From UMR5099, Laboratoire de Biologie
Moléculaire Eucaryote du Centre National de la Recherche
Scientifique, Université Paul-Sabatier, 118 route de Narbonne,
Toulouse 31062, France, ¶ UPR 9023 CNRS, Mécanismes
Moléculaires des Communications Cellulaires, 34094 Montpellier
Cedex 05, France, and
Institute for Experimental Pathology,
Zentrum für Molekulare Biologie der Entzündung, 48149 Münster, Germany
Received for publication, April 20, 2001
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ABSTRACT |
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Antisense box C/D small nucleolar RNAs (snoRNAs)
guide the 2'-O-ribose methylations of eukaryotic rRNAs and
small nuclear RNAs (snRNAs) through formation of a specific base
pairing at each RNA methylation site. By analysis of a box C/D snoRNA
cDNA library constructed from rat brain RNAs, we have identified a novel box C/D snoRNA, RBII-36, which is devoid of complementarity to
rRNA or an snRNA and exhibits a brain-specific expression pattern. It
is uniformly expressed in all major areas of adult rat brain (except
for choroid plexus) and throughout rat brain ontogeny but exclusively
detected in neurons in which it exhibits a nucleolar localization. In
vertebrates, known methylation guide snoRNAs are intron-encoded and
processed from transcripts of housekeeping genes. In contrast, RBII-36
snoRNA is intron-encoded in a gene preferentially expressed in the rat
central nervous system and not in proliferating cells. Remarkably, this
host gene, which encodes a previously reported noncoding RNA, Bsr,
spans tandemly repeated 0.9-kilobase units including the
snoRNA-containing intron. The novel brain-specific snoRNA appears to
result not only from processing of the debranched lariat but also from
endonucleolytic cleavages of unspliced Bsr RNA (i.e. an
alternative splicing-independent pathway unreported so far for
mammalian intronic snoRNAs). Sequences homologous to RBII-36 snoRNA
were exclusively detected in the Rattus genus of rodents,
suggesting a very recent origin of this brain-specific snoRNA.
In eukaryotic cells, ribosome biogenesis takes place mainly in the
nucleolus, through a series of intricate steps including (i) rDNA
transcription by the cognate RNA polymerase I, (ii) processing of the
pre-rRNA transcript by endo- and exonucleases and covalent modification
of a subset of pre-rRNA nucleotides, (iii) packaging of pre-rRNA by
ribosomal proteins, and (iv) cytoplasmic export of ribosomal subunits.
Pre-rRNA maturation involves a large number of small nucleolar
ribonucleoprotein particles
(snoRNPs),1 which can be
defined as a metabolically stable association between small nucleolar
RNA (snoRNA) and a specific set of proteins (1). All snoRNAs to date
(except the RNA component for RNase MRP) fall into two major classes,
box C/D and box H/ACA snoRNAs, based on the presence of short consensus
sequence motifs (2). Whereas a few of them are involved in pre-rRNA
nucleolytic cleavages, most box C/D and box H/ACA snoRNAs play a key
role in specifying the two major types of rRNA nucleotide modification
(2'-O-ribose methylations and pseudouridylations,
respectively) through formation of a specific RNA duplex at the
modification site (3-6). While function(s) of these nucleotide
modifications are still elusive they are proposed to fine tune rRNA
folding and interactions with ribosomal proteins. Thereby, they could
play a role in the biogenesis and activity of mature cytoplasmic
ribosomes, particularly with regard to their peptidyl transferase
activities (7). Antisense box C/D snoRNAs contain conserved sequence
motifs box C (5'-RUGAUGA-3') and box D (5'-CUGA-3'), generally brought
together by a short 5'-3'-terminal stem, and one (sometimes
two) antisense element(s) (8). Each antisense element, which exhibits
an extended (10-21-nt) complementarity to a site of rRNA ribose
methylation, is located immediately upstream from box D (or another
copy of this motif, box D', in the 5' half of the snoRNA). In the
corresponding snoRNA/rRNA duplexes, the 2'-O-ribose
methylated nucleotide is systematically paired to the fifth nucleotide
upstream from box D (or D') (9, 10). Expression of an artificial box
C/D snoRNA carrying an appropriate antisense element is sufficient to
target a novel ribose methylation on the predicted pre-rRNA nucleotide
and also, to a lesser extent, to RNA-polymerase II transcripts (4, 11), showing that the antisense element associated with box D (or D') is the
sole determinant of the site of methylation. Antisense box C/D snoRNAs
able to direct the formation of three distinct ribose methylations in
U6 and U5 snRNA have been reported (12-14) as well as several other
box C/D snoRNAs likely to represent bona fide guides for
additional snRNA ribose methylations in U6, U2, and U4 snRNAs (15).
Moreover, several novel mammalian box C/D snoRNAs devoid of
complementarity to rRNA or snRNA remain without a presumptive RNA
target (15, 16). Finally, the intriguing detection of one H/ACA- and
three C/D-box snoRNAs exclusively expressed in the brain and also
devoid of rRNA or snRNA complementarity opens new perspectives as to
the potential range of functions of this unexpectedly large snoRNA
family (17). Remarkably, one of the novel brain-specific snoRNA
displays an 18-nt antisense element that could play a key role in the
processing of a brain-specific mRNA.
Methylation guide snoRNAs are also intriguing by their unusual genomic
organization and modes of biosynthesis. In vertebrates, they are
encoded within introns and are not independently transcribed but
processed from the pre-mRNA introns, in most cases by
exonucleolytic digestion of the debranched lariat (18-21). In
contrast, in Saccharomyces cerevisiae, only a few of them
are intronic, while most of them are synthesized from independent
mono-, di-, or polycistronic RNA transcripts processed by endo- and
exonucleases (22-26). Polycistronic snoRNAs have also been reported in
plants (27-29) and in Trypanosoma brucei (30-32). Finally,
the detection of archaeal homologs of box C/D snoRNAs also points to
novel aspects in their biosynthesis, with the coding sequences of
several of them partially overlapping upstream and/or downstream open
reading frames (33, 34). Earlier observations that snoRNA host genes
coded for proteins involved in ribosome biosynthesis and/or nucleolar
function (e.g. ribosomal proteins, translation
factors, nucleolin) suggested that this gene organization might provide
a regulatory link between partners in the same biological process (1,
35). However, it is now clear that this hypothesis cannot be
generalized. First, some intronic snoRNAs are hosted by different genes
in different eukaryotic species, thus appearing as mobile genetic
elements. Second, the function of some snoRNA host genes lack any
obvious direct relationship with translation. Finally, an increasing
number of snoRNAs have been found in introns of non-protein-coding
genes of elusive function (36-40). However, all snoRNA host genes
known to date are housekeeping genes exhibiting hallmarks of 5'
TOP genes (i.e. a C residue at the +1-position
followed by a 5-15-nt-long polypyrimidine tract in a short
5'-untranslated region), which could provide the basis for a
coordination of snoRNA biosynthesis at the transcriptional level (37,
40).
In the present study, we have focused our attention on the
identification of novel brain-specific box C/D snoRNAs, in order to
better assess the complexity of their repertoire and provide insights
into their biosynthetic pathway and function(s) in brain cells.
Following the construction of a rat brain cDNA library specific for
box C/D snoRNAs, we have identified and further characterized a novel
brain-specific specimen, RBII-36, without apparent equivalent in mouse
or human brain cells. Also devoid of complementarity to rRNA or snRNAs,
RBII-36 shares with two brain-specific box C/D snoRNAs recently
identified in mouse and human (17) an outstanding feature; the snoRNA
is transcribed from a complex transcription unit spanning an array of
tandemly repeated snoRNA-containing units. Moreover, RBII-36 snoRNA
appears to be processed from intronic RNA through a superimposition of
splicing-dependent and splicing-independent processing events.
Unless otherwise noted, all techniques for cloning and
manipulating nucleic acids were performed according to standard protocols.
Primers--
All oligonucleotides were synthesized on a
PerSeptive Biosystems Expedite apparatus: MBII-52,
5'-TCAGCGTAATCCTATTGAGCAT-3'; MBII-85,
5'-ACAGAGTTTTCACTCATTTGTTC-3'; IPLIB-36,
5'-TCAGACTC(G/C)CAATGTTGTGGTCATCAT-3'; IPLIB36-2,
5'-TGGACCTCAGACTCGCAATGT-3'; IPLIB36-3, 5'-TTTT(T/A)TG(G/T)TCATCAATGAT; IPLIB36-4, 5'-TGGATGT(C/T)TCAGA(G/C)GACTCCCAGATG-3'; IPLIB36-5, 5'-TCAGACTC(G/C)CAATGTTGTGGTCATCATG(G/C)ATGT(T/C)TCAGA(G/C)GACTCCCA(G/C)A-3'; primer 1, 5'-AATAAAGCGGCCGCGGATCCAA-3'; primer 2, 5'-TTGGATCCGCGGCCGCTTTATTNNNNTCAG-3'; primer 3, 5'-AATAAAGCGGCCGCGGATCCAANNNNRTGATGA-3'; U3,
5'-AAATGATCCCTGAAAGTATAGTCTT-3'; U40, 5'-AGTCAGACATTGATACTGATTCAGG-3';
U21, 5'-TGCCATCAGTCCCGTCTTGAAAC-3'; 5.8 S,
5'-TCCTGCAATTCACATTAATTCTCGCAGCTAGC-3'; 5' Bsr1,
5'-CGGGATCCAGAATTGTGAGAGTGTGAGTTCATCC-3'; 3' Bsr1,
5'-CGGAATTCTCTCCCAAGATT ATCAACTCATAGGG-3'; 5' RBII-36, 5'-CGCGGATCCGATCATTGATGACCCGAAAAAAA-3'; 3' T7RBII-36,
5'-CCGGAATTCTAATACGACTCACTATAGGGTGGACCTCAGACTCCC-3'; 5' Bsr
exon,
5'-CGGGATCCTC(T/C)TGAGGT(T/G)TGGGATTCCCGGT(G/T)T(G/T)TGT-3'; 3' Bsr exon,
5'-CGGAATTCTGAGGAAGAACT(T/C)(G/T)GA(G/A)ATCACCTTGGAGG-3'; Bsr junction, 5'-GAGGTAT(G/C)TATTACTCTC(G/A)(C/A)AATT-3'.
Restriction sites are underlined.
Construction of a Rat Brain snoRNA cDNA Library--
Half a
dissected rat brain was resuspended in 6 ml of NET-150 buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.05%
Nonidet P-40) and homogenized at 1.53 kilobar using the One Shot Model (Constant Systems Ltd.). Brain extract was then clarified by
centrifugation (10 min, 10,000 rpm in a Sorvall SS34 rotor at 4 °C),
and the supernatant was submitted to immunoprecipitation as described below. RNA extracted from 0.5 ml of PAS-Ig pellet was ligated to
phosphorylated primer 1 (10 pmol) in the presence of T4 RNA ligase as
described (10). Ligation product was used as template for cDNA
synthesis with primer 2 and Superscript II reverse transcriptase (Life
Technologies, Inc.). The cDNA product was then used as template for
PCR by Taq polymerase (Appligene) with primers 2 and 3, and the PCR product was digested by BamHI and cloned in pKS
vector. Individual clones were manually sequenced (T7 sequenase version 2.0, U.S. Biochemical Corp.).
Immunoprecipitations--
Ten microliters of rabbit
antitrimethyguanosine (anti-TMG R1131 (7 mg/ml), kindly provided by R. Lührmann) or monoclonal anti-fibrillarin 72B9 (kindly provided by
M. Pollard) were incubated with gentle agitation for 120 min at room
temperature with 2.5 mg of swollen protein A-Sepharose (Sigma) in 0.5 ml of NET-150 buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.05% Nonidet P-40). PAS-Ig pellets were washed
eight times with 1 ml of NET-150 buffer. Ten micrograms of total RNA
purified from rat brain or 0.5 ml of total brain extract (prepared as
described above for construction of the rat cDNA library) was then
added to PAS-R1131 (in 0.5 ml of NET-150) or PAS-72B9, respectively,
and incubated for 60 min at 4 °C with gentle agitation. PAS pellet
was then collected and washed eight times with 1 ml of NET-150 buffer.
RNAs from pellet was recovered by SDS/phenol extraction and analyzed by
Northern blot as described below.
Primer Extension and RNA Sequencing--
Primer extension was
carried out as described previously (41) using avian myeloblastosis
virus reverse transcriptase (Promega) at 42 °C for 60 min. Direct
RNA sequencing by primer extension was performed using the same
conditions with dideoxynucleotides elongation mixes.
Isolation and Fractionation of RNA from Tissues and Rat Brain
Areas--
Rat tissue freshly prepared from a dead animal
according to French institutional guidelines and rat brain areas
manually dissected were quickly frozen in liquid nitrogen and stored at
Cloning of Spliced Bsr cDNAs--
A rat brain cDNA
library (Lamba ZAP II library from Stratagene) has been screened by
hybridization as recommended by the manufacturer with a
32P-labeled oligonucleotide specific for spliced Bsr RNA.
Probes and Northern Blots--
A 0.9-kb-long repeat unit was
PCR-amplified from rat genomic DNA with 5'Bsr1/3'Bsr1 primers and
cloned into pBluescriptII plasmid opened by
BamHI/EcoRI. This vector was used to clone by PCR
RBII-36 snoRNA downstream from a T7 promoter (using 5' RBII-36/3' RBII-36T7 primers). A spliced Bsr riboprobe spanning four ligated exons
was cloned by RT-PCR in pBluescriptII vector using 3' Bsr exon and
5' Bsr exon primers as indicated in Fig. 5B. Internally labeled antisense RBII-36 snoRNA and spliced Bsr RNA riboprobes were
synthesized by standard in vitro transcription of linearized vectors using T3 or T7 RNA polymerases, respectively, and
[ Processing of RBII-36 snoRNA from Intronic Sequences--
The
0.9-kb-long Bsr repeat unit was PCR-amplified from genomic DNA with 5'
Bsr exon and 3' Bsr exon primers and inserted into eukaryotic vector
pRCEN downstream from the cytomegalovirus promoter. Transfection of the
construct pCMV/RBII-36 into mouse cell line L929 and processing assays
were as described (11).
In Situ Hybridization--
Detection of RBII-36 snoRNA and Bsr
RNAs by in situ hybridization was performed on adult rat
brain 10-15 µm cryostat sagittal sections as described (44) using
digoxigenin-labeled antisense snoRNA or a spliced Bsr RNA riboprobe
spanning four spliced exons.
Search for Novel Rat Brain-specific Box C/D snoRNAs in
Rat--
Taking advantage of the fact that nucleolar protein
fibrillarin specifically binds all members of this large snoRNA family, we first constructed a rat brain cDNA library specific for box C/D
snoRNAs (Fig. 1). Briefly, box C/D
snoRNPs were immunoprecipitated from a total rat brain extract by an
anti-fibrillarin antibody (72B9). Extracted RNAs were
oligonucleotide-tagged at both ends by T4 RNA ligase and amplified by
RT-PCR as described (10). RT-PCR primers were designed in such a way as
to only amplify RNA molecules containing box C (RUGAUGA) and D (CUGA)
motifs at the appropriate distance from their extremities (Fig. 1).
Amplified DNAs were then cloned, and individual clones were
systematically sequenced. A large proportion of the clones corresponded
to rat homologues of ubiquitous box C/D snoRNAs already identified in humans, mice, or other mammals (3, 15). Another substantial fraction
corresponded to novel, ubiquitously expressed rat snoRNAs either able
to target rRNA or snRNA ribose methylations or devoid of any obvious
presumptive RNA target. Finally, a few clones corresponding to
brain-specific box C/D snoRNAs were also isolated, three of them
corresponding to rat homologues of brain-specific snoRNAs previously
detected in mouse, MBII-52, MBII-85, and MBII-48 (15, 17). In this
study, we have focused our attention on three closely related clones
without equivalent among previously detected mouse snoRNAs.
Three Related Clones Correspond to a New Box C/D snoRNA--
Data
base searches revealed that closely related sequences of clones
IPLIB-36, IPLIB-76, and IPLIB-256 (Fig.
2A) shared 93-100% identity
with four EST sequences (GenBankTM accession numbers
AA900746, BE099838, AI599685, and AW919728) and also, interestingly,
with the 5'-end of a brain-specific non-protein-coding RNA, Bsr,
recently identified in rat (45). As depicted in Fig. 2B
(top), this region of Bsr contains a series of 0.9-kb-long
tandemly repeated units. Within the snoRNA-like sequences of Bsr, the
hallmark box motifs (C, D', C', and D, in that order) are generally
conserved among the different repeated units, with polymorphism
essentially restricted to length variation of a 5-19-nt-long adenosine
tract located a few nt downstream from box C (Fig. 2B).
Moreover, segments immediately upstream from box D or D' do not exhibit
a complementarity to rRNA or an snRNA of at least 9 bp, thus making it
unlikely that the snoRNA could direct a 2'-O-methylation of
one of these potential targets (41). Examination of all specimens of
the snoRNA-like sequence of Bsr available in GenBankTM
showed that a perfect 5-bp-long 5'-3'-terminal stem could
form in all cases at the expected location relative to the C and D motifs (Fig. 2B). This reinforces the notion that part of
the Bsr transcript actually encoded a box C/D snoRNA. To test this possibility, a Northern blot analysis of total rat brain RNA was performed with 32P-labeled probe IPLIB36-2. Three distinct
radioactive signals were detected at about 76, 80, and 88 nt (Fig.
2C, left), in good agreement with the size
expected for an RNA exhibiting the above-mentioned length polymorphism
and ending at the expected 5'-3'-terminal stem. Intriguingly,
we also observed a radioactive band of much faster mobility, at about
40 nt. Reverse transcription performed on total rat brain RNAs using
IPLIB36-2 primer confirmed the expected location of the snoRNA 5' end
and revealed a snoRNA size heterogeneity consistent with its being
expressed from multiple units of the Bsr repetitious array (Fig.
2C, right). In agreement with the presence of the
40-nt RNA band detected by Northern blot, an intense cDNA band
doublet was also detected at 39-40 nt, indicating that the 5'-end of
the shortened form was located 3-4 nt upstream from box C'. As
expected, the RNA bands of 76, 80, and 88 nt detected in Northern blot
with the IPLIB36-2 probe were all specifically immunoprecipitated with
the 72B9 anti-fibrillarin antibody (Fig. 2D,
left) and also with anti-hNop58 and to a lesser extent with anti-Nop140 antibodies (data not shown). This was also the case for the
5' truncated 40-nt-long species. Taken together, these data indicate
that the three closely related sequences isolated from our cDNA
library correspond to a bona fide box C/D snoRNA encoded in the Bsr locus. This snoRNA is devoid of a 5'-trimethyl cap,
as indicated by its lack of immunoprecipitation with an anti-trimethyl cap (R1131) antibody (Fig. 2D, right). In the
course of their study, Komine et al. (45) detected with the
0.9-kb Bsr cDNA probe, in addition to the long Bsr transcript, an
intriguing cross-hybridizing 80-nt-long RNA of elusive origin. In
hindsight, it is now clear that this 80-nt-long RNA must correspond to
the novel box C/D snoRNA identified in the present study, termed
RBII-36 (for rat brain fraction II 36).
RBII-36 Is Mainly Expressed in Rat Brain--
To gain more
information about the pattern of expression of RBII-36, a panel of
total RNA samples extracted from various adult rat tissues was analyzed
by Northern blot using the IPLIB36-2-specific probe (Fig.
3A). We detected a signal only
in the brain RNA sample (lane 1), although very
low levels of RBII-36 snoRNA could also be detected in ovaries and
stomach after much longer exposures (data not shown). This
tissue-specific expression pattern is in striking contrast to what has
been observed for previously identified snoRNAs guiding rRNA or snRNA
methylation. Thus, as shown in Fig. 3A, U40 and U21 snoRNA
were detected at roughly similar levels in all of the rat tissues
checked. The same membrane was also probed with sequences derived from
two brain-specific box C/D snoRNAs recently identified in mice, MBII-52
and MBII-85 (17). As expected, rat homologs of these snoRNAs were
detected, which were also specifically expressed in the brain, although
a very weak signal could also be observed in other tissues for RBII-85 snoRNA after much longer exposures (not shown). We conclude that RBII-36 snoRNA, like MBII-52 and MBII-85 snoRNAs, belongs to a novel
subset of box C/D snoRNAs specifically expressed in the brain. However,
the abundance of RBII-36 snoRNA in rat brain is much lower than that of
rat homologs of MBII-52 and MBII-85 snoRNAs, both present at levels
similar to U3 snoRNA, and is in the same range of magnitude as
ubiquitously expressed box C/D snoRNAs U21 and U40 (Fig.
3B). The expression of RBII-36 snoRNA was also studied during rat brain development (Fig. 3C). Rat brains were
dissected from embryos at days 15, 17, and 19 (E15,
E17, and E19) or newborns at days 2, 5, 13, and
21 (P2, P5, P13, P21) and
analyzed by Northern blot. Using 5.8 S rRNA as an internal reference,
quantification of the RBII-36 Northern blot signal indicated that the
RBII-36 snoRNA expression profile is remarkably constant throughout rat brain development.
RBII-36 snoRNA Expression in Different Brain Areas--
We
next investigated the distribution of RBII-36 snoRNA within various rat
brain areas. In a first step, several brain areas were isolated from
adult rat brain, total RNA was extracted and analyzed by Northern blot
using IPLIB36-2 as a probe. Fuji Bas-1000 imager analysis (Molecular
Dynamics, Inc., Sunnyvale, CA) of Northern blot signals revealed
that RBII-36 snoRNA was nearly uniformly expressed in all brain areas
checked (Fig. 4A,
lanes 2-9) with the notable exception of choroid
plexus (Fig. 4A, lane 10), which corresponds to highly vascular epithelial structures arising from the
wall of ventricles. Such a brain-regional distribution is highly
reminiscent of that observed for mouse brain-specific MBII-52 and
MBII-85 snoRNAs (17). To gain more information at the cellular and
subcellular level, we next performed in situ hybridization to rat brain sections using a digoxigenin-labeled snoRNA riboprobe. Results clearly confirm that RBII-36 snoRNA is expressed in most brain
areas (Fig. 4B and data not shown). Moreover, RBII-36 snoRNA was mainly detected within neurons and more pronounced within granular
and pyramidal layers of the hippocampus and granular layers and
Purkinje cells in the cerebellum. About 60 and 80% of neurons were
labeled in thalamus and in cortex, respectively, while no snoRNA signal
was detected within the most external cortical layers. RBII-36 snoRNA
localization within the brain thus closely parallels that reported
previously for unspliced Bsr RNA (45) and also for spliced Bsr RNA (see
below).2 We next investigated
the subcellular localization of RBII-36 snoRNA by in situ
hybridization (Fig. 4C). The position of the nucleolus was
detected by using antibodies to nucleolar protein nucleolin, and
RBII-36 snoRNA was visualized with a digoxigenin-labeled antisense
riboprobe. As shown in Fig. 4C (top), the RBII-36
snoRNA signal in Purkinje cells colocalized with the nucleolin signal, showing that the brain-specific snoRNA is mainly located within the
nucleolus. Intriguingly, a punctate secondary staining was also
systematically detected within the nucleoplasm (indicated by an
arrowhead), not only with the snoRNA antisense probe but also with the riboprobe specific for spliced Bsr RNA (Fig.
4C, bottom, and see below). This extranucleolar
signal was typically detected only once per nucleus and was generally
surrounded by tractlike structures.
RBII-36 snoRNA Is Intron-encoded in Bsr RNA--
Bsr RNA is a
noncoding RNA expressed in rat central nervous system only and consists
of a direct tandem repetition of 0.9-kb-long units that are 87-100%
identical to each other (45). Since comparison of genomic and cDNA
clones did not suggest the presence of intervening sequences in the Bsr
transcript, our detection of RBII-36 snoRNA within the Bsr RNA locus
was quite unexpected. Analysis of various Bsr cDNA sequences by the
NetGene neural network program predicted several potential donor and
acceptor splice sites (not shown). In order to assess whether Bsr RNA
was actually spliced, we performed RT-PCR using primers positioned over
the predicted exon with the highest score (Fig.
5A). Analysis of DNA products
on ethidium bromide-stained agarose gel revealed a ladder-like pattern
of at least four bands differing by about 100 bp increments
(data not shown), a result in agreement with the presence of a tandem repetition of spliced exons of the shortest predicted size
(i.e. 86 nt). This notion was confirmed by Southern blot
hybridization of the RT-PCR product using a 32P-labeled
oligonucleotide probe specific for religated exons (as depicted in Fig.
5A), which revealed the same ladder-like pattern (Fig.
5B, left). Finally, we sequenced the three
shortest DNA bands of the ladder-like pattern generated by RT-PCR,
which confirmed the predicted location of exonic junction, as shown for
the shortest, 86-nt RT-PCR product, band I (Fig. 5B,
right). Taken together, these data indicate that Bsr RNA
harbors a previously uncharacterized 807-nt-long intron containing
RBII-36 snoRNA. By screening a rat brain cDNA library with the
probe specific for spliced Bsr, we were able to isolate 12 independent
Bsr cDNA clones, which have been partially sequenced from both ends
(Fig. 5C). They all differ from each other in the number of
spliced exons, utilization of cryptic donor and acceptor splice sites
(see legend to Fig. 5, A and C), and occasional
presence of unspliced intron(s), indicating that Bsr is an
alternatively spliced RNA. Interestingly, seven of the positive clones
have identical 3'-ends. Within the common 3'-terminal region and
immediately downstream from the 3'-most copy of repeated exons E1 (Fig.
5C), we identified by Blast search an 82-nt-long tract
matching precisely a sequence located about 700 bp downstream from the
repeated Bsr units in the rat genomic clone described previously (45).
As expected, genomic sequences flanking the matching segment exhibit
hallmarks of splice junction (Fig. 5C, top
right), indicating that the 82-nt-long segment, unrelated to
repetitive exon E1 described above, is a novel exon, E2. Moreover, the
sequence of cDNA clones revealed that the 3'-end of exon E2 is
spliced to a 700-bp sequence, absent from the genomic clone mentioned
above, which exhibits homology with Lx7/LINE1 repetitive elements.
Since Bsr RNA had been proposed to be a nontranslated RNA (45),
identification of the previously undetected repeated introns prompted
us to reexamine the validity of this conclusion. No sizeable open
reading frame was detected in the spliced Bsr sequence, confirming the
notion that Bsr is a non-protein-coding RNA.
A Complex Processing Pattern for the Bsr Transcript--
To gain
more information about RBII-36 snoRNA processing from Bsr transcripts,
RNAs extracted from rat brain were assayed by Northern blot
hybridization with a riboprobe specific for spliced Bsr RNA (Fig.
6A, right). A
heterodisperse signal (from about 0.5 to more than 10 kb) was detected
for total RNA (lane 3) as well as for
poly(A) RBII-36 snoRNA Is Only Detected in Rats--
Most box C/D snoRNAs
have homologues in distant vertebrate species that can be detected by
Northern cross-hybridizations using oligonucleotide probes directed to
their conserved antisense element (9, 43, 46). Given that the 3' half
of RBII-36 snoRNA sequence is preferentially conserved among the
different tandemly repeated units (Fig. 2B), we first
selected the IPLIB36-2 probe to look for homologues in other vertebrate
species. At moderate hybridization stringency, RBII-36 snoRNA was
detected in rat only, whereas homologs of brain-specific snoRNAs
MBII-52 and MBII-85 (17) could be detected in distantly related
placental mammals but not in chickens or even in marsupials, as shown
in Fig. 3D. At very low stringency (27 °C), weak signals
were detected for both human and mouse brain RNA (Fig. 3D),
but their significance remains uncertain because they were not
reproducible (data not shown). Moreover, in these very low stringency
conditions we could not detect any signal for humans or mice with four
other RBII-36 probes, IPLIB36-1, IPLIB36-3, IPLIB36-4, and IPLIB36-5
(not shown). We also tried to detect the potential presence of
RBII-36-like, tandemly repeated sequences in the genome of various
rodent species (a generous gift from F. Catzeflis) by PCR amplification
performed on genomic DNA with a pair of divergently oriented primers
spanning the most conserved part of snoRNA sequence among the rat
repeated units. A PCR product of roughly similar size was obtained with
each rodent belonging to the genus Rattus sensu
lato (47), such as Rattus rattus, Niviventer
rapit, Maxomys hylomyoides, and
Leopoldamys sabanus. Conversely, no signal was detected for
other rodent species outside this genus, such as Mus
musculus, Mus pahari, and Mus saxicola (not
shown). We therefore conclude that either RBII-36 is a rat-specific
snoRNA or that its mouse/human counterparts are very poorly
expressed and/or evolutionarily divergent.
RBII-36 snoRNA, a Novel Rat Brain-specific C/D Box snoRNA Lacking
an Obvious RNA Target--
By sequencing a cDNA library specific
for rat brain C/D box snoRNAs (Fig. 1), we have isolated a novel C/D
snoRNA, RBII-36, which, in contrast to the vast majority of C/D
snoRNAs, is only expressed in the central nervous system (Fig.
3A). RBII-36 accumulates within nucleoli of neuronal cell
types, with a particularly strong expression in pyramidal layers of the
hippocampus and Purkinje cells of the cerebellum (Fig. 4, B
and C). The lack of any potential antisense element able to
direct a 2'-O-ribose methylation to rRNA or an snRNA raises
puzzling questions about its function and the identity of its potential
RNA target in neuronal cells, like for the few brain-specific C/D
box snoRNAs recently identified in mouse (17). A growing list of C/D
box snoRNAs devoid of likely rRNA or snRNA targets has been recently
identified (15, 16), suggesting the possibility that the repertoire of
cellular RNAs targeted by methylation guide snoRNAs could be larger
than anticipated. In this regard, several stable ubiquitous noncoding
RNA species transit through the nucleolus, which seems to be involved
in the metabolism of a variety of cellular RNAs in addition to rRNA
(48, 49). Moreover, archaeal homologs of C/D box snoRNAs direct
methylation of not only rRNAs but also
tRNAs.3,4
The detection of brain-specific C/D box snoRNAs without rRNA or snRNA
complementarity might reflect their targeting of brain-specific RNAs,
possibly messenger RNAs, although no internal ribose-methylated nucleotide modification has been reported so far in mRNA. In line with this notion, one of the brain-specific C/D box snoRNAs recently reported in mouse, MBII-52, contains a long (18-nt) phylogenetically conserved antisense element directed against a brain-specific mRNA
encoding a serotonin receptor (17). Remarkably, the mRNA nucleotide
predicted to be methylated by MBII-52 snoRNA is an adenosine undergoing
conversion to inosine (50), suggesting that the snoRNA is involved in a
control of the editing process, since methylation of an adenosine to be
edited strongly inhibits its deamination in vitro (51). In
this hypothesis, the presence of inosine at tissue-specific levels,
particularly elevated in brain mRNAs (52), hints at the possibility
that additional brain-specific box C/D snoRNAs with long
complementarities to brain mRNAs still await identification.
Searches of genomic data bases have not provided so far any indication
as to the identity of a potential mRNA target for RBII-36 snoRNA
among a large number of candidate sequences in mammals. The punctate
extranucleolar signal detected within nucleoplasm by in situ
hybridization with the snoRNA probe (Fig. 4C,
top) could correspond to the nuclear site where RBII-36 snoRNA interacts with its still unknown cellular RNA target.
Alternatively, it could represent the site of either transcription or
early processing of Bsr RNA, in line with our detection of a similar
signal with a probe specific for spliced Bsr (Fig. 4C,
bottom). Ongoing analyses with additional probes within the
Bsr locus should provide further insight into this question.
Bsr, an Intriguing snoRNA-containing Host Gene--
Eukaryotic
cells contain an unexpectedly large number of noncoding RNAs, which
usually have a narrow expression pattern and seem to play pivotal
role(s) in many aspects of gene expression (53). Strikingly, several
noncoding RNAs of elusive function belonging to the 5' TOP family of
ubiquitous vertebrate genes host one or several intronic C/D snoRNAs:
U19HG (36), U50HG (38), U17HG (40),
gas5 (37), and UHG (39). Noncoding RNA Bsr
was first described (45) as an intronless transcript isolated upon
characterization of genes involved in cerebellar long term depression.
It is mainly expressed in the rat central nervous system and consists
of tandem repeats of 0.9-kb-long sequences (45). In this study, we have
shown that the tandemly repeated units within Bsr RNA actually contain
an intron from which RBII-36 snoRNA is processed (Fig. 5) and that the
intriguing 75-nt-long RNA species detected during the initial
characterization of Bsr RNA corresponds to the novel RBII-36 snoRNA.
Curiously, the tandemly repeated organization of snoRNA genes detected
for RBII-36 is highly reminiscent of that of two other brain-specific
snoRNAs also encoded within noncoding RNA genes in mice and humans (17, 54). The relationship, if any, of this particular type of gene organization to the expression of brain-specific snoRNAs remains elusive. The Bsr RNA gene could be identified only within the Rattus genus (Ref. 45 and data not shown), and its function is unknown. The absence of the long open reading frame and the exclusively nuclear location of spliced Bsr RNA (Fig. 4C)
strongly argue against its protein coding potential. In this regard, it is noteworthy that genes of noncoding RNAs hosting a ubiquitous snoRNA
within one or several of their introns, such as UHG,
gas5, and U19HG, generally exhibit highly
divergent exons among mice and humans, suggesting that the
intron-encoded snoRNAs could well be the only functional entities of
these complex transcripts (36, 37, 39). However, we cannot rule out the
possibility that spliced Bsr RNAs have a function per se,
like Xlsirt noncoding RNAs, a family of interspersed repeat RNAs of
about 80 nt required for the proper intracellular location of Vg1 RNA
in Xenopus oocyte (55). Finally, another tantalizing
question concerns the potential link between noncoding RNAs and
imprinted chomosomal regions, since many noncoding RNAs are imprinted
(see Ref. 56 for a review), and imprinted loci are often associated
with repeated sequences (57).
An Unusual Processing Pathway for RBII-36 snoRNA--
Most
intronic snoRNAs are produced by a splicing-dependent
processing pathway involving exonucleolytic trimming of the debranched lariat (for a review, see Ref. 6). Another minor, splicing-independent pathway involving endonucleolytic cleavage within the intron followed by exonucleolytic trimming has been identified for U16 and U18 snoRNAs
in Xenopus oocytes and for U18 in S. cerevisiae
(58-61). In mouse, brain-specific MBII-52 snoRNA is also encoded in
tandemly repeated introns, but boundaries and fate of cognate
transcript(s) have not been assessed so far (17). We have identified
spliced Bsr RNA products containing at least eight ligated exons (Fig. 5C) together with hemiprocessed snoRNA-containing Bsr RNA
precursors (Fig. 6, A and B; Ref. 45), strongly
arguing for the superimposition of the two mutually exclusive snoRNA
biosynthetic pathways mentioned above (Fig.
7). The heterodisperse Bsr RNA signal
(ranging from about 500 bp to more than 10 kb) could reflect
transcription from multiple promoters as well as complex alternative
splicing of a common transcript synthesized from a unique promoter. The
heterodisperse signal, also reported for brain-specific noncoding RNAs
IPW and PWCR1, which carry a snoRNA-containing
tandem repetition (54, 62), might also reflect nuclear degradation of
untranslatable transcripts via NMD (63). Boundaries of available Bsr
cDNA clones suggest that endonucleolytic cleavages of the Bsr
transcript preferentially occur within a few nucleotide positions
upstream from the RBII-36 snoRNA sequence in the intron, within an
irregular 27-bp-long secondary structure involving both snoRNA flanking
sequences in the intron (Fig. 6C). Interestingly, a long
base-paired structure in C/D snoRNA-containing introns promotes
intronic endonucleolytic cleavages (64, 65). The truncated form of
RBII-36 snoRNA lacking the box C-containing 5'-end (Fig. 2C)
could result from an impaired arrest of 5'-3' exonucleolytic trimming,
possibly due to a noncanonical 5'-3'-terminal stem-box C/D
structure. Alternatively, it could reflect the presence of some
truncated Bsr gene copies, possibly separate from the regularly
repeated array, as reported for snoRNA-containing PWCR1 genes in humans (54). In line with this notion,
we have identified several rat ESTs (GenBankTM accession
numbers BF389774, BF386393, and BF390839) exhibiting the
truncated snoRNA sequence at their 3'-end, while their immediately
upstream sequences do not match any known Bsr sequence. Accumulation of
the relatively abundant truncated form of the snoRNA (Fig.
2C) and its efficient immunoprecipitation by antibodies
against C/D box snoRNA-binding proteins (Fig. 2D, left, and data not shown) suggests that C' box might
substitute for C box to form the terminal stem-box structure,
recognized by snoRNP core proteins, which acts as nucleolar
localization signal (66). The detection of RBII-36 snoRNA, the first
mammalian intron-encoded snoRNA likely to result from a
splicing-independent processing pathway, adds another level of
diversity to the strategies used to produce snoRNAs in eukaryotic
cells.
Implications of the Tandemly Repeated snoRNA Gene
Organization--
While exhibiting structural hallmarks of a
bona fide methylation guide snoRNA, RBII-36 strikingly
departs from known mammalian C/D box snoRNAs by being exclusively
detected in the Rattus genus, suggesting it might have
originated rather recently in the evolution of rodents (Ref. 45; Fig.
3D and data not shown). In contrast, known ubiquitous
intronic C/D box snoRNAs, whether they are encoded in protein genes or
in noncoding RNAs, are conserved over a much larger span of vertebrate
phylogeny (9, 37, 39, 43, 46). The three previously reported
brain-specific C/D box snoRNAs also seem substantially more ancient
than RBII-36, since their origin must predate the primates/rodents
split during mammalian evolution (17). Due to both its recent origin
and outstanding gene organization, the novel brain-specific snoRNA
should represent a valuable system to catch a glimpse of the genetic
processes underlying the evolution of the large family of box C/D
snoRNAs in vertebrates. Although a large fraction of rRNA
2'-O-methylations are conserved among yeast and vertebrates,
the vertebrate pattern is definitely more complex. Moreover, this
pattern exhibits some substantial differences among distantly related
vertebrates, with the presence of a few additional methylations in
mammals as compared with amphibian Xenopus laevis (7),
suggesting that cognate ubiquitous snoRNAs have originated at
relatively late stages during vertebrate evolution. RBII-36 genes could
represent a paradigm of an intermediate step in the expansion and
functional diversification of the C/D box snoRNA family in vertebrate
genomes. Their tandemly repeated organization could provide the
structural basis for the generation of a large reservoir of novel C/D
box snoRNA sequences among which some might eventually acquire the
ability to recognize entirely novel RNA targets. Analysis of the
RBII-36-containing repeat unit and derivation of the snoRNA sequence in
other representatives of the Rattus genus could provide
hints as to the actual function of the recently created snoRNA.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
80 °C. Total cell RNA was isolated by the method of Chomczynski
and Sacchi (42) adapted to our conditions (43). Poly(A)+
RNA purification was performed as recommended by manufacturer (Promega). Fractionation of RNAs was carried out by electrophoresis in
1.2% agarose, 2.2 M formaldehyde or 6% acrylamide,
7 M urea gels. Fractionated RNAs were transferred
electrophoretically (120 min in 0.5× TBE at 1 mA) to nylon membrane
(Hybond N+; Amersham Pharmacia Biotech) and cross-linked by
UV irradiation (120,000 µJ/cm2, Stratalinker, Stratagene,
La Jolla, CA) before Northern blot hybridization.
-32P]CTP. Northern blot hybridizations were carried
out with oligodeoxynucleotide probes 5'-end-labeled with
[
-32P]NTP (5·106 cpm/ml). Incubations
were performed in 5× SSPE, 1% SDS, 5× Denhardt's solution, 150 µg/ml yeast tRNA at 50 °C or at 30 °C (low hybridization stringency conditions) either overnight or for 3 h. Membranes were
washed twice for 15 min with 0.1× SSPE, 0.1% SDS or with 2× SSPE,
0.1% SDS at room temperature (low hybridization stringency conditions). For riboprobe hybridization, Northern blots were carried
out through overnight incubation at 65 °C in 5× SSPE, 1% SDS, 5×
Denhardt's solution, 150 µg/ml yeast tRNA, 50% (v/v) deionized
formamide, and membranes were washed twice for 15 min in 2× SSPE,
0.1% SDS at 65 °C and then for 30 min in 1× SSPE, 0.1% SDS and
finally for 15 min at room temperature in 0.1× SSPE, 0.1% SDS.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Construction of a cDNA library for box
C/D snoRNAs in rat brain. A, experimental strategy. A
rat brain extract was immunoprecipitated by an anti-fibrillarin
antibody (72B9), and extracted RNAs were tagged at both ends by
oligonucleotide primer 1 using T4 RNA ligase. Tagged RNAs were
amplified by RT-PCR using primers 2 and 3, designed so as to amplify
RNAs containing box C (RUGAUGA) and box D (CUGA) motifs at the
appropriate distance from their 5'- and 3'-ends; the two primers
included 4 degenerated positions between the primer 1-matching sequence
and their 3'-terminal sequence (matching either box C or box D).
Amplified DNAs were cloned into pKS vector, individual clones were
systematically sequenced, and new snoRNAs were assayed by Northern blot
as described in the legend to Fig. 2B.
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Fig. 2.
IPLIB clones representing a novel rat box C/D
snoRNA. A, sequences of the three related cDNA
clones isolated in this study. B, alignment of EST and Bsr
clones exhibiting homology with the three related cDNA clones
described in the legend to Fig. 2A. Top,
schematic representation of a previously characterized Bsr
genomic clone (GenBankTM accession number AB014883)
containing four 0.9-kb-long repeated units (45). The position of the
IPLIB snoRNA-like sequence (80-90 nt) within 1 unit is indicated by a
black box. EST and Bsr clones are designated by
their GenBankTM accession numbers, with the letter in
parentheses referring to a particular repeat within the
clone according the previously used terminology (45). Box C and D
motifs are boxed, and nucleotides presumably involved in the formation
of a 5'-3'-terminal stem are delineated by
horizontal arrows in opposite orientation.
Below the alignment, nucleotide positions conserved in at
least 16 of the 18 Bsr repeats are denoted by asterisks, and
the location of the different oligonucleotide probes or primers is
shown by bars. The arrowheads denote the 5'-end
of the truncated 40-nt-long snoRNA form. C, detection of the
cognate snoRNA in total rat brain. Left, Northern blot
hybridization with 32P-labeled probe IPLIB36-2 using 10 µg of total brain RNA (lane M, size marker).
Right, 5'-end identification by primer extension with
32P-labeled oligonucleotide IPLIB36-2 using 10 µg of
total brain RNA. Top, cDNA bands corresponding to
full-size snoRNA. Bottom, 5'-truncated, 40-nt-long cDNA.
In both cases, the corresponding RNA sequencing pattern is also shown.
Correspondences between Northern bands and primer extension products
are denoted by arrows. D, immunoprecipitation by
anti-fibrillarin 72B9 (left) and anti-trimethyl cap R1131
(right) antibodies. RBII-36 snoRNA was detected by Northern
blot hybridization with IPLIB36-2 probe, and membranes were also probed
with U3- or U2-specific probes, respectively, as positive controls, and
with a 5.8 S rRNA probe as a negative control. Lanes
1 and 3, input RNA; lanes 2 and 5, RNA recovered from the pellet; lane
4, RNA recovered from the supernatant.
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Fig. 3.
Brain-specific expression of RBII-36
snoRNA. A, RBII-36 snoRNA abundance in various adult
rat tissues. Equal amounts of total RNA prepared from adult rat tissues
(as specified at the top of each lane) were
fractionated in a 6% polyacrylamide gel, and RBII-36 snoRNA was
detected by Northern blot as in Fig. 2A. The membrane was
also probed for MBII-52 and MBII-85 and for U3, U21, and U40 snoRNAs
(M, DNA size marker in nt). B, abundance of
RBII-36 snoRNA in rat brain. Decreasing amounts of total rat brain RNA
(15, 5, and 1 µg, in lanes 1, 2, and
3, respectively) were separated on a 6% polyacrylamide gel,
and RBII-36 snoRNA was detected by Northern blot as in Fig.
2A. The same membrane was also hybridized with
oligonucleotide probes specific to snoRNAs MBII-52, MBII-85, U3, U21,
and U40, 32P-end-labeled to the same specific activity (all
blots were exposed for identical times). C, RBII-36 snoRNA
expression pattern in the developing rat brain. Ten micrograms of total
RNA from developing rat brains were fractionated in a 6%
polyacrylamide gel, and RBII-36 snoRNA was detected by Northern blot as
in Fig. 2A. The various developmental stages (embryonic
days, E15-19; postnatal days, P2-21) are
indicated in each lane. Signal intensities were quantified
with a Fuji Bas-1000 imager. D, search for vertebrate
homologs of RBII-36 snoRNA. Northern blot analysis was performed at
high (50 °C) or low (27 °C) hybridization stringency on 10-µg
RNA samples from the brain of the indicated species (top of
each lane) using IPLIB36-2 as a probe. Membranes were also
probed with 5.8 S rRNA-specific oligonucleotides for gel loading
control.
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Fig. 4.
RBII-36 snoRNA expression pattern in rat
brain. A, Northern blot hybridization of RNA from major
rat brain areas. Five micrograms of total RNA extracted from manually
dissected brain areas from adult rat were analyzed by Northern blot as
indicated in Fig. 2A, and the same membrane was also
hybridized with a 5.8 S rRNA probe for gel loading controls. Signal
intensities were quantified with a Fuji Bas-1000 imager. B,
in situ hybridization on brain sections with a
digoxigenin-labeled snoRNA probe. Panel of different brain areas
in sagittal sections of adult rat brain, as indicated in each
image. DG, dentate gyrus; CGC,
cerebellar granular cells; PC, Purkinje cells.
CA1, CA2, and CA3 represent different
areas from of hippocampus. A bar represents 100 micrometers.
Bottom, 4',6-diamidino-2-phenylindole (DAPI)
staining (only shown for hippocampus) to visualize position of the
cells. C, subcellular location of RBII-36 snoRNA
(top) or Bsr RNA (bottom) within Purkinje cells.
Field was 20 × 20 µm. 4',6-Diamidino-2-phenylindole staining
indicates the position of the nucleus, while nucleoli are visualized by
an anti-nucleolin antibody. RBII-36 snoRNA or spliced Bsr RNA are
detected as described in the legend to Fig. 4B.
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Fig. 5.
Identification of an intron in Bsr RNA.
A, sequence of a single arbitrarily chosen Bsr RNA repeat
unit (GenBankTM accession number AB01879). Exon sequences
are in uppercase type, intron sequence in
lowercase, and the snoRNA sequence is underlined.
Major spliced sites are underlined, whereas minor ones,
identified by sequence analysis of a rat brain cDNA library, are
boxed. The location of RT-PCR primers used in B)
(filled bars) and the two moieties of the probe
specific for spliced exons (hollow bars) are also
shown. B, detection of the Bsr spliced exons by RT-PCR
analysis. Total rat brain RNA (10 µg) was annealed with 3' Bsr exon
primer, and cDNA was synthesized with avian myeloblastosis virus
reverse transcriptase before amplification by PCR with 5' and 3' Bsr
exon primers. PCR products were fractionated on a 2% agarose gel and
analyzed by Southern blotting with 32P-labeled Bsr junction
oligonucleotide probe specific for spliced exons (lane
1, control experiment performed without reverse
transcriptase). Right, sequence analysis at the spliced
junction for band I. A nucleotide polymorphism relative to the repeat
unit sequence shown in Fig. 5A is denoted by a
star. C, schematic representation of 12 partially
sequenced Bsr cDNAs clones isolated upon screening of a rat brain
cDNA library. Portions of cDNA clones that have not been
sequenced are schematized by dots. The size of each Bsr
cDNA insert (estimated by enzymatic digestion analysis) is
indicated in parenthesis (a star denotes the
presence of RBII-36 snoRNA sequence within the unsequenced portion of
the clone, as assessed by dot blot hybridization). Repeated Bsr exons
E1 are represented by open boxes, and an
additional unrelated exon, E2, is represented by a black
box (its sequence is in capital
letters (top)). An unspliced intron I1
(thick line, with the snoRNA coding region
depicted by an open arrow) is occasionally
detected. Other portions of the clones exhibiting homologies to rat
ESTs are also indicated. Gray box, Lx7#LINE/L1
repetitive element (GenBankTM accession numbers AI072426
and BE105131); hatched box, GenBankTM
accession numbers AI180055, BF551987, and AI574801), The
wavy line denotes the 5' part of an unspliced
intron, I2, located between E1 and E2 exon (GenBankTM
accession number AB014882).
and poly(A)+ RNAs (lanes
4 and 5, respectively), reflecting the presence
of a complex spectrum of Bsr RNA processing products. Northern blot analysis with a snoRNA-specific probe also revealed an heterodisperse signal in the same size range in addition to mature RBII-36 snoRNA (Fig. 6A, left). However, superimposed on
the heterodisperse signal, discrete bands at about 0.9, 1.8, and 2.7 kb
were also apparent, which could result from endonucleolytic cleavages
of unspliced Bsr transcripts taking place at a specific site within the
0.9-kb repeat unit. It is noteworthy that among the 33 Bsr cDNA
clones described previously (45) seven of them have their 5'-end
mapping within the same 16-bp segment of the 0.9-kb repeat unit,
immediately upstream from the snoRNA coding region. Remarkably, 5'
boundaries of two of these clones, AB014878 and AB01881, map exactly at the 5'-end of mature and truncated RBII-36 snoRNAs, respectively. Moreover, the 3'-terminal region of one of the Bsr cDNAs identified in the present work, clone 11.5, corresponds to the entire portion of
the intron upstream from the mature snoRNA coding region (Figs. 5C and 6C). Likewise, the 5' boundary of two
other previously reported clones (AB014877 and AB014879) maps only 10 nt upstream from the 5' end of mature RBII-36 snoRNA (Fig. 6,
B and C). Moreover, the 3'-end of clones
AB014877, AB014879, and AB014880 also maps only a few nt upstream from
the expected location of the 5'-end of a RBII-36 snoRNA copy in the
downstream repeat unit (Fig. 6, B and C). Taken
together, these observations strongly suggest that the 0.9-, 1.8-, and
2.7-kb-long RNA species might represent hemiprocessed intermediates of
RBII-36 snoRNA biogenesis (as depicted in Fig. 6A,
left). A single Bsr 0.9-kb repeat unit spanning the
snoRNA-containing intron and two flanking exons was transiently
expressed under the control of a strong viral promoter in mouse L929
cells. As shown in Fig. 6D, faithfully processed RBII-36
snoRNA was detected in transfected mouse cells. This strongly suggests
that brain specificity of the snoRNA expression is exclusively
determined at the transcriptional level and that snoRNA processing from
brain-specific Bsr transcripts is not dependent upon brain-specific
factors. It is also noteworthy that the 5'-truncated, 40-nt-long form
of RBII-36 was not detected in transfected mouse cells, consistent with
the possibility that this form originates from a subset of truncated
repeat units.
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Fig. 6.
RBII-36 snoRNA biosynthetic pathway.
A, detection of unspliced and spliced Bsr RNA by Northern
blot hybridization. Ten-microgram samples of total RNA from rat brain
(lanes 1 and 3), rat liver
(lanes 2 and 6), rat brain
poly(A) RNA (lane 4), and rat brain
poly(A)+ RNA (lane 5) were
fractionated on 1.2% formaldehyde-agarose gels. Membranes were probed
with either a snoRNA RNA riboprobe (left) or with a probe
specific for spliced Bsr (right). Probes specific for 5.8 S
rRNA and actin mRNA were also used as controls for gel loading and
recovery of poly(A)+ RNA, respectively. B,
schematic representation of previously reported Bsr cDNA clones
(45). Exons are represented by black boxes, and
the intronic snoRNA is represented by an open
arrow. The numbering of each repeat unit in Bsr
cDNA is according to Ref. 45. C, location of the ends of
several Bsr cDNA clones within the secondary structure of Bsr
transcript. Sequence polymorphisms among units are shown in
parenthesis. D, expression of RBII-36 snoRNA from
a Bsr repeat unit transfected into mouse L929 cells. Left,
schematic representation of the construct expressing a repeat unit
under the control of the strong cytomegalovirus promoter.
Right, Northern blot analysis of transfected cell RNA using
a 32P-labeled oligonucleotide probe specific for RBII-36
snoRNA, IPLIB36-2. Lane 1, total rat brain RNA;
lane 2, total RNA from untransfected mouse L929
cell; lanes 3-5, total RNA from mouse L929 cells
transfected with three independent pCMV-RBII-36 clones; lane
M, DNA size markers (size in nt).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 7.
A model for RBII-36 snoRNA biosynthesis.
Bsr genes are transcribed as long RNA precursors from one or several
promoters. In the splicing-dependent model
(left), Bsr RNAs are alternatively spliced, and RBII-36
snoRNA is processed from the debranched intron 1 lariat. In the
splicing-independent model (right), Bsr transcripts are
cleaved endonucleolytically within each repeated intron (immediately
upstream from the snoRNA coding region), as reflected by the
ladder-like pattern of RNA fragments of about 0.9, 1.8, and 2.7 kb
detected by Northern blot (Fig. 6A, left). The
snoRNA-containing debranched lariat and the product of endonucleolytic
cleavages are both further processed by exonucleolytic trimming.
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ACKNOWLEDGEMENTS |
---|
We thank N. Joseph for technical assistance in DNA sequencing, Y. de Preval for oligonucleotide synthesis, E. Bertrand for technical advice on in situ hybridization, Gerard Alonso for advice on rat brain anatomy, and P. Veyrac, S. Mazan, J. M. Zajac, S. Sordello, S. Chatelain, P. Fons, and V. Perez for assistance in animal dissection. We are also grateful to J. Demmer and F. Catzeflis for the gift of an oppossum brain and rodent tissues belonging to the R. sensu lato species (from the collection of preserved mammalian tissues of the Institut des Sciences de l'Evolution, Montpellier II), respectively. We thank R. Lührmann for anti-trimethyl cap antibody (R1131), M. Pollard for anti-fibrillarin antibody (72B9), and G. Joseph for anti-nucleolin antibody (134).
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FOOTNOTES |
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* This work was supported by a grant from the Association pour la Recherche sur le Cancer (to J. P. B.), by laboratory funds from the Center National de la Recherche Scientifique and Université Paul-Sabatier, Toulouse, and by the German Human Genome Project through Bundesministerium für Bildung, Wissenschaft, Forschung Grant 01KW9966 and an Interdisziplinäres Zentrum für Klinische Forschung grant (Teilprojekt F3, Münster) (to A. H.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence may be addressed. Tel.: 33 5 61 33 59 34; Fax: 33 5 61 33 58 86; E-mail: cavaille@ibcg.biotoul.fr.
** To whom correspondence may be addressed. Tel.: 33 5 61 33 59 34; Fax: 33 5 61 33 58 86; E-mail: bachel@ibcg.biotoul.fr.
Published, JBC Papers in Press, May 9, 2001, DOI 10.1074/jbc.M103544200
2 E. Basyuk, et al., unpublished data.
3 P. Dennis, personal communication.
4 B. Clouet, C. Gaspin, and J. P. Bachellerie, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: snoRNP, small nucleolar ribonucleoprotein particle; snoRNA, small nucleolar RNA; snRNA, small nuclear RNA; nt, nucleotide(s); PCR, polymerase chain reaction; RT-PCR, reverse transcriptase-PCR; kb, kilobase(s); EST, expressed sequence tag; bp, base pair(s).
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