Next Article 
Mol Cell Biol, August 1998, p. 4409-4417, Vol. 18, No. 8
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Three Small Nucleolar RNAs Identified from the
Spliced Leader-Associated RNA Locus in Kinetoplastid
Protozoans
T. Guy
Roberts,1
Nancy R.
Sturm,1
Billy K.
Yee,1
Michael C.
Yu,1
Toinette
Hartshorne,2
Nina
Agabian,3 and
David A.
Campbell1 *
Department of Microbiology and Immunology,
UCLA School of Medicine, Los Angeles, California
90095-17471;
Biochemistry & Molecular
Biology, Albany Medical College, Albany, New York
122082; and
Program in Molecular
Pathogenesis, University of California, San Francisco, California
94143-04223
Received 24 February 1998/Returned for modification 29 April
1998/Accepted 1 May 1998
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ABSTRACT |
First characterized in Trypanosoma brucei, the spliced
leader-associated (SLA) RNA gene locus has now been isolated from the kinetoplastids Leishmania tarentolae and Trypanosoma
cruzi. In addition to the T. brucei SLA RNA, both
L. tarentolae and T. cruzi SLA RNA repeat units
also yield RNAs of 75 or 76 nucleotides (nt), 92 or 94 nt, and ~450
or ~350 nt, respectively, each with significant sequence identity to
transcripts previously described from the T. brucei SLA RNA
locus. Cell fractionation studies localize the three additional RNAs to
the nucleolus; the presence of box C/D-like elements in two of the
transcripts suggests that they are members of a class of small
nucleolar RNAs (snoRNAs) that guide modification and cleavage of rRNAs.
Candidate rRNA-snoRNA interactions can be found for one domain in each
of the C/D element-containing RNAs. The putative target site for the
75/76-nt RNA is a highly conserved portion of the small subunit rRNA
that contains 2'-O-ribose methylation at a conserved
position (Gm1830) in L. tarentolae and in vertebrates. The
92/94-nt RNA has the potential to form base pairs near a conserved
methylation site in the large subunit rRNA, which corresponds to
position Gm4141 of small rRNA 2 in T. brucei. These data
suggest that trypanosomatids do not obey the general 5-bp rule for
snoRNA-mediated methylation.
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INTRODUCTION |
Posttranscriptional modifications to
the rRNAs, including endonucleolytic cleavage, 2'-O-ribose
methylation and pseudouridinylation, are mediated by small,
nucleolar RNAs (snoRNAs). The most abundant snoRNA, U3, has been
identified in numerous eukaryotes, including the kinetoplastids
(27) and Euglena sp. (23). A
requirement for U3 snoRNA in endonucleolytic cleavages of precursor
rRNA transcripts has been established in yeast and vertebrate systems
(31, 36). Multiple other snoRNAs have been identified that
are involved in the 2'-O-ribose methylation and
pseudouridinylation of the 18S and 25/28S rRNAs (3, 22, 42,
43), although the precise function of many snoRNAs remains
unclear.
Several criteria have been used to identify snoRNAs (24, 36,
50), including the presence of conserved sequence motifs, association with nucleolar proteins (fibrillarin, Gar1p, and Pop1p), complementarity to pre-rRNAs, and localization to the nucleolus. Like
other cellular RNAs, snoRNAs are believed to exist as ribonucleoprotein complexes (36, 50) and have been divided into two major
classes based on conserved sequence elements and protein associations. The box C/D snoRNAs usually contain box C and box D elements near their
5' and 3' ends, respectively; these elements are required for snoRNP
interaction with the abundant nucleolar protein, fibrillarin. In
vertebrates, box C/D snoRNAs are frequently processed from the introns
of protein-encoding mRNAs (54), while in plants and yeasts
they may be transcribed polycistronically (32, 50). The
other class, the box H/ACA snoRNAs, contains two conserved sequence
motifs: box H resides in a hinge region between two stem-loop structures, and the "ACA" trinucleotide motif resides 3 nucleotides (nt) from their 3' ends (3, 22). Box H/ACA snoRNAs of yeasts associate with the essential Gar1 protein (22). Most members of each class of snoRNAs function in modification of rRNA sequences: box C/D snoRNAs guide 2'-O-ribose methylation, and box H/ACA
snoRNAs are involved in pseudouridinylation (21). Box C/D
snoRNAs direct modification of rRNAs through stretches of sequence
complementarity. The region of base pairing between box C/D snoRNAs and
the site of methylation in the rRNA always lies immediately upstream
from a D box or a D' box, which is a slightly degenerate version of the
D box (29, 43, 55). A number of other snoRNAs containing box
C and D elements (U3, U8, U14, and U22) or box H/ACA elements (for
example, U17/E1 [19] and snR30 [42])
do not appear to play roles in rRNA nucleotide modification, but they
may be required for pre-rRNA cleavages or have roles in pre-RNA folding
and ribosome assembly (50).
A number of small nuclear RNAs have been described in the
kinetoplastids: the spliced leader (SL) RNA (12, 30, 38), four U small nuclear RNAs involved in trans splicing (U2,
U4, and U6 [39, 51, 52] and U5 [18,
60]), the SL-associated (SLA) RNA (44, 46, 58), the
mitochondrial guide RNAs (6, 49), the 7SL RNAs
(37), the tRNAs (11, 40), the six large-subunit (LSU) rRNA fragments (15, 28), and the U3 snoRNA (27,
39). The U3 RNA is the only snoRNA identified in this group of
organisms.
We have characterized four small RNAs in Leishmania
tarentolae and Trypanosoma cruzi that are similar to
those originally identified as transcripts of unknown function from the
SLA RNA repeat in T. brucei (46). We present
evidence that three transcripts fractionate with nucleolar markers. Two
of the transcripts are bona fide snoRNAs: the 75/76- and 92/94-nt RNAs
contain box C, D, and D' elements and 10- to 14-bp stretches with
complementarity to sites in the small-subunit (SSU) or LSU rRNAs. We
show that one nucleotide within each of these blocks is ribose
methylated. The positions of methylated nucleotides relative to
the D or D' box in the complementary snoRNA differ from the
canonical 5-bp spacing seen in higher eukaryotes; in the two sites
described here the methylated ribose is the first in the stretch of
complementarity. This suggests that the 5-bp rule that directs rRNA
methylation in the higher eukaryotes may not be conserved among the
early-diverging kinetoplastids.
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MATERIALS AND METHODS |
Cloning, sequencing, and computer analyses.
Cosmid clones
containing the L. tarentolae SLA locus were selected by
hybridization with the oligonucleotide probe TGR10 (Table 1) from a library as described previously
(20). PvuII, XbaI, and XhoI
fragments of one cosmid were subcloned into pBluescript (Stratagene)
for further analysis. DNA sequencing was performed with Sequenase
(Amersham), and the sequence was submitted to GenBank (accession number
AF016399).
The
T. cruzi SLA locus-containing clone (51H20) was selected
from filters provided by the
T. cruzi genome project cosmid
library
(
26) and analyzed as a
XhoI fragment
(GenBank accession number
AF016400).
Cell culture.
L. tarentolae (UC strain) was grown at
27°C with slow rotation in brain heart infusion medium supplemented
with hemin (10 µg/ml), penicillin (100 U/ml), and streptomycin (100 µg/ml). Procyclic-form T. brucei (IsTat1.1 or strain 427)
was grown in BSM (5) or SDM-79 (8) containing 5%
fetal bovine serum. Cell lysates of T. cruzi Tulahuèn
were a kind gift from Jaime M. Santana, University of Brasilia,
Brasilia, Brazil.
DNA and RNA extraction and analysis.
DNA and RNA were
isolated with TRIzol reagent (Gibco-BRL) as directed by the
manufacturer except that the cell density was increased to
approximately five times that recommended by the manufacturer in order
to compensate for the smaller cell volume of the kinetoplastids. DNA
was isolated with DNAzol (Gibco-BRL) as directed by the manufacturer.
Electrophoresis of DNA and RNA on polyacrylamide and agarose gels and
transfer to nylon membranes for hybridization analyses
were performed
as described previously (
47) unless otherwise
indicated.
Hybridizations with oligonucleotides were performed
at 42°C. Wash
conditions were 2× SSC (1× SSC is 0.15 M NaCl plus
0.015 M sodium
citrate)-0.1% sodium dodecyl sulfate at 39°C unless
otherwise
specified. The oligonucleotides used in this study are
listed in Table
1.
Primer extension on RNA templates was performed with Superscript
reverse transcriptase (Gibco-BRL) as described earlier (
46).
3'-end determination of the SLA RNA was performed with total RNA
that
had been tailed by poly(A) polymerase (Gibco-BRL) and copied
into a
cDNA with an oligo(dT) primer and Moloney murine leukemia
virus reverse
transcriptase (Gibco-BRL); this was followed by
PCR with the sense
oligonucleotide SLAs1 (Table
1).
Cell fractionation.
The isolation of cytoplasmic,
nucleoplasmic, and nucleolar fractions was adapted from published
methods (27, 53). Leishmania cultures were grown
to a density of 108 cells/ml, and the cells were harvested
after centrifugation at 5,000 × g for 10 min at 4°C.
Cell pellets were resuspended in phosphate-buffered saline and washed
twice. The pellet was then resuspended in 3 volumes of ice-cold
hypotonic buffer (10 mM HEPES, pH 7.9; 1.5 mM MgCl2; 10 mM
KCl; 0.5 mM dithiothreitol [DTT]; pepstatin, 1 µg/ml; leupeptin,
0.5 µg/ml; 0.5 mM phenylmethylsulfonyl fluoride) and incubated on ice
for 10 min. The cell suspension was transferred to a 15-ml glass Dounce
homogenizer (Wheaton), Nonidet P-40 was added to a final concentration
of 0.2%, and the cells were lysed by repeated strokes with a type A
pestle. The extent of lysis was monitored by staining 10 µl of the
lysate with DAPI (4',6-diamidino-2-phenylindole) followed by
fluorescence microscopic examination to determine the release of
nuclei. The lysate was centrifuged at 5,000 × g for 20 min at 4°C. The supernatant was removed, flash frozen in an
ethanol-dry ice bath, and stored at 
80°C as "cytoplasm."
The nuclear pellet was suspended in 5 volumes of sucrose buffer (0.25 M
sucrose; 10 mM HEPES, pH 7.9; 3.3 mM MgCl
2; 10 mM
KCl; 0.5 mM DTT; pepstatin, 1 µg/ml; leupeptin, 0.5 µg/ml; 0.5
mM
phenymethylsulfonyl fluoride) and centrifuged in a swinging
bucket
rotor at 1,100 ×
g for 15 min at 4°C. The
supernatant was
discarded, and the pellet was suspended in 2.5 volumes
of sucrose
buffer and layered over an equal volume of 0.35 M sucrose
buffer
(i.e., sucrose buffer adjusted to 0.35 M sucrose) for
centrifugation
at 1,100 ×
g for 15 min at 4°C. The
supernatant was discarded,
and the pellet was suspended in 2.5 volumes
of 0.35 M sucrose
buffer. One-tenth of the volume was removed, flash
frozen, and
stored at
80°C as "nuclei."
The remaining suspension was sonicated on ice to disrupt the nuclear
structures by using a Bransonic sonicator with a microtip
at setting 3 and with five 10-s pulses followed by 30-s intervals
or until the
organellar structures were fully destroyed. The sonicate
was layered
over an equal volume of 0.88 M sucrose buffer and
centrifuged at
2,000 ×
g for 20 min at 4°C. The upper two-thirds
of
the volume was removed, aliquoted into small volumes, flash
frozen, and
stored at
80°C as "nucleoplasm," and the lower third
was stored
as "nucleoli" at
80°C.
Protein analysis.
Immunoblotting of the cellular fractions
was performed following sodium dodecyl sulfate-polyacrylamide gel
electrophoresis of samples from each fraction and electrotransfer to
the nitrocellulose membrane (2). Anti-fibrillarin
monoclonal antibody P2G3 (the kind gift of M. Christensen, University
of Kentucky [14]) was used as the primary antibody and
was followed by donkey anti-mouse immunoglobulin G (Jackson
Laboratories) as the secondary antibody. Antibody reactions were
detected with the ECL system (Amersham).
Mapping of 2'-O-methylation sites.
Primer
extension reactions on SSU rRNA (L. tarentolae) or LSU rRNA
(T. brucei) used total L. tarentolae or T. brucei cellular RNA as substrate and 75/SSU or 92/LSU as primers,
respectively. For both species, ribose methylations in the vicinity of
snoRNA-rRNA complementarity were detected by the deoxyribonucleotide
titration method (35). Oligonucleotides complementary to
L. tarentolae SSU rRNA (75/SSU; see Table 1) or T. brucei LSU rRNA (94/LSU) were used as primers. For each primer,
four extension reactions were performed with concentrations of each
dNTP of 1, 0.2, 0.04, or 0.004 mM. Reactions were carried out in a
solution containing 50 mM Tris (pH 8.0), 25 mM KCl, 5 mM
MgCl2, and 5 mM DTT with 0.5 pmol of
32P-labeled oligonucleotide primer and 5 U of avian
myeloblastosis virus reverse transcriptase at 43°C. The ribose
methylation sites in the vicinity of RNA/SSU rRNA complementarity in
L. tarentolae were also mapped by primer extension with
radiolabeled primer 75/SSU on partially hydrolyzed RNA, which was
generated by alkaline hydrolysis as described previously
(55).
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RESULTS |
The L. tarentolae and T. cruzi SLA RNA
genes are tandemly arrayed.
The cloned SLA RNA loci were isolated
from cosmid libraries of L. tarentolae and T. cruzi (26). The genomic organization of the L. tarentolae and T. cruzi SLA RNA loci (Fig.
1) was similar to that described for
T. brucei (46): the SLA RNA genes were in tandem
arrays containing greater than 10 copies of approximately 1.45-kb units
in L. tarentolae and 0.85-kb units in T. cruzi
(data not shown). Thus, the organization of the SLA RNA gene in tandem arrays appears to be common among the kinetoplastids. The difference in
the unit length of the three SLA RNA repeats was almost entirely due to
variation in the spacer between the SLA RNA and the 75/76-nt RNA.
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FIG. 1.
The SLA RNA locus organization is conserved in L. tarentolae, T. brucei, and T. cruzi. Repeat
units from each species are aligned relative to the 75/76-nt RNA.
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Multiple conserved RNAs are transcribed from the SLA RNA
locus.
Sequence alignments of the entire SLA RNA repeat units from
L. tarentolae, T. cruzi, and T. brucei
prepared by using the PILEUP routine revealed an overall sequence
similarity of 41% between L. tarentolae and T. brucei. Further alignments between the individual T. brucei SLA RNA repeat transcripts and the entire L. tarentolae and T. cruzi SLA repeat sequences revealed
more pronounced similarities (66 to 82% [Fig.
2]) that included short blocks of
complete identity. These conserved blocks invariably corresponded to
regions within the transcripts mapped in T. brucei
(46). The transcripts from the SLA RNA repeats showed no
obvious similarity to sequences with known function as determined from
the major databases with the BLAST algorithm (1); however,
blocks of identity within the 75/76- and 92/94-nt RNAs contained box D
motifs, CUGA, which had been described for the box C/D class of snoRNAs
in higher eukaryotes (36); sequences with near consensus to
the box C were also found (Fig. 2).
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FIG. 2.
Conservation of sequence blocks and motifs in the SLA
RNA repeat. Shown is a sequence alignment of L. tarentolae
(Lt: accession number AF016399) and T. cruzi
(Tc: AF016400) repeats with the T. brucei
(Tb: Z50171) transcripts. Alignments were generated with the
Wisconsin package PILEUP (17) by using a gap weight of 1 and
a length weight of 0.6. The 75/76-nt RNAs are aligned with U25 snoRNAs
from human (Hs: U40580), mouse (Mm: U40654), and
Xenopus sp. (Xl: U72853). Periods denote sequence
matches. Gaps introduced into sequences are denoted by dashes.
Asterisks indicate positions conserved between phyla in the 75/76-nt
RNA alignment. Box C, D, and D' elements are overlined; no potential
box C was obvious within the 250/270-nt RNAs.
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To determine whether the
L. tarentolae SLA RNA repeat
yielded transcripts homologous to those seen in
T. brucei
(
46), oligonucleotides
with complementarity to the core of
each of the four regions of
interspecies similarity were used to probe
total RNA blots from
both organisms. At least four small RNAs
hybridized to these oligonucleotide
probes in both
T. brucei
and
L. tarentolae (Fig.
3A).
On 8% polyacrylamide
gels the
L. tarentolae RNAs have
apparent lengths of 75, 92, >400,
and 69 nt, the smallest being the
SLA RNA. The four
L. tarentolae transcripts are located
within 750 nt of the repeat and are more
tightly clustered than in the
T. brucei SLA RNA repeat (Fig.
1).
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FIG. 3.
Four stable RNAs from the L. tarentolae SLA
RNA repeat. (A) RNA blot analysis of total RNA from T. brucei (lanes Tb) and L. tarentolae (lanes Lt)
hybridized with the  -32P-labeled antisense
oligonucleotides Lt75.2, Lt92, Lt250, and LtSLA.1. Sizes are relative
to pUC19/HaeIII fragments. (B) Aberrant electrophoretic
mobility of the L. tarentolae 250-nt RNA. Total L. tarentolae RNA was separated on 8 M urea and polyacrylamide gels
of either 7.5 or 4.5% (denoted at the top of each panel). Lanes: M,
pBR322 MspI digest; Lt, L. tarentolae RNA. The
position of the 250-nt RNA is denoted by arrowheads.
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The
L. tarentolae >400-nt transcript migrated through
polyacrylamide gels with aberrant mobility, as indicated by a
discrepancy
between the apparent size of the RNA and the size available
for
the transcript within the SLA RNA repeat. Direct comparison of
the
electrophoretic mobilities on 4.5 and 7.5% gels revealed that
this RNA
migrated as ~400 nt on 7.5% gels and as 250 nt on 4.5%
gels (Fig.
3B), while other RNAs transcribed from this locus migrated
consistently
with the denatured DNA markers. The effect on relative
mobility of
modified RNAs (
34) appeared to be minimized in gels
with
lower concentrations of polyacrylamide; therefore, this transcript
was
subsequently referred to as the 250-nt RNA. The nature of
this
modification has not been pursued; its biological relevance
is
uncertain, since the
T. brucei counterpart did not show
anomolous
migration (data not shown). A high-molecular-weight species
(>622
nt) in
L. tarentolae that appeared in the RNA blot of
the 4.5%
gel may represent an unprocessed precursor as proposed for
T. brucei (
46).
The corresponding transcripts were also detected in
T. cruzi
total RNA (data not shown) and were approximately the same size
as in
T. brucei (
46), except for the 270-nt RNA that
migrated
at 350 nt in 7.5% gels (see below). The
T. cruzi
transcripts possessed
regions of extensive identity with the
corresponding transcripts
from
T. brucei and
L. tarentolae (Fig.
2). The order of the transcript
sequences in the
repeats was conserved (Fig.
1), and the
T. cruzi transcripts
were more closely spaced than in either
L. tarentolae or
T. brucei. The homologous RNAs showed multiple blocks of
sequence
conservation (Fig.
2). The region upstream of the D' box was
extensively
conserved in the 74/75- and 92/94-nt RNAs, whereas the
region
upstream of the D box was better conserved between the two
Trypanosoma species than between either of the
Trypanosoma spp. and
L. tarentolae.
The 5' end of each
L. tarentolae RNA was mapped by primer
extension (Fig.
4). The primer extension
termination products from
all four RNAs mapped to a position similar to
that of the corresponding
T. brucei transcripts
(
46). The 3' end of the
L. tarentolae SLA RNA
transcript was mapped by using a PCR strategy (see Materials
and
Methods) to give a precise size of 69 nt (data not shown).
Whether
these RNAs are primary transcripts or derived from a precursor
RNA as
suggested by the detection of lower-mobility species remains
to be
determined.
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FIG. 4.
Determination of the 5' ends of the RNA transcripts from
L. tarentolae. Antisense oligonucleotide primers for each
RNA (see Table 1; Lt75.2, Lt92, Lt250.2, and LtSLA.1) were used in both
primer extensions on total L. tarentolae RNA (PX) and
sequencing reactions on the repeat-containing plasmid pLtSLA. The
sequence leading up to the primer extension stop position is shown in
the left margin of each panel.
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The candidate snoRNAs partition to the nucleolus.
We examined
how these small RNAs were distributed in the cytoplasmic, nuclear,
nucleoplasmic, and nucleolar compartments of L. tarentolae
and T. brucei (Fig. 5). The
75-, 92-, and 250-nt L. tarentolae transcripts and the
T. brucei homologs were recovered primarily in the nucleolar
fractions, as predicted for snoRNAs. The profiles of the negative
controls, U2 and SL RNA (which were predominantly cytoplasmic, although
the majority of the nuclear material is nucleoplasmic) (27),
and the nucleolar positive controls, U3 snoRNA (27) and the
snoRNA-associated protein, fibrillarin (13, 53), were
consistent with successful subcellular fractionation.
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FIG. 5.
Candidate snoRNAs localize to the nucleolus. The
cellular fractions generated for L. tarentolae or T. brucei are abbreviated as follows: Tot, whole cell; Cyt,
cytoplasmic; Nuc, nuclear; Npl, nucleoplasmic; and Nli, nucleolar. RNA
blots were hybridized with antisense oligonucleotides against RNAs
designated in the center margin. Homologous RNAs were hybridized with
the same antisense oligonucleotide and are shown in adjacent panels. A
protein blot containing L. tarentolae fractions was
incubated with the monoclonal antibody P2G3 against Physarum
fibrillarin (bottom left panel). Control blots showing nucleoplasmic
localization of T. brucei SL RNA and nucleolar localization
of U3 and fibrillarin have been published elsewhere (27).
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Two of the small RNAs have sequence complementarity with
rRNAs.
In higher eukaryotes, the region of snoRNA interaction with
the rRNA lies within the 25-nt region upstream of a D or D' box (29, 43, 55). By using a modified matrix (6),
regions of complementarity to the kinetoplastid rRNAs were found for
both the 75/76- and 92/94-nt RNAs. Invariably, complementarity fell within the 25 nt upstream of the potential D or D' boxes in these RNAs
(Fig. 6). An 11-nt stretch of
complementarity was present upstream of box D' in the 75/76-nt RNA
(Fig. 6A) that corresponded to helix 36' of the SSU rRNA
(16). A region of complementarity (9 to 14 bp) upstream of
box D in the 92/94-nt RNA (Fig. 6B) indicated an interaction within
domain VI of the LSU rRNA that corresponded to small rRNA 2 in
kinetoplastids (10, 59). The sequences of the SSU rRNAs from
L. tarentolae, T. brucei, and T. cruzi
and those of the LSU rRNAs from T. brucei and T. cruzi were available; thus, a complete analysis could not be made
for L. tarentolae. The rRNA sequences in helix 36' and
domain VI are conserved throughout the kinetoplastid, vertebrate, and
fungal phyla and are documented sites of 2'-O-ribose
methylation in the vertebrates (LSU and SSU) and yeasts (SSU)
(34). The vertebrate U25 snoRNAs implicated in the
methylation of this SSU rRNA site have been described and are shown in
alignment with the 75/76-nt RNA in Fig. 2. These experiments were
controlled by using sequences outside of the 25-nt region upstream of
boxes D and D' and throughout the 250/270-nt RNAs and the SLA RNAs in
searches for complementarity to rRNA sequences. These searches
consistently resulted in less than 10 nt of complementarity (data not
shown).
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FIG. 6.
The 75/76- and 92/94-nt RNAs are complementary to the
SSU and LSU rRNAs. Each candidate snoRNA sequence was compared to the
respective SSU and LSU rRNA by using the Wisconsin package BESTFIT
(17) and allowing for G-U base pairing with a gap weight of
5 and a length weight of 0.3. Schematics of the 75/76-nt RNA (A) and
the 92/94-nt RNA (B) indicate the regions of complementarity with rRNA.
For each region of complementarity the rRNA is shown above the reversed
partial sequence of the putative snoRNA. Box D and D' elements are
underlined. Standard Watson-Crick base pairs are indicated by colons
(:); G-U base pairs are indicated by periods. Filled circles represent
2'-O-methylation sites mapped in this study that are
conserved among higher eukaryotes. The open circle represents a
nonconserved methylation site in T. brucei. Y
represents the equivalent position in kinetoplastid rRNAs of
2'-O-methylation sites conserved among higher eukaryotes
(Gm1448 in X. laevis SSU rRNA [33] in panel
A and Am3713 in X. laevis and Am4550 in human LSU rRNA
[34] in panel B). The accession numbers for the rRNA
sequences were as follows: T. brucei SSU, M12676; T. cruzi SSU, M31432; L. tarentolae SSU, M54225; T. brucei LSU, X14553; and T. cruzi LSU, L22334.
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Ribose methylations occur within the regions of complementarity
between the kinetoplastid snoRNAs and rRNAs.
The significance of
complementarity between the putative snoRNAs and the rRNAs depends
on the presence of a ribose methylation on the rRNA in the appropriate
regions; therefore, methylation mapping studies were done.
2'-O-Methylations inhibit reverse transcription at low
nucleotide concentrations (35), so primer extension
reactions were performed from sites 3' of the complementarity in the
SSU and LSU rRNAs with decreasing concentrations of dNTPs (Fig.
7). These reaction mixtures were
electrophoresed alongside a sequencing reaction mixture to allow the
bases to be assigned. In the case of the 75/76-nt RNA (Fig. 7A),
extension reactions on L. tarentolae RNA with the
oligonucleotide primer 75/SSU produced two sets of bands. One, a stop
(Fig. 7, open arrowhead) at all nucleotide concentrations at position
G1835 of the L. tarentolae SSU rRNA (7), is most
likely template sequence dependent. The other set was a doublet (solid
arrowheads) that is most likely due to a ribose-methylated site because
of concentration dependence of the stops (increased abundance at low
concentrations of nucleotide). The larger arrowhead denotes the
position of the methylated nucleotide G1830. The small arrowhead
appears to be "stuttering," a common outcome of reverse
transcription across ribose-methylated nucleotides (35). To
determine whether the complete stop at position G1835 was due to a
methylation site, a second method of 2'-O-methylation mapping was done. This method takes advantage of the fact that the 2'
modification prevents the hydrolysis of the phosphodiester bond of RNA
under heated alkaline conditions. L. tarentolae RNA was
partially hydrolyzed and used as a substrate for primer extensions with
the same primer (75/SSU). The extension reaction produced a ladder of
stops at each nucleotide, including U1836, but with a strongly
diminished stop at G1831, a finding consistent with 2'-O-methylation of G1830 and no ribose methylation at
G1835.
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FIG. 7.
Regions of complementarity are associated with conserved
2'-O-methylated positions. Primer extension mapping of the
ribose methylation sites in the region of complementarity between the
L. tarentolae (L.t.) 75-nt RNA and the SSU rRNA
(A) and between the T. brucei (T.b.) 94-nt RNA
and the LSU rRNA (B) is shown. Primer extension reactions were done
with decreasing concentration of dNTPs. Concentration-dependent stops
are denoted by filled arrowheads, with the larger arrowhead marking the
position of the ribose methylation. An open arrowhead marks a
concentration-independent stop. Positions of modified nucleotides are
shown by sequencing reactions on rDNA-containing plasmids (pL8,
L.t. SSU rRNA [9]; pRB3, T.b.
LSU rRNA [10]) primed with the same oligonucleotides
used in the primer extension reactions (as labeled at the top of each
panel). Positions of ribose methylations in the L. tarentolae SSU rRNA were additionally determined by primer
extension of partially hydrolyzed total L. tarentolae RNA
(alk-rRNA). RNA was exposed to heated alkaline conditions for 2 min
(2') or 4 min (4') as labeled. Positions of
2'-O-methylations are indicated by circles to the right of
the rRNA sequences; filled circles denote positions that are methylated
in the higher eukaryotes (see Fig. 6 for references).
|
|
The region of complementarity between the
T. brucei LSU rRNA
and the 94-nt RNA corresponded to a ribose methylation site conserved
in higher eukaryotes (
34). Due to the unusual processing of
the
T. brucei LSU, Gm4141 (Fig.
7B) corresponds to position
40
of srRNA 2 (
59). In addition, ribose methylation of
Am4150,
which is not phylogenetically conserved, was observed in this
experiment.
| |
DISCUSSION |
Characterization of the SLA RNA locus revealed that the three
additional small transcripts were conserved in the kinetoplastids. We
present evidence identifying three of these transcripts as snoRNAs. The
gene organization is similar in the related kinetoplastids L. tarentolae, T. brucei, and T. cruzi; the SLA
RNA genes are tandemly arrayed, with 
12 copies per haploid genome.
The relative order of the four transcripts templated within the SLA RNA
repeat is identical: 75/76-, 92/94-, and 250/270-nt and SLA RNA (Fig. 1). The 250/270-nt RNA is a candidate snoRNA based on subcellular localization in L. tarentolae and T. brucei,
although conserved elements or structural features that would allow it
to be categorized among the known groups of snoRNAs are not apparent.
The 75/76- and 92/94-nt RNAs contained box C, D, and D' motifs and
fractionated with nucleolar markers. Consistent with this observation,
RNAs of similar size are immunoprecipitated from T. brucei
extracts by antifibrillarin antiserum (27). The sequence
immediately upstream of the D' box in the 75/76-nt RNA shows conserved
complementarity with SSU rRNA sequences in L. tarentolae,
T. brucei, and T. cruzi, which corresponds to a
2'-O-methylation site that is known to also be present in
human, mouse (54), and yeast (29) SSU rRNA and in
Xenopus sp. (33). In these higher organisms this
ribose methylation (equivalent to nucleotide Gm1448 in Xenopus
laevis) is guided by the U25 snoRNA (55). Likewise,
sequences upstream of the D box in the 92/94-nt RNA appear to interact
with the T. brucei LSU rRNA to guide the methylation of the
position equivalent to nucleotide Am3717 in the Xenopus LSU
rRNA (34). Potential snoRNA-rRNA interactions upstream of
box D in the 75/76-nt RNA (Fig. 5A) and upstream of box D' in the
92/94-nt RNA are predicted only for T. cruzi. The lack of
support for homologous interactions in T. brucei and
L. tarentolae may be due either to interaction with variable
regions of the rRNA or to divergence of functional domains within the
snoRNAs. The homologous modifying domains may be coupled with different
functional domains in the various trypanosomatids.
The kinetoplastid LSU rRNA is processed through a cleavage pathway
analogous to the processing seen in higher eukaryotic rRNAs. It is
further cleaved into two large and four small fragments that remain
associated in the ribosome (28) and form the same core LSU
structure found in higher eukaryotes (48). Methylation of
rRNA is also a conserved phenomenon: an estimated 1.4% of the Crithidia fasciculata rRNA is conserved compared to 3.0% of
human and 2.1% of yeast rRNA (34). snoRNAs appear to have
two functional domains (43); thus, the divergence between
the sequences upstream of the D' and D boxes in the different
kinetoplastid species may represent the independence of function in the
two halves of each molecule. While many of the
2'-O-methylated positions seen in the rRNAs of higher
eukaryotes are conserved among vertebrates and fungi, others are
conserved only among vertebrates and still others appear to be specific
to particular taxa (34). The populations of snoRNAs may
reflect these differences, and a similar variation in methylation
patterns among the divergent kinetoplastid species may account for the
variation in sequences of these small RNAs.
The rRNA 2'-O-methylations identified in the proposed 75/76-
and 92/94-nt RNA interaction sites correspond to sites within the rRNA
of vertebrates (34). In vertebrates and yeasts the site of
methylation is typically complementary to 5 nt downstream of the D or
D' box; these 5 nt generally have 4 to 5 bp of complementarity with the
rRNA. It is notable that the corresponding methylated nucleotides in
the kinetoplastids are positioned at the first base pair of
complementarity proximal to the D or D' box and are 1 nt downstream of
the D' box (75/76-nt RNA) or 6 nt downstream of the D box (92/94-nt
RNA). Thus, the mechanism of recognition (snoRNA base pairing with
rRNA) is probably conserved in all eukaryotes; however, the selection
of the specific modification site may follow an altered rule in
trypanosomatids. We cannot rule out the possibility that
as-yet-unidentified snoRNAs bring about the rRNA modifications we have
mapped or that these snoRNAs modify RNA molecules other than rRNA.
The definitive assignment of snoRNA function awaits deletion and/or
modification of these multicopy genes and rescue with mutated or
episomal snoRNAs.
Base and ribose methylations can have a wide range of effects, both
stabilizing and destabilizing RNA secondary structure (25,
34). Although individual methylations are generally nonessential for viability or rRNA processing, their importance may be cumulative or
synergistic. The role of box C/D snoRNAs in 2'-O-ribose
methylation involves base pairing with the target rRNA site, in
contrast to bacterial methyltransferase activities, which require only
the secondary structure of the rRNA (4, 61). The secondary
structure may also be the determining feature for eukaryotic
nonnucleolar structural RNAs that are methylated outside of their
7-methyl-G cap structures, as in some snRNAs. These internal
methylation sites on small nuclear RNAs are located in regions that are
functionally important and postulated to facilitate RNA-RNA
interactions within the spliceosome (25). An RNA
methyltransferase activity has been identified in trypanosomatids
(56), and the elucidation of the enzymatic components will
allow comparisons with higher eukaryotes.
The clustering and conserved order of the four transcripts may reflect
constraints such as polycistronic transcription (45). The
major difference in size among the repeats occurs in the region between
the SLA RNA transcript and the 75-nt RNA (725 bp in L. tarentolae, 34 bp in T. brucei, and 115 bp in T. cruzi). This spacing and the presence of the 75-nt RNA as the
first transcript in the array (Fig. 1) creates a working model for the
order of transcripts within the repeat. In higher eukaryotes the box
C/D-containing class of snoRNAs are often processed from the introns of
protein-encoding genes in vertebrates (43, 54) or are
transcribed polycistronically in some yeast genera and plants (32,
50). Kinetoplastids do not have typical introns in their
protein-coding genes; given the precedents of polycistronic
transcription of protein-coding genes, divergent transcription of small
RNA genes in trypanosomatids (41, 57), and the unusual
nature of transcription of snoRNAs in higher eukaryotes, in vivo
expression studies will be necessary to determine whether the
SLA-repeat RNAs are transcribed individually or they are processed from
a common precursor molecule, as suggested by the observation of larger,
potential precursor molecules in RNA blots (Fig. 3) and S1 nuclease
protection assays (46).
The multicopy, tandemly repeated nature of the trypanosomatid SLA RNA
locus-derived snoRNAs may not represent the only arrangement for
snoRNAs in this lineage. Identification and analysis of additional snoRNAs will reveal if this is a characteristic feature of the kinetoplastids or whether other snoRNAs can be found in the introns of
polycistronic pre-mRNAs, analogous to the vertebrate arrangement. Why
does the cell maintain 10 to 12 copies of the genes for these snoRNAs yet only one copy of the U3 snoRNA gene? The linkage of the
snoRNAs and the SLA RNA may be indicative of the SLA RNA function; the
SLA RNA was identified by psoralen cross-linking to a methylated molecule, the SL RNA.
| |
ACKNOWLEDGMENTS |
We thank Shula Michaeli and Jan Dungan for helpful discussions
and Jörg Hoheisel, Jaime M. Santana, Marc Christensen, and Roberto Hernández for providing invaluable reagents and
information for this study. We also thank Aparche Yang for
contributions to the project.
This work was funded by NIH grants AI34536 to D.A.C., AI21975 to N.A.,
and AI34093 to T.H. The Microbial Pathogenesis Training Grant,
5-T32-AI-07323, supported trainees N.R.S. and B.K.Y.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Microbiology and Immunology, UCLA School of Medicine, 10833 Le Conte Ave., Los Angeles, CA 90095-1747. Phone: (310) 825-4195. Fax: (310)
206-3865. E-mail: dc{at}ucla.edu.
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Abstract
Introduction
