                rbd-1(RNAi) animals, such as the molting defect and cuticle
tion of the linkage of ribosome biogenesis with several                      malformation, seem to be closely related to protein synthesis.
developmental events. BjoÈrk et al. (12) described brie¯y that               During the molting and subsequent growth processes, drastic
RNAi of rbd-1 causes various developmental phenotypes in                     and rapid changes occur in the nematode surface structures,
C.elegans, as we observed in this study. However, they did not               and thus rapid massive production of proteins should be
show any evidence for the RBD-1 function in 18S rRNA                         required for such processes. Accordingly, it appears that an
processing in C.elegans. In this respect, our results provide                insuf®cient quantity of 40S ribosomal subunits caused by
direct evidence for the ®rst time that ribosome biogenesis is                RBD-1 depletion disturbs such rapid protein synthesis,
involved in developmental regulation in multicellular organ-                 resulting in severe defects in molting and cuticle formation.
isms. Previous studies in bacteria and yeast have demonstrated               Since it is well known that RNAi ef®ciency varies with the
that cell growth (increase in cell size and number) is correlated            dosage of dsRNA delivered in individual animals, target genes
closely with an increase in both protein synthesis and                       and cell types (35,36), it is not surprising that various
Nucleic Acids Research, 2004, Vol. 32, No. 3                1035

phenotypes emerge upon RNAi of rbd-1. More complete                           10. Jin,S.B., Zhao,J., BjoÈrk,P., Schmekel,K., Ljungdahl,P.O. and
                                                                                  Wieslander,L. (2002) Mrd1p is required for processing of pre-rRNA and
inhibition of 18S rRNA synthesis by ef®cient RNAi of rbd-1
                                                                                  for maintenance of steady-state levels of 40 S ribosomal subunits in
seems to lead to fatal effects, such as larval arrest and lethality               yeast. J. Biol. Chem., 277, 18431±18439.
that we observed in this study. Consistent with this, RNAi of                 11. Gavin,A.C., Bosche,M., Krause,R., Grandi,P., Marzioch,M., Bauer,A.,
®b-1 showed only such severe phenotypes (our unpublished                          Schultz,J., Rick,J.M., Michon,A.M., Cruciat,C.M. et al. (2002)
data). An alternative explanation for the phenotypes caused by                    Functional organization of the yeast proteome by systematic analysis of
                                                                                  protein complexes. Nature, 415, 141±147.
RNAi of rbd-1 is that RBD-1 may have a speci®c role for                       12. BjoÈrk,P., Bauren,G., Jin,S., Tong,Y.G., Burglin,T.R., Hellman,U. and
cuticle formation in addition to the role for 18S rRNA                            Wieslander,L. (2002) A novel conserved RNA-binding domain protein,
processing, like yeast Nop7p and Nop15p that are not only                         RBD-1, is essential for ribosome biogenesis. Mol. Biol. Cell, 13,
required for 28S rRNA processing but also have critical                           3683±3695.
functions in DNA replication and cytokinesis, respectively                    13. Mayer,A.N. and Fishman,M.C. (2003) Nil per os encodes a conserved
                                                                                  RNA recognition motif protein required for morphogenesis and
(37,38).                                                                          cytodifferentiation of digestive organs in zebra®sh. Development, 130,
   Another important ®nding of this study is that RBD-1 is not                    3917±3928.
present in early embryos, although 18S rRNA synthesis should                  14. MacQueen,A.J. and Villeneuve,A.M. (2001) Nuclear reorganization and
require RBD-1. We have also shown similar limitation of the                       homologous chromosome pairing during meiotic prophase require
expression to late embryogenesis and thereafter for a                             C. elegans chk-2. Genes Dev., 15, 1674±1687.
                                                                              15. Tollervey,D., Lehtonen,H., Carmo-Fonseca,M. and Hurt,E.C. (1991) The
component of U3 snoRNP, FIB-1, which is thought to be                             small nucleolar RNP protein NOP1 (®brillarin) is required for pre-rRNA
required for 18S rRNA synthesis in C.elegans. These obser-                        processing in yeast. EMBO J., 10, 573±583.
vations imply that processing of 18S rRNA may be repressed                    16. Tollervey,D., Lehtonen,H., Jansen,R., Kern,H. and Hurt,E.C. (1993)

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in early embryos. It is likely that only the maternal stock of                    Temperature-sensitive mutations demonstrate roles for yeast ®brillarin in
                                                                                  pre-rRNA processing, pre-rRNA methylation and ribosome assembly.
ribosomes is utilized for protein synthesis in early embryos                      Cell, 72, 443±457.
and that such maternal ribosomes would have been fully                        17. Epstein,H.F. and Shakes,D.C. (eds) (1995) Caenorhabditis elegans:
consumed in late embryos. Thus, zygotic RBD-1 should start                        Modern Biological Analysis of an Organism. Academic Press, San
to be expressed in late embryos to newly synthesize 18S                           Diego, CA.
rRNA. Interestingly, it was reported that zygotic transcription               18. Brenner,S. (1974) The genetics of Caenorhabditis elegans. Genetics, 77,
                                                                                  71±94.
of pre-rRNA starts as early as the one-cell stage and persists                19. Ellis,R.E., Sulston,J.E. and Coulson,A.R. (1986) The rDNA of C.
thereafter in C.elegans (39). Thus, the apparent absence of                       elegans: sequence and structure. Nucleic Acids Res., 14, 2345±2364.
RBD-1 and FIB-1 in early embryos raises the possibility for                   20. Fujita,M., Takasaki,T., Nakajima,N., Kawano,T., Shimura,Y. and
the ®rst time that transcription and processing of pre-rRNA are                   Sakamoto,H. (2002) MRG-1, a mortality factor-related chromodomain
uncoupled during early embryogenesis.                                             protein, is required maternally for primordial germ cells to initiate
                                                                                  mitotic proliferation in C. elegans. Mech. Dev., 114, 61±69.
                                                                              21. Hope,I. (1999) C. elegans: A Practical Approach. Oxford University
                                                                                  Press, Oxford.
ACKNOWLEDGEMENTS                                                              22. Aris,J.P. and Blobel,G. (1988) Identi®cation and characterization of a
                                                                                  yeast nucleolar protein that is similar to a rat liver nucleolar protein.
We thank Dr Andrew Fire for the GFP expression vector,                            J. Cell Biol., 107, 17±31.
Dr Yuji Kohara for C.elegans EST clones, Dr John Aris for                     23. Ahnn,J. and Fire,A. (1994) A screen for genetic loci required for body-
anti-FIB-1 antibody, and Dr Elizabeth Nakajima for reading                        wall muscle development during embryogenesis in Caenorhabditis
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the manuscript. This work was supported in part by grants                     24. Epstein,H.F., Berliner,G.C., Casey,D.L. and Ortiz,I. (1988) Puri®ed thick
from MEXT to H.S.                                                                 ®laments from the nematode Caenorhabditis elegans: evidence for
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