From the Department of Biological Chemistry, Weizmann
Institute of Science, Rehovot 76100, Israel and the ¶ Faculty of
Life Sciences, Bar-Ilan University, Ramat-Gan 52900, Israel
Received for publication, August 3, 2000, and in revised form, January 2, 2001
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ABSTRACT |
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We analyzed three chromosomal loci of the
trypanosomatid Leptomonas collosoma encoding box C/D small
nucleolar RNAs (snoRNAs). All the snoRNAs that were analyzed here carry
two sequences complementary to rRNA target sites and obey the +5 rule
for guide methylation. Studies on transgenic parasites carrying the
snoRNA-2 gene in the episomal expression vector (pX-neo)
indicated that no promoter activity was found immediately adjacent to
this gene. Deleting the flanking sequences of snoRNA-2 affected the
expression; in the absence of the 3'-flanking (but not 5'-flanking)
sequence, the expression was almost completely abolished. The snoRNA
genes are transcribed as polycistronic RNA. All snoRNAs can be folded into a common stem-loop structure, which may play a role in processing the polycistronic transcript. snoRNA B2, a member of a snoRNA cluster,
was expressed when cloned into the episomal vector, suggesting that
each gene within a cluster is individually processed. Studies with
permeable cells indicated that snoRNA gene transcription was relatively
sensitive to The nucleolus of eukaryotic cells contains numerous small
nucleolar RNAs (snoRNAs)1
that play a regulatory role in rRNA biogenesis (1-5). The snoRNAs can
be divided into two major groups: the box C/D snoRNAs and H/ACA snoRNAs
(6-8). Although some of the snoRNAs are involved in pre-rRNA cleavage,
most function in site-specific modification, i.e.
2'-O-ribose methylation and pseudouridine formation.
Ribose methylations are guided by box C/D snoRNAs that
contain two sequence motifs, box C (5'-PuUGAUGA-3', where Pu is
purine) and box D (5'-CUGA-3') (7, 9, 10), which are required for their processing and stability and are important for binding the
nucleolar protein fibrillarin (11, 12). The box C/D snoRNAs contain
long stretches (>10 nucleotides) that perfectly complement the
universal core regions of the mature rRNA. Studies in both yeast and
mammals indicate that the fifth nucleotide (+5) upstream from box D or
D' within the domains of interaction between the guide RNA and the rRNA
target site is methylated; this is known as the +5 rule (7, 10,
13).
snoRNA genes have been of interest because of their distinct mode of
expression. The essential and abundant snoRNAs such as U3, U8, and U13
are expressed from their own promoters, whereas most vertebrate and
some yeast snoRNAs are encoded by introns of host genes (1, 5). In
mammalian cells, most of the snoRNAs are encoded by introns of host
genes that encode proteins involved in ribosome biosynthesis and
function (14-16). However, in yeast, only a few snoRNAs are encoded by
introns, and most of them are independently transcribed (1, 4). The
maturation of most of the intron-encoded snoRNAs involves debranching
of the lariat, followed by exonucleolytic trimming (17). The
self-transcribed snoRNAs are processed from a precursor by
endonucleolytic cleavage and exonucleolytic trimming (18). In yeast,
the exonucleases Rat1p and Xrn1p were shown to play a role in 5' to 3'
trimming (18-20). In addition, the processing of the independently
transcribed box C/D or H/ACA snoRNAs in yeast requires the endonuclease
Rnt1p, which is the yeast ortholog of bacterial RNase III.
Interestingly, Rnt1p specifically cleaves the duplex region of
pre-snoRNAs (21, 22). The cleaved precursor is then processed by
exonuclease trimming. The splicing-independent processing mechanism
also functions in processing the clustered plant snoRNAs, which are
processed from a precursor molecule (23). Protein factor(s) were found to bind the snoRNA termini, including boxes C and D (24, 25), which
protect the snoRNAs from further degradation by the processing exonucleases.
Trypanosomatids are protozoan parasites that diverged very early in the
eukaryotic lineage (26). These organisms possess some unique
RNA-processing pathways, including pre-mRNA
trans-splicing (27, 28) and RNA editing (29). So far, there
is no evidence that polymerase (pol) II-regulated promoters exist; the
genes are regulated post-transcriptionally by mRNA stability (30, 31). Trypanosomatid rRNA also has a peculiar processing pathway; the
large subunit rRNA (28 S) is further processed into two large and six
small rRNA fragments (32).
Very little is known so far about ribosome biogenesis in
trypanosomatids. Previously, we reported the identification of the first box C/D snoRNA, which was termed snoRNA-2 in
Leptomonas collosoma (33). We suggested that
trypanosomatids obey the canonical +5 rule for snoRNA-mediated
methylation. The snoRNAs located within the spliced leader-associated
(SLA1) RNA loci were studied in several trypanosomatids,
including Trypanosoma brucei, Trypanosoma cruzi, and Leishmania tarentolae. It was found
that the trypanosomatid snoRNAs do not obey the general methylation
rules. More specifically, the methylation sites corresponding to the
duplex formed between the snoRNA and the target site are located either
1 or 6 nucleotides upstream from box D or D' (34). More recently, 17 box C/D snoRNAs were analyzed from T. brucei (35) by
immunoprecipitation using antibodies against the cloned T. brucei fibrillarin protein. Mapping of the methylation sites
potentially guided by eight snoRNAs suggested that the T. brucei snoRNAs obey the +5 rule.
To further elucidate the trypanosomatid snoRNA structure-function
relationship and the mode of transcription and processing, we
specifically studied four box C/D snoRNAs encoded by three chromosomal
loci. Our analyses indicate that there is no irregularity in the guide
methylation rule of the trypanosomatid snoRNAs and that the L. collosoma box C/D snoRNAs obey the +5 rule. The genes we analyzed
were transcribed as polycistronic precursors. Proper expression of a
tagged snoRNA-2 gene cloned into the episomal vector pX-neo
requires at least 20-nucleotide flanking sequences. Expression,
although at a lower level, was detected even in the absence of an
upstream sequence, suggesting the lack of a conventional promoter
adjacent to the gene. However, we cannot exclude the possibility that a
promoter may lie upstream from the gene cluster. Transcriptional
studies performed in permeable cells suggest that snoRNA gene
transcription is relatively sensitive to Oligonucleotides--
The oligonucleotides for snoRNA-2 (33)
were as follows: 16865, 5'-ATCAGATGCCGGTAGTC-3', antisense from
positions 67 to 83 of the coding region; 22075, 5'-CGGGATCCAATGATGCACCAGGCTCGCG-3', sense upstream from
positions
The oligonucleotides for snoRNA B2 were as follows: 26553, 5'-CGGGATCCCCGTTGCGCTATTCGGTCTT-3', sense upstream from
positions
The oligonucleotides for snoRNA TS1 were as follows: 20936, 5'-CCGAAAGGTGATTGTT-3', antisense to the 3'-end of the coding region
from positions 67 to 82; and 26551, 5'-AGGCCTCCACCGGGGTTTCC-3', antisense to 28 S rRNA from positions 958 to 977 for mapping the methylation site guided by snoRNA TS1.
The oligonucleotides for snoRNA G2 were as follows: 20405, 5'-TGAGCTGAGTTGAAGTT-3', antisense to the 3'-end from positions 56 to
72 of the gene; 22078, 5'-CGGGATCCAAAGAGAGGGACGCGACA-3', antisense downstream from positions 40 to 57; 26263, 5'-TGGATGGGTTTAAATATCCC-3', antisense to the small subunit of rRNA (18 S) from positions 560 to 570 for mapping the methylation site guided by
snoRNA G2; and 26552, 5'-CAATCTCCAGCCAAGTAGGG-3', antisense to 28 S
rRNA from positions 3746 to 3765 for mapping the methylation site
guided by snoRNA G2.
The oligonucleotides for snoRNA TS2 were as follows: 20935, 5'-ACCTGTTGAGTTTTGT-3', antisense to the 3'-end from positions 67 to 82 of the gene; 22077, 5'-CGGGATCCACGAAGTGGTGTGCGTAT-3', sense
upstream from positions
Other oligonucleotides were as follows: 21995, 5'-GAGGGAGGAATGAGGTGAGC-3', complementary to neo
mRNA complementary to positions Growth and RNA Preparation--
L. collosoma cells
were grown as previously described (38). Cultures were harvested at log
phase, and total RNA was prepared with TRIzol Reagent (Life
Technologies, Inc.) according to the manufacturer's protocol.
Cloning and Sequencing--
L. collosoma small
ribonucleoproteins were enriched by DEAE chromatography and
fractionation on sucrose gradients (33, 39). The RNA obtained from
10-20 S particles was extracted and separated on a preparative
denaturing gel, and two RNA bands of ~85 and ~90 nucleotides,
designated RNAs B2 and G2, respectively, were eluted from the gel,
labeled at the 5'-end, and used to screen a phage genomic library (33).
Two Plasmid Construction--
Tagging of the snoRNA-2 and B2 genes
was performed by PCR mutagenesis using primers carrying the tag. The
sequences of the tagged constructs were confirmed by DNA sequencing.
The deletions of the 5'- and 3'-flanking sequences of snoRNA-2 were
constructed by PCR and cloned into the pX-neo episomal
vector, which carries a neomycin resistance gene (neo)
upstream from the cloning site. Stable cell lines carrying the
different constructs were established as previously described (40).
Northern and Primer Extension Analyses--
The RNA samples (5 µg) were fractionated on a 10% polyacrylamide with 7 M
urea gel and electroblotted onto a nylon membrane (Hybond, Amersham
Pharmacia Biotech). Hybridization with labeled oligonucleotides was
performed at 42 °C in 5× SSC, 0.1% SDS, 5× Denhardt's solution,
and 100 µg/ml salmon sperm DNA. Primer extension was performed using
end-labeled oligonucleotide (100,000 cpm/pmol). After annealing at
60 °C for 15 min, the sample was kept on ice for 1 min; 1 unit of
reverse transcriptase (Expand RT, Roche Molecular Biochemicals) and 1 unit of RNase inhibitor (Promega) were added; and extension was
performed at 42 °C for 90 min. The reaction was analyzed on a 6%
denaturing polyacrylamide gel next to a DNA sequencing reaction with
the same primer. Primer extension sequencing of the rRNA was carried
out as previously described (41).
Determination of 2'-O-Methylated Nucleotides--
The primer
extension reaction for mapping the 2'-O-methylation
nucleotides was performed in the presence of different concentrations of dNTPs using 5'-end-labeled oligonucleotides complementary to the
3'-end downstream from each methylated position (42, 43). Total
RNA (0.5 µg/µl) was annealed with end-labeled primer at 60 °C
for 5 min; the annealing mixture was chilled on ice. The reaction was
carried out in buffer (50 mM Tris-Cl (pH 8.6), 60 mM NaCl, 9 mM MgCl2, and 10 mM dithiothreitol), 1 µg of RNA, 10,000 cpm labeled
primer, and 1 unit/µl reverse transcriptase (Expand RT) at 0.05-5.0
mM dNTP. After incubation at 42 °C for 1 h, the reaction was analyzed on 6% polyacrylamide with 7 M urea
gel next to primer extension rRNA sequencing with the same primer. The same experimental procedure was used to determine the
2'-O-methylated sites in yeast (43).
RT-PCR--
Total RNA (20 µg) was extensively treated with RQ1
RNase-free DNase I (5 units; Promega), and the RNA was extracted with
phenol/chloroform and precipitated in ethanol. One-tenth of the
precipitated RNA was used to prepare cDNA as described above,
except that two unlabeled oligonucleotides (10 pmol) complementary to
the sequence downstream of the snoRNA-2 and G2 coding region
(oligonucleotides 22076 and 22078, respectively) were used. One-tenth
of the sample was further used as templates for the PCRs with primers
located in the intergenic or coding region.
Transcription Analysis in Permeable Cells--
L.
collosoma cells were made permeable generally as previously
described (44), except that the transcription buffer contained an
additional 20 mM potassium glutamate. The radiolabeled RNA was purified with TRIzol reagent and was used for hybridization. RNA
was hybridized with blotted plasmid DNA (using a Bio-Rad dot-blot apparatus). Hybridization was performed in 5× saline/sodium
phosphate/EDTA, 0.1% SDS, 5× Denhardt's solution, 50% formamide,
and 100 µg/ml salmon sperm DNA at 45 °C. The blots were washed at
55 °C in 2× SSC and 0.1% SDS.
Sequence, Structure, and Genomic Organization of Two Loci Encoding
Four snoRNAs--
We previously described the enrichment of L. collosoma small ribonucleoproteins by fractionation on a
DEAE-Sephacel column and sucrose gradient (33, 39). RNA enriched by
these fractionations was separated on a denaturing gel, and
size-selected RNAs were labeled and used as probes to clone the
corresponding genes from a genomic library. Using such a scheme, we
previously identified and isolated the first trypanosomatid box C/D
snoRNA, snoRNA-2 (33). In this study, we isolated and characterized two
additional
To demonstrate the existence of these snoRNA transcripts, we used an
antisense oligonucleotide to each of the snoRNAs in Northern analyses
and primer extensions. The results, presented in Fig. 2, indicate that the sizes of B2, G2,
TS1, and TS2 are 92, 84, 91, and 90 nucleotides, respectively. Position
+1 of the RNA (Fig. 1A) was obtained from primer extension
(data not shown). The intensity of the hybridization signals reflects
the abundance of the snoRNAs; snoRNA-2 is more abundant than the other
snoRNAs examined.
Trypanosomatid Box C/D Methylation snoRNAs Obey the Canonical +5
Rule--
Computer sequence analysis was performed to determine
whether these snoRNAs belong to the family of box C/D methylation
snoRNAs by searching for the potential base pairing interaction between the snoRNAs and the rRNA sequences. Fig.
3 presents the results. snoRNA B2 has the
potential to guide ribose methylation of G3403 and
C3484 on 28 S rRNA, TS1 to guide ribose methylation of
U910 and A921 on 28 S rRNA, G2 to guide ribose
methylation of A3697 and A3709 on 28 S rRNA,
and TS2 to guide ribose methylation of G893 and
G2981 on 28 S rRNA. In each case, the potential for base
pairing presented perfect complementarity across a duplex of 10-12 bp.
One exception was that the sequence downstream of box C in
snoRNA G2 can potentially interact with 18 S rRNA by perfect base
pairing (Fig. 3). However, no methylation sites were found in the
region of rRNA that forms base pairing with the snoRNA G2 sequence
(data not shown). Our data suggest that there are methylated sites on
rRNA that are conserved through trypanosomatids, yeast, and man. The
methylation of adenosine at position 921, proposed to be guided by TS1,
is guided by U32/U51 in humans and by snR39/snR59 in yeast, and
the methylation of adenosine at position 3709, proposed to be guided by
G2, is guided by human U29 and by yeast snR71. These yeast/human snoRNAs carry exactly the same guiding sequences as the trypanosomatid homologs.2 However, the other
methylation sites that we analyzed are trypanosomatid-specific. Of
interest is the potential of these snoRNAs to form a very conserved structure with their adjacent target sites on the rRNA (Fig. 3). In
this proposed model, boxes C and C' were found in proximity.
The finding that the methylated positions are conserved among
trypanosomatids, yeast, and mammals supports the notion that the same
guiding rule is shared by these organisms. Indeed, our previous studies
using the mapping of partial alkali hydrolysis supported the +5 rule
since the methylated site on 5.8 S rRNA was located at position 5 upstream from box D' of snoRNA-2 (33). Here, we determined the
2'-O-methylated nucleotides by a reverse transcriptase
primer extension assay in the presence of dNTPs from low to high
concentrations (42), and the results agree well with the previous data
detected by partial alkali hydrolysis. In the presence of low dNTPs,
the reverse transcriptase stops 1 nucleotide before the methylation
site. The same method was used to map modified nucleotides in yeast
(43). Here, we present the mapping of modified nucleotides potentially
guided by snoRNA B2 using the primer extension assay (Fig.
4). The data, presented in Fig. 4,
support the +5 rule. Moreover, we have mapped all the methylation sites
proposed to be guided by the snoRNAs in this study, and all obey the +5
rule (Fig. 3 and data not shown).
Structural Requirement for Expression of snoRNA-2 and B2 Genes in
the pX-neo Episomal Vector--
As a first step toward understanding
the elements that control the expression of snoRNA genes, we analyzed
the snoRNA-2 gene (33). snoRNA-2 is encoded by a reiterated repeat unit
and is flanked by 300-bp upstream and 279-bp downstream sequences (Fig. 1B). The gene was tagged by inserting a linker of 8 nucleotides in a position located between boxes D' and C'. The tagged
gene was cloned into the pX-neo episomal vector, which
carries a neo gene upstream from the cloning site and
confers resistance to neomycin. The stable transgenic parasites
carrying the vector were obtained. The expression of the tagged gene
compared with the wild-type transcript was assayed by primer extension
using an oligonucleotide complementary to the 3'-end of the RNA. The results, presented in Fig. 5B
(lane 1), indicate that tagged snoRNA-2 was highly
expressed. To further determine the elements that control the
expression of the gene and especially to find out whether a specific
promoter drives the transcription of this snoRNA gene, we generated
constructs carrying variable upstream and downstream sequences and
cloned them into the pX-neo vector in two orientations with
respect to the neo gene on the vector (Fig. 5A).
The results demonstrate that high expression of the tagged gene was
dependent on its orientation in the vector. Efficient expression was
obtained only when the direction of transcription of the snoRNA-2 gene coincided with the neo gene (Fig. 5B, lanes
1-9). When the snoRNA gene was transcribed in the opposite
orientation, the expression was very poor (Fig. 5C,
lanes 1-9). The expression of the tagged gene was reduced
only when the entire upstream sequence was deleted (Fig. 5A,
construct 7), but was unaffected when only 10 nucleotides of
upstream sequence were present, suggesting that snoRNA genes may lack
conventional upstream promoters adjacent to the gene. Deletion of the
3'-flanking sequence had a more profound effect on expression;
expression was reduced when only 15 nucleotides of downstream sequence
were present, but was completely abolished in the absence of downstream
sequence (Figs. 5, B and C, lanes 8 and 9). These results suggest that proper expression of the snoRNA gene is dependent on both the 5'- and 3'-sequences immediately flanking the coding region of the gene. These flanking sequences (10-20 nucleotides) were probably needed to form the stem structure that could potentially be generated with sequences flanking the snoRNA
gene (see below). The poor expression of the tagged gene in the
opposite orientation with respect to the neo gene was likely to depend on transcription activity derived from the opposite strand.
It has been known for some time that transcription, at different rates,
takes place from both strands of the Leishmania episomal
vector (36).
The level of the wild-type snoRNA was dependent on the expression of
the tagged RNA since the same amount of RNA was used in the primer
extension assay (based on the U6 control) (Fig. 5B), yet the
level of the wild-type snoRNA-2 was reduced by 30-60% (compare
lane W with lanes 1-6). This demonstrates that
the overexpression of the tagged RNA gene repressed the level of the
wild-type transcript. This repression could originate from competition
for an RNA-binding protein (for example, fibrillarin) that binds to
this snoRNA. Examining the level of other snoRNAs such as snoRNA B2 in
these cell lines suggests that the effect was gene-specific since there was little effect on the level of snoRNA B2 (Fig. 5B). The
repression therefore did not originate from a reduction in the level of
a common binding protein for all snoRNAs.
Since the data presented in Fig. 5 suggest that the
expression of the snoRNA genes may be regulated mainly at the
processing level, we examined the potential for the flanking sequences
to form a structure that may play a role in the processing event. Folding the flanking sequences of the snoRNA genes using the MFOLD program showed that the sequences could be folded into a stem-loop structure where the snoRNA gene either forms the loop (in the cases of
snoRNA-2, B2, G2, and TS2) or participates also in the stem
structure (like TS1) (Fig. 6). The
minimal proposed stem is composed of 20-50 nucleotides from the 5'-
and 3'-flanking sequences. Although the conservation of these stem-loop
structures seems to be at the secondary structure, the sequence of all
the stems is GT- and GC-rich (Fig. 1A). Based on the data
presented in Fig. 5, complete disruption of the stem by deleting
sequences either from the 5'- or 3'-flanking sequence reduced the
production of mature RNAs, suggesting that this stem structure may
serve as a recognition site for the processing machinery.
To examine whether the snoRNA gene located in a gene cluster encoding
multiple snoRNAs could also be autonomously expressed when removed from
its neighboring genes, we tagged the B2 and B2' genes by inserting an
EcoRI linker between boxes D' and C' (Fig.
7A). Since we observed a
difference in the upstream sequence between the B2 and B2' genes (an
insertion of a sequence indicated in Fig. 7A), we tagged
both genes and examined their expression. The genes were flanked by 93- or 101-bp upstream and 64-bp downstream sequences. Transgenic parasites
were generated, and the expression of the tagged genes was compared
with that of the wild-type transcript by a primer extension assay. The
results, presented in Fig. 7B, suggest that both genes were
efficiently expressed, indicating that each gene harbors elements
necessary for its expression in vivo when cloned in the
episomal vector. However, we cannot rule out the possibility that
expression of the gene in its authentic chromosomal locus is not
dependent on a distant classical promoter element located in the
5'-flanking sequence of the gene cluster. As already mentioned, the
overexpression of a tagged snoRNA B2 gene from the episomal vector also
repressed the level of the wild-type transcript.
snoRNA Genes Are Transcribed as Polycistronic snoRNA
Precursors--
Sequences upstream from each of the snoRNA genes did
not reveal common motifs that could potentially serve as internal
promoters. In addition, the finding that snoRNA-2 was properly
expressed in the absence of the 5'-flanking sequence may suggest that
either the promoter for the entire snoRNA cluster is present upstream from the first copy or that such a promoter does not exist, like pol
II-transcribed protein-coding genes in this family (31). However, in
both scenarios, we would expect to detect a polycistronic snoRNA
precursor that is processed to generate the individual mature
snoRNAs.
An RT-PCR assay was performed to investigate whether such a precursor
snoRNA might exist. An antisense oligonucleotide 85 nucleotides
downstream from the snoRNA-2 gene was used to produce a cDNA that
was amplified by PCR using a sense oligonucleotide located 282 bp
upstream from the snoRNA-2 coding region (Fig. 8A). To avoid DNA
contamination, the RNA sample was extensively treated with DNase I
(RNase-free). As a control, PCR was carried out on the RNA sample
without reverse transcription; and in this case, no product was
produced (Fig. 8B, lane 1), suggesting that the
PCR product was generated from the cDNA template. A PCR product of
490 bp was detected using the cDNA as a template (Fig.
8B, lane 5), demonstrating that the snoRNA was
processed from a longer precursor carrying sequences from both the 5'-
and 3'-flanking sequences. The existence of a polycistronic snoRNA
precursor was also observed for the g2 locus. Using a primer located in
the coding region of G2 and a primer situated in the intergenic region between TS1' and TS2 (Fig. 8A), we detected a PCR product of
407 bp (Fig. 8B, lane 4), indicating the
existence of an RNA molecule carrying both coding regions of G2 and TS2
and the intergenic region between them. Likewise, a product of 389 bp
was observed (Fig. 8B, lane 3) using a primer
located in the coding region of TS1' and a primer in the upstream
sequence of B2' (Fig. 8A). In addition, we could also detect
a 1.0-kb product that covers the entire g2 locus using a primer located
downstream from the G2 gene and a primer upstream from the B2' gene
(Fig. 8B, lane 2). Moreover, the data support the
existence of a precursor snoRNA that covers the entire g2 gene cluster.
The differences in intensity of the RT-PCR products reflect the length
of transcripts to be extended. Weak PCR products were observed in
lanes 2 and 3 because the products either cover
the entire g2 locus (lane 2) or are located at a distal
portion of a long cDNA (lane 3). The longer the
cDNA, the weaker is the PCR product made from the template.
Analysis of snoRNA-2 Precursors That Accumulate in Transgenic
Parasites--
To further characterize the snoRNA precursor, we
performed primer extension on RNA from wild-type cells and cell lines
carrying the different snoRNA constructs (Fig. 5A). When
primer extension was performed using an oligonucleotide located at the
3'-end of snoRNA-2, different extension products were detected that
were dependent on the extent of the 5'-flanking sequence. The sizes of
the extension products were always ~110 nucleotides longer than those
of the upstream sequence present in the constructs (Fig.
5A). The additional sequence beyond the snoRNA 5'-upstream region originated from the vector sequence since the same pattern of
extension products (shorter by 80 nucleotides) was obtained when primer
extension was performed with an oligonucleotide located 20 nucleotides
upstream from the gene (Fig.
9B, lanes 1-9). No extension product was detected with the wild-type RNA, which may reflect the short half-life of the precursor carrying the 5'-flanking sequence. Without an efficient system for processing the nascent snoRNA
transcripts, it is difficult, at this point, to demonstrate a
precursor-product relationship and to unequivocally prove that the
stable snoRNAs are derived from the large precursor RNA molecules we
detected in this study.
Box C/D snoRNA Genes Are Transcribed by In this study, we present evidence that L. collosoma
box C/D snoRNAs, proposed to function in ribose methylation, obey the canonical +5 rule established for yeast and mammals. The three snoRNA
loci we have studied, which encode a single snoRNA (snoRNA-2) or
clusters of snoRNAs (g2 and b2), are transcribed as polycistronic RNAs.
Deletion analysis of sequences in the 5'- and 3'-flanking sequences of
the snoRNA-2 gene suggests that only the immediate 20-nucleotide
flanking sequences are most critical for the snoRNA expression.
However, more dramatic effects on expression were observed when the
entire 3'-flanking sequence was removed as opposed to the same deletion
of the 5'-flanking region. Moreover, a single snoRNA gene from a
cluster could be self-expressed when cloned in an episomal vector. The
data may suggest that snoRNA genes lack a conventional promoter that
lies immediately adjacent to the genes. However, we cannot exclude the
possibility that a promoter exists upstream from the gene cluster. The
transcription analyses suggest that the snoRNA genes are transcribed by
RNA pol II.
Based on the location of the methylation site thought to be
guided by snoRNA-2, we have shown that L. collosoma snoRNA
obeys the canonical rule for guide methylation established for yeast and mammals (33). However, a related study by Roberts et al. (34) on snoRNAs that are associated with the SLA1 loci in
several trypanosomatid species suggested that the selection of the
specific modification site may follow an altered rule. This discrepancy led us to map all the methylation sites that have been proposed to be
guided by the b2- and g2-associated snoRNAs. We always found that in
L. collosoma, the +5 rule holds true. This finding is supported by the fact that two of the methylation sites
(A921 and A3709) identified in this study are
conserved in yeast and mammals. Our conclusion regarding the +5 rule in
trypanosomatids was recently confirmed by a study on T. brucei snoRNAs, in which seven methylation sites were mapped, and
the corresponding guide-methylating snoRNAs were identified (35). In
addition, 6 of the 15 guide snoRNAs identified in that study are
potential homologs of the snoRNAs from yeast and vertebrates that obey
the +5 rule.
All the snoRNAs we have analyzed contain two domains complementary to
the target sites and are therefore double-guide snoRNAs. This may be
expected since ~100 sites of 2'-O-ribose methylation were
mapped in Crithidia fasciculata, which is almost as numerous as in humans (48). The need to methylate so many sites and the relatively small genome size of the parasite may have forced many more
snoRNAs to guide two sites. In comparison, only 7 of 41 yeast snoRNAs
were found to be double guides (43). Interestingly, as in
trypanosomatids, in Archaea, numerous small RNAs appear to have
the ability to guide methylation from boxes D and D' and are therefore
double guides (49). In both trypanosomatids and Archaea, the predicted
target sites of double-guide snoRNAs are within the same rRNA molecule
and are located in adjacent sites. It still remains to be seen whether
the proximity of the two sites to be directed by the double-guide
snoRNAs, as well as the structure we proposed between the snoRNAs and
their two target sites, may have any functional significance.
Studies in yeast, vertebrates, and plants suggest different strategies
for box C/D snoRNA transcription and maturation (5). In vertebrates,
snoRNAs encoded by introns of host genes involved in ribosome
biogenesis or ribosomal functions are not transcribed by their own
promoters (14, 16). Exonucleolytic degradation plays a major role in
their processing by trimming the debranched lariat (24, 25). A minor
alternative pathway involves endonucleolytic cleavages within the
pre-mRNA intron (50). In yeast, most of the snoRNAs are dispersed
as independent singlets or within clusters carrying two to seven
snoRNAs (20). These snoRNAs are transcribed as polycistronic RNAs from
an upstream promoter and are processed by the endonuclease Rnt1p and
degraded by 5' to 3' exonuclease, Rat1p, and, to a lesser extent by
Xrn1p (18-20, 22). In plants, the genes for both box C/D and H/ACA
snoRNAs are transcribed as a polycistronic pre-snoRNA transcript from
an upstream promoter (51). The plant snoRNAs are processed by
endonucleolytic activity, followed by trimming (23). The three
chromosomal loci of box C/D snoRNAs described in this study represent a
novel organization of snoRNA genes. Although we provide evidence for
the existence of pre-snoRNAs, as for several genes in yeast and plants,
we have no evidence for the existence of a promoter that regulates the expression of these genes. We cannot, however, exclude the possibility that a promoter exists upstream from the snoRNA gene cluster. We favor
the hypothesis that snoRNA gene expression is not regulated at the
level of transcription, but, like many protein-coding genes in these
organisms, is mainly regulated by RNA processing and stability
(30).
This is the first study that shows that the expression of a small RNA
gene in trypanosomatids is affected by its orientation in the
pX-neo plasmid. The expression of SL RNA (40), 7SL RNA (52), and U snRNAs in L. collosoma (53) was shown to be
dependent on extragenic promoter elements and was not affected by their orientation on the plasmid. The previous studies suggested that the
SLA1 locus, which also codes for box C/D methylation snoRNA, is transcribed by a modified RNA pol II, similar to the polymerase that
transcribes the SL RNA gene locus. The results presented in this study,
however, demonstrate that the snoRNA genes we have analyzed are
transcribed most likely by the same RNA polymerase that transcribes
protein-coding genes. It is therefore possible that there are at least
two types of snoRNA genes in trypanosomatids, those that are linked to
other small RNAs with a different cellular function (like
SLA1) and those that are flanked by protein-coding genes. It
will be of great interest to determine whether the snoRNAs described
here are flanked by protein-coding genes that are transcribed in the
same direction.
The components of the RNA degradation machinery that may play a role in
rRNA processing, mRNA stability, or snoRNA maturation have not so
far been characterized in trypanosomatids. The yeast RNase III
ortholog, Rnt1p, was shown to participate in the processing of snRNA
(U5 snRNA) (54) and polycistronic yeast snoRNAs (21, 22). The site of
cleavage was always found in a double-stranded stem present upstream or
downstream from or flanking the coding region of the small RNAs.
Because of the potential of the trypanosomatid snoRNA gene to form a
similar structure with its flanking sequences, we examined the ability
of the yeast recombinant Rnt1p enzyme to cleave the trypanosomatid
snoRNA substrates. In experiments performed with the yeast U5 substrate
as a control, we failed to observe any specific endonucleolytic
cleavages in the trypanosomatid snoRNA flanking sequences using the
yeast enzyme (data not shown). This result suggests that the
trypanosomatid substrates lack the sequence specificity required for
Rnt1p cleavage (21).
The lack of conventional promoters for protein-coding genes and for the
snoRNA genes adjacent to the genes may imply that snoRNA processing and
mRNA stability mediated by the exosomal functions should be tightly
regulated in these organisms. Another possibility for co-regulating
ribosomal function and its biogenesis with the level of snoRNAs in
trypanosomatids may involve genomic clustering. The answers to these
questions await the completion of the trypanosomatid genome projects.
-amanitin, thus supporting transcription by RNA
polymerase II. We propose that snoRNA gene expression, similar to
protein-coding genes in this family, is regulated at the processing level.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-amanitin, similar to pol
II-transcribed genes. The data in this study suggest that the
expression of trypanosomatid snoRNAs is mainly regulated at the
processing level.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
282 to
300 carrying a BamHI site at the 5'-end
(underlined); 22076, 5'-CGGGATCCCTGCCAGAATTGTCCCGTGC-3', antisense downstream from positions 85 to 104; 22178, 5'-CTGCGCGACGACAAACGGAATTCCGACCAACGATAACAT-3', sense from
positions 38 to 66 of the coding region with an EcoRI site
in the insertion between positions 52 and 53; 24358, 5'-CGGGATCCCTGACGCGCCCATGCGAGCT-3', sense from positions
241 to
260; 24357, 5'-CGGGATCCCAGATGGACAACCGAGTCAA-3', sense from positions
190 to
209; 24356, 5'-CGGGATCCAACTCCATGGTGTCGGCTG-3', sense from positions
120 to
138; 24355, 5'-CGGGATCCTTGTGCGTCGACGTGCAGT-3', sense from positions
51 to
69; 25788, 5'-CGGGATCCGTGTTGCCACGGCTGCAAC-3', sense from positions
10 to
28; 26709, 5'-CGGGATCCCGCCAGGTGCTCAATT-3', sense
from positions 6 to
10; 26710, 5'-CGGGATCCTCAATTGATGATGAA-3', sense from positions 1 to 15 of the coding region; 17853, 5'-CGGGATCCAACAACAACAGGTGCTC-3', antisense for deletion of
the 3'-flanking sequence from position 84 of the coding region to
position 15 downstream from the gene; 28505, 5'-CGGGATCCTCATCAGATGCCGGTAGTC-3', antisense from positions 63 to 85 of the coding region; and 30355, 5'-GCACCTGGCGTTGCAGCCGT-3', antisense upstream from positions
1 to
19.
63 to
82; 26554, 5'-CGGGATCCCAGGTACGCAGGTACGCAGG-3', antisense downstream
from positions 46 to 65; 26680, 5'-GCACGAGCATCCGAATTCAAAGGTGTCCTAA-3', sense from positions
23 to 65 of the coding region with an EcoRI insertion
between positions 35 and 36; 20406, 5'-TTTCACATGCACGAGCATCC-3', antisense from positions 35 to 54 of the coding region; 28508, 5'-TACGCTTGTGTTCCACGTGA-3', antisense to 28 S rRNA from positions 3435 to 3454 for mapping the methylation site guided by box D' of snoRNA B2;
and 22268, 5'-ATTTCCTGTTCTTCGCAAAG-3', antisense to 28 S rRNA from
positions 3517 to 3536 for mapping the methylation site by box D of
snoRNA B2.
39 to
56; and 22269, 5'-ACACCTCCAAAGTCGCCGCA-3', antisense to 28 S rRNA from positions 3008 to 3027 for mapping the methylation site guided by snoRNA TS2.
1 to +19 (positions 4996-5016
of the pX-neo vector and position
1 of the 5'-untranslated
region) (36); and 12407, 5'-AGCTATATCTCTCGAA-3', antisense to the
3'-region from positions 83 to 98 of the U6 snRNA gene (37).
phage for RNAs B2 and G2 were isolated and further analyzed by
subcloning ~500- and ~600-bp Sau3AI fragments into the
pBluescript plasmid, respectively. A 1.2-kb HpaII fragment
overlapping the RNA G2 Sau3AI fragment was also subcloned.
All clones were sequenced with T3 and T7 primers.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
phage encoding small RNA genes. A 520-bp
Sau3AI subclone generated from one
phage that hybridized
strongly with RNA B2 was termed the b2 locus. Two fragments, 560-bp
Sau3AI and 1.2-kb HpaII subclones derived from
the other phage, which hybridized with the labeled RNA G2, were termed
the g2 locus. The b2 locus carries two putative snoRNAs that were
termed B2 and TS1. The g2 locus carries four snoRNAs. The first two are
shared with the b2 locus and were therefore termed B2' and TS1'; the
other two were designated G2 and TS2. Fig.
1A presents the sequence of
the 1.2-kb g2 genomic locus. The coding regions of B2 and B2' as well
as TS1 and TS1' are identical. However, the sequences flanking the
genes are different (Fig. 1A), suggesting that the two
cloned genes represent two different chromosomal loci. Indeed, Southern
blot analyses using g2 and b2 as probes showed that these genes are
duplicated in the genome since digestion with restriction enzymes of a
6-bp recognition site always resulted in at least two fragments, and
partial digestion with Sau3AI produced a duplicated ladder
(data not shown). Southern blot analyses also indicated that the gene
is repeated at least five times in each locus, as previously
demonstrated for the snoRNA-2 gene (33). The genomic organization of
the b2 and g2 loci is illustrated in Fig. 1B. One
characteristic of these genomic loci was the presence of runs of GT and
GC dinucleotides in the regions flanking the snoRNA genes (Fig.
1A).
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Fig. 1.
DNA sequence of the g2 gene cluster and the
genomic organization of the snoRNA genes. A, DNA
sequence of the g2 locus (GenBankTM/EBI accession number
AF331656). The coding regions of snoRNAs are presented in
boldface uppercase letters, and the +1 positions are
indicated. The direction of transcription of the snoRNA genes is marked
with arrows. The conserved boxes C/C' and D/D' of the
snoRNAs are boxed. The intergenic upstream sequence
difference between snoRNA B2/TS1 and B2'/TS1' is boxed. The
oligonucleotide sequences used for RT-PCR analysis as described in the
legend to Fig. 8A are underlined and indicated.
B, schematic representation of the snoRNA gene genomic
organization. The snoRNA genes are boxed, and the sizes of
the intergenic regions are indicated in base pairs.
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Fig. 2.
Expression analysis of the snoRNA genes.
Total L. collosoma RNA (3 µg) was separated on a 10%
polyacrylamide with 7M urea gel and electroblotted onto a
nylon membrane. The membrane was hybridized with end-labeled antisense
oligonucleotides specific for the different snoRNAs as indicated:
lane 1, snoRNA-2 (oligonucleotide 16865); lane 2,
snoRNA B2 (oligonucleotide 20406); lane 3, snoRNA G2
(oligonucleotide 20405); lane 4, snoRNA TS1 (oligonucleotide
20936); lane 5, snoRNA TS2 (oligonucleotide 20935).
Lane M, end-labeled pBR322 HpaII digest.
nt, nucleotides.
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Fig. 3.
Schematic representation of potential base
pairing interactions between the snoRNAs and their target sites on
rRNA. The conserved boxes C/C' and D/D' are marked. The potential
guided methylation sites are marked with asterisks. The
identities of the RNAs and the sequence positions on rRNA are
indicated.
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Fig. 4.
Mapping of the ribose-methylated nucleotides
potentially guided by snoRNA B2. A, primer extension
analysis. Primer extension was performed in the presence of increasing
dNTP concentrations (0.05, 0.5, 1.0, and 5.0 mM) with
antisense oligonucleotide 22268, specific to the sequence downstream
from the target site guided by snoRNA B2 box D. rRNA sequencing was
performed with the same oligonucleotide by primer extension. Part of
the cDNA sequence is indicated on the left. The position of the
reverse transcriptase stop site (1 nucleotide before the methylated
site) is indicated by an arrow. The methylated site is
marked with an asterisk. B, same as described for
A, but with oligonucleotide 28508, specific to the sequence
downstream from the target site guided by snoRNA B2 box D'.
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Fig. 5.
Expression of the tagged snoRNA-2 gene.
A, schematic representation of snoRNA-2 constructs. The
lengths of the 5'- and 3'-flanking sequences are indicated in
bp. The tag sequence is indicated by a flag. Boxes
C/C' and D/D' are boxed and indicated. The constructs were
cloned into the BamHI site of the pX-neo
expression vector. B, primer extension analysis for the
expression of tagged snoRNA-2. Primer extension was performed with the
end-labeled antisense oligonucleotide to the 3'-end of snoRNA-2
(oligonucleotide 16865) using the RNA extracted from transgenic cell
lines carrying the constructs as in A in the same
orientation as the neo gene (lanes 1-9). The
direction of transcription is indicated by the arrow. The
tagged snoRNA-2 and wild-type snoRNA-2 (WT) are marked on
the left. C, same as described for B, but using
the cell lines with the constructs in the opposite orientation as
indicated by the arrow to the left. As a control, primer
extension was performed in the same cell lines, but with antisense
oligonucleotides specific for U6 snRNA (oligonucleotide 12407) and
snoRNA B2 (oligonucleotide 20406). The U6 and snoRNA B2 extension
products are indicated. Lane w, wild-type cells.
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Fig. 6.
Secondary structure comparison of snoRNA
genes and the flanking sequences. The structures were folded using
MFOLD (55). The identities of the snoRNAs are indicated, and
their coding regions are framed.
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Fig. 7.
Expression of the tagged snoRNA B2/B2' genes
in transgenic parasites. A, schematic representation of
two different snoRNA B2 loci: snoRNA B2 (locus 1) and snoRNA
B2' (locus 2). The upstream sequence difference between the
two loci is indicated. The tag sequence is marked with a
flag, and the lengths of the flanking sequences are
indicated in base pairs. B, primer extension analysis to
determine the expression of tagged snoRNA B2/B2'. Both primer extension
and DNA sequencing were carried out with the same 3'-end
oligonucleotide for snoRNA B2 (oligonucleotide 20406). The positions of
the tagged and wild-type (WT) snoRNA B2 and the DNA sequence
at the 5'-end are indicated. Lane w, wild-type cells;
lanes 1 and 2, cell lines carrying the constructs
in A, respectively.
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Fig. 8.
RT-PCR analyses for the detection of snoRNA
precursors. A, shown is a schematic representation of
the snoRNA-2 and g2 genomic loci and corresponding PCR products. The
coding regions of snoRNAs and the lengths (in base pairs) of the
flanking sequences are indicated. The oligonucleotides are numbered and
marked with small shaded rectangles. Reverse transcription
(RT) was performed with oligonucleotides 22078 and 22076 for
the g2 and snoRNA-2 loci, respectively. The PCR products are numbered,
and their sizes are indicated in base pairs and kilobases.
B, RT-PCR products were separated on 1% agarose gel. The
primers and corresponding PCR products are indicated in A.
In lane 1, a negative control was carried out with the DNase
I-treated RNA, but without reverse transcription, using the set of
primers as in lane 5. In lane 6, a positive
control was performed with genomic DNA using the same primers as in
lane 5. Lane M, 1-kb DNA ladder.
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Fig. 9.
Mapping snoRNA precursors by primer
extension. Primer extension was carried out on the RNAs extracted
from the cell lines carrying the constructs shown in Fig.
5A. The end-labeled antisense oligonucleotides used in the
primer extension analyses were oligonucleotide 16865 (specific to the
3'-coding region; A) and oligonucleotide 30355 (specific to
the 5'-flanking sequence; B). The extension products are
marked with short arrows. The long arrows above
the panels indicate the direction of transcription (the same as the
neo gene on the plasmid). Lane w, wild-type
cells; lanes 1-9, cell lines with the constructs in Fig.
5A, respectively; M, end-labeled pBR322
HpaII digest. nt, nucleotides.
-Amanitin-sensitive RNA
Polymerase--
The results presented in Fig.
10A indicate that the
expression of the snoRNA gene was dependent on the expression of the
neo gene. Interestingly, as observed in Fig. 5A,
the high expression of the tagged snoRNA resulted in repression of the
authentic chromosomal gene. We have previously demonstrated that the
elevation in neo expression is correlated with the episomal
copy number (40). Because of the dependence of the tagged snoRNA gene
expression on neo expression, the elevated level of
neo mRNA resulted in increased expression of tagged
snoRNA-2. The data suggest that snoRNA-2 can be transcribed by pol II
or that transcription of an upstream pol II gene enhances snoRNA-2
transcription. However, the dependence of snoRNA-2 expression on the
presence of the upstream neo gene does not unequivocally
prove that pol II transcribes this gene. To gain further support for
the transcription of snoRNA-2 by pol II, we used permeable cells, which
efficiently and accurately transcribe endogenous genes (44). The
permeable cells were incubated with various concentrations of
-amanitin, and the transcription of the different genes was examined
by hybridizing the RNA with the tested genes immobilized on a filter.
As presented in Fig. 10B, the transcription of the snoRNA-2
gene was compared both with the pol II-transcribed gene that codes for
the SL RNA core-binding protein SmE (45) and with the pol
III-transcribed gene, the U6 snRNA. In addition, we also examined and
compared the transcription of the SL RNA, which is the most efficiently
transcribed gene in this system. The results indicate, as in the
previous study (46), that the level of inhibition of SL RNA by
-amanitin was intermediate between those of pol II- and pol
III-transcribed genes. The transcription pattern of the snoRNA-2 gene
resembled that of pol II-transcribed genes since its transcription was
severely inhibited at 50 µg/ml (5% of the level in the absence of
the drug), compared with SL RNA, whose transcription was reduced to
40% at the same concentration (Fig. 10B, panel
b). Our results differ from those reported for the SLA1
locus (47) since, in that study, the transcription data suggested that
the SLA1 locus was transcribed by a pol II that was
slightly more resistant to
-amanitin than pol II which transcribes
protein coding genes.
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Fig. 10.
A, linkage between neo and
snoRNA-2 gene expression. A cell line carrying construct 5 in Fig. 5A was established and selected for growth at the
increased G418 concentrations as indicated. The expression of the
snoRNA and neo genes was detected by primer extension on the
RNA from the same cell culture with oligonucleotides 16865 (specific
for snoRNA-2) and oligonucleotide 21995 (specific for neo
mRNA). The extension products of the tagged and wild-type
(WT) snoRNA-2 and neo mRNA are indicated.
B, in vivo transcription analyses in permeable
cells in the presence of -amanitin. Panel a, dot-blot
analysis. DNA (5 µg/dot) encoding for the genes indicated was
immobilized on a nylon membrane and hybridized with
[
-32P]UTP-labeled RNA transcribed in permeable cells
in the presence of
-amanitin. The concentrations of
-amanitin are
indicated. The differential transcription efficiency of the various
genes required several exposures. Panel b, quantitative
analyses of transcriptional inhibition by
-amanitin. The percentage
of inhibition was relative to the transcription activity without
-amanitin. The data were obtained by densitometric analyses of the
dot-blot data shown in panel a.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
---|
We thank Todd Lowe for help in assigning the yeast and mammalian homologs to the trypanosomatid snoRNAs reported in this study and Guillaune Chanfreau for providing the recombinant Rnt1p enzyme.
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FOOTNOTES |
---|
* This work was supported by a grant from the Israeli Ministry of Health.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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF331656
§ The first two authors contributed equally to this work.
To whom correspondence should be addressed. Tel.:
972-3-531-8068; Fax: 972-3-535-1824; E-mail:
michaes@mail.biu.ac.il.
Published, JBC Papers in Press, February 1, 2001, DOI 10.1074/jbc.M007007200
2 T. M. Lowe, personal communication.
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ABBREVIATIONS |
---|
The abbreviations used are: snoRNAs, small nucleolar RNAs; pol, polymerase; snRNA, small nuclear RNA; bp, base pair(s); kb, kilobase; PCR, polymerase chain reaction; RT-PCR, reverse transcription-polymerase chain reaction; SL, spliced leader.
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