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J Biol Chem, Vol. 275, Issue 4, 2259-2264, January 28, 2000
From the U4 small nuclear RNA is essential for
trans-splicing. Here we report the cloning of U4 snRNA gene
from Leptomonas collosoma and analysis of elements
controlling its expression. The trypanosome U4 RNA is the smallest
known, it carries an Sm-like site, and has the potential for extensive
intermolecular base pairing with the U6 RNA. Sequence analysis of the
U4 locus indicates the presence of a tRNA-like element 86 base pairs
upstream of the gene that is divergently transcribed to yield a stable
small tRNA-like RNA. Two additional tRNA genes, tRNAPro and
tRNAGly, were found upstream of this element. By stable
expression of a tagged U4 RNA, we demonstrate that the tRNA-like gene,
but not the upstream tRNA genes, is essential for U4 expression and
that the B box but not the A Box of the tRNA-like gene is crucial for expression in vivo. Mapping the 2'-O-methyl
groups on U4 and U6 small nuclear RNAs suggests the presence of
modifications in canonical positions. However, the number of modified
nucleotides is fewer than in mammalian homologues. The U4 genomic
organization including both tRNA-like and tRNA genes may represent a
relic whereby trypanosomatids "hired" tRNA genes to provide
extragenic promoter elements. The close proximity of tRNA genes to the
tRNA-like molecule in the U4 locus further suggests that the tRNA-like
gene may have evolved from a tRNA member of this cluster.
In trypanosomes all pre-mRNAs are produced by
trans-splicing. In this process a common short spliced
leader (SL)1 derived from a
small RNA, the SL RNA, is added to each mRNA. trans-splicing is mechanistically related to
cis-splicing (1).
As in cis-splicing, U snRNAs are required. Trypanosome
counterparts to U2, U4, and U6 have been characterized and shown to function in trans-splicing (2, 3). Trypanosome snRNAs are generally smaller than their cis-splicing counterparts. Only
recently was the trypanosome U5 homologue identified and its gene
cloned and sequenced (4, 5). Evidence supports the presence of a
trypanosome tri-snRNP complex carrying the U4/U6·U5 snRNAs (5). However, the trypanosome U5 snRNA may have a unique role in
trans-splicing based on its potential to interact with the
SL RNA intron region by base pairing, as supported by in
vivo cross-linking experiments (4) and phylogenetic conservation
(5).
The majority of trypanosome snRNAs carry a divergent Sm site. The Sm
site was shown in other eukaryotes to bind core proteins that are
common to all snRNPs and are recognized by sera from autoimmunue
patients (6). The trypanosome snRNPs are the only ones so far that are
not recognized by anti-Sm sera that recognize Sm proteins from yeast to
man. Surprisingly, however, trypanosomes do possess Sm proteins,
because recently an SmE homologue was identified among the core
proteins that bind the SL RNA (7).
In mammals and yeast, U snRNA genes, except U6, are transcribed by RNA
polymerase II (8). The promoters of these genes include distal and
proximal elements and TATA boxes. In trypanosomes, in contrast, all
small RNAs including U2, U3, U6, and 7SL RNA are transcribed by
polymerase III (9). In the last three cases, A and B box elements
located upstream of the gene were shown to be essential for
transcription in vivo. These control elements are part of
the internal control regions of divergently oriented tRNA genes or in
the case of the U2 snRNA, of a tRNA-like gene (10).
To study further the assembly of trypanosome U snRNAs in
vivo, we have cloned and sequenced the U4 gene from the
monogenetic trypanosomatid Leptomonas collosoma. This
completes the set of spliceosomal RNAs from this trypanosomatid
species. The U4 gene locus represents a new mode of genomic
organization, harboring both tRNA-like and tRNA genes upstream of the
coding region. Like U2 snRNA, but unlike U6 snRNA, the B box sequence
of the tRNA-like molecule plays a major role in the expression of the gene.
Oligonucleotides--
Oligonucleotide 424, 5'-GCACTAAATGAACAATCACTGAGG-3'; 425, 5'-ATACTAAAAACTCCAGTACTCCC-3'; 25000, 5'-CCCCACCCTTGGTGGATGCA-3' antisense to positions 94-113 of the U4 RNA; 25002, 5'-GGGAATCGAACCCGGGCCTC-3' antisense complementary to positions Cloning of the Gene Encoding the U4 RNA--
L.
collosoma DNA (10 µg) was digested to completion with
Sau3AI, and the digest was electrophoresed on 1% agarose
gel. Fragments between 0.5 and 1.2 kilobases were excised and
electroeluted. The eluted DNA was extracted with phenolchloroform and
precipitated in ethanol. The concentrated DNA (0.25 µg) was ligated
in 500 µl of buffer containing 50 mM Tris-HCl, pH 7.4, 10 mM MgCl2, 10 mM dithiothreitol, 1 mM ATP, 10 mg/ml gelatin, and 1 unit/ml ligase (New England
Biolabs) at a DNA concentration of 0.5 µg/ml. The ligation mixture
was further extracted with phenolchloroform, precipitated in ethanol,
and used as inverse PCR template. The PCR was performed with the
primers, 424 and 425, at 94 °C for 1 min, 50 °C for 1 min,
72 °C for 2 min for 30 cycles. The PCR product (600 bp) was
gel-purified and then cloned in pCRTMII cloning vector
(Invitrogen). The clone termed as U4-1 was sequenced by SP6 and T7
primers and was found containing the U4 coding region and only 157 bp
upstream sequence. To obtain the full sequence of the upstream
regulatory region, a Plasmid Construction--
Tagging of the U4 gene and
site-directed mutagenesis were generated by PCR using primers carrying
the mutation and oligonucleotides from the 5'- and 3'-ends of the gene
(indicated above). The mutations were confirmed by sequencing. The
mutated genes were cloned to the pX vector (11). L. collosoma cells were transformed with the constructs, and cell
lines were selected in the presence of G418 as described previously
(11).
RNA Isolation and Analysis--
Total RNA was prepared with
TRIzol (Life Technologies, Inc.). The RNA samples were fractionated on
a 10% polyacrylamide-7 M urea gel and electroblotted onto
a Nytran 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). Reactions were performed as described previously
(12) and were analyzed on a 6% polyacrylamide-7 M urea gel
next to DNA sequencing reaction performed with the same primer. Primer
extension reaction for mapping of 2'-O-methylations was
performed at 0.02 and 5 mM concentrations of dNTPs using
5'-end-labeled oligonucleotides complementary to the 3'-end of U4, U5,
and U6 snRNAs (13, 14).
The U4 Gene Is Linked to tRNA-like and Two tRNA Genes--
By
inverse PCR using primers derived from the Trypanosoma
brucei U4 snRNA sequence (2), a Sau3AI 600-bp genomic
fragment was cloned and sequenced. An RNA probe synthesized from this
clone was used to screen a genomic library. A 1.2-kilobase
AluI fragment carrying the gene was subcloned and sequenced.
The sequence contains the U4 coding region 432 bp upstream and 253 bp
downstream of sequences (Fig. 1).
Sequence analysis indicated the presence of two tRNA genes,
tRNAPro and tRNAGly, located at positions
The genomic arrangement of the L. collosoma U4 locus does
not resemble the loci of T. brucei U3 (15), L. collosoma U5 (5), and L. collosoma U6 (11, 16, 17);
each of which has a divergently transcribed tRNA gene located 95-98 bp
upstream of the U snRNA sequences (Fig.
2). We therefore examined a tRNA-like
element upstream of the U4 gene, analogous to that described for U2
snRNA (10). Indeed, bona fide A and B boxes were found
(5'-TCGCGGAGTGGG-3' and
5'-GGTTCGATCCC-3',
respectively), both of which agree with the consensus sequences
(underlined). In comparing the consensus sequences with the
corresponding boxes in the U2 locus (10), the A box of U2 is located at
position
The U4 gene organization is unique when compared with other U snRNAs
(Fig. 2), because it not only contains a tRNA-like element like the
T. brucei U2 locus, but also carries two additional upstream tRNA genes, similar to U3, 7SL RNA, U6, and U5 snRNA gene loci. However, the companion tRNA located 95-98 bp upstream of the snRNAs is
always divergently transcribed, whereas the tRNA gene adjacent to the
tRNA-like sequence is transcribed from the same strand as the U4 gene.
The presence of tRNA-like and tRNA sequences suggests that the former
may have originated from a bona fide tRNA gene that had accumulated
major changes and lost its identity. This tRNA may have been part of a
tRNA cluster because most tRNA genes in trypanosomatids are clustered.
In the case of 7SL, U5, U6, and U3, the identity of tRNA gene was kept,
whereas in the case of U2 and U4, tRNA gene mutations accumulated but
the identity of the A and B boxes was preserved including their proper
spacing (Fig. 3A).
Because the tRNAs upstream of small RNA genes are transcribed (16, 18),
it was of interest to examine whether the tRNA-like gene is also
transcribed. Primer extension analysis was performed using an
oligonucleotide (26264), and a discrete extension product was obtained,
suggesting the presence of tRNA-like RNA transcript (Fig.
3B). Comparison of the tRNA-like structure to the
tRNAPro secondary structure is presented in Fig.
3A. Deviations of the tRNA-like structure from the canonical
are mainly in the D stem and T
To examine the genomic organization of the U4 gene, L. collosoma DNA was subjected to Southern blot analysis. A single
hybridization band was observed after digesting the DNA using
restriction enzymes with 4-bp and 6-bp recognition sites, suggesting
that the U4 gene, like all other U snRNA genes in trypanosomatids (2),
is a single copy gene (Fig.
4A). Northern blot analysis
indicates a single transcript of 114 nt (Fig. 4B).
The U4 RNA and the Relatedness to Its Homologues--
The U4
coding sequence is highly conserved among trypanosomatid species: 87%
identity with T. brucei (2) and 89% with Leishmania ternatolae (GenBankTM/EBI Data Bank
Accession no. X97621) U4 RNA (Fig. 5).
The RNA is shorter (114 nt) compared with metazoans (142-145 nt) (20), higher plants (150-154 nt) (21), and budding yeast (160 nt) (22). The
predicted secondary structure of U4 snRNA contains four stem-loop
structures (I-IV) similar to higher eukaryotic U4 snRNAs. Unlike other
unicellular organisms such as Physarum polycephalum,
Saccharomyces cerevisiae, and Schizosaccharomyces pombe, the trypanosomatid U4 snRNAs possess a full stem-loop (IV) at the 3'-end (Fig. 5) (21). The U4 secondary structure is conserved among the three trypanosomatids. High sequence conservation exists in
the 5'-region (Fig. 5), whereas variability occurs in the loops and the
single-stranded region between stem-loops II and III.
The potential for base pair interactions between L. collosoma U4 and U6 is presented in Fig.
6. The first interacting domain (Stem II)
is composed of 17 perfect base pairs as in T. brucei (2).
This stem is more stable than in yeast, where 11 perfect base pairs are
disrupted by a bulged C and followed by a perfect 5 base-paired duplex
(23). Stem I is composed of a 9-bp duplex disrupted by two bulged nt in
both L. collosoma and T. brucei. The position of
the bulged A in U4 snRNA is conserved in both trypanosomatid species,
whereas the position of the bulged nt in the U6 duplex is different
(the bulged A in T. brucei is located between positions 4 and 5 of the duplex, whereas the bulged U in L. collosoma is
located between positions 6 and 7, Fig. 6). In yeast, stem I is
composed of 8 perfect base-paired nt (23). Interestingly, the single
sequence difference (U at position 57) between T. brucei and
L. collosoma U4 RNAs is compensated for by the A at position
51, which is bulged in the T. brucei U4-U6 duplex.
The majority of changes between the trypanosomatid U4 RNAs are in the
central domain and central loop (Fig. 6). The central domain and
3'-stem-loop in the yeast U4 RNA is quite tolerant to mutation (24).
Interestingly, however, when the T. brucei stem-loop
structure, which is homologous to the 3'-stem-loop of the yeast RNA was
used to replace this homologous domain in yeast, the chimeric RNA could
not complement a null allele of the yeast U4 snRNA (25). This chimeric
U4 RNA did not associate well with the U6 snRNA, suggesting that in
addition to base pair interactions in stem I and II, other interactions
are required to promote U4/U6 complex formation. It was found that only
a single mutation in the 3'-stem-loop in yeast resulted in a
cold-sensitive phenotype (24). This suggests that the overall structure
of the 3'-stem-loop, rather than its particular sequence, may be
important for the U4 function.
A third potential for base pair interactions between U4 and U6 exists
in the Sm-like site of U4 positions 90-95 (Fig. 6), similar to a more
stable potential interaction for the T. brucei U4 snRNA at
positions 86-91 (also in the Sm-like site) (2). The significance of
this potential is currently unknown. Recently, a phylogentically
conserved stem structure (Stem III) was discovered, which has a
counterpart in the highly diverged U4atac and U6atac snRNAs (26). In
T. brucei this domain involves nt 22-27 of U6 and 86-91 of
U4 snRNA; the L. collosoma duplex would be formed by nt
90-95 of the U4 and 24-30 of the U6. It was suggested that at a
particular stage of splicing stem III could sequester a specific stretch of the U6 before it base pairs with U2. The potential for
interaction between the U6 and U2 (helix III) also exists for the
trypanosome snRNAs (27). Interestingly, the U6 sequence block involved
in the formation of the stem III between U6 and U4 is also involved in
the formation of the helix III between U6 and U2. So far no genetic
evidence exists to support these phylogentically conserved stem III interactions.
The central domain is the most variable region in the U4 molecule.
Indeed, deletion of almost the entire region in yeast yielded only a
mild cold-sensitive phenotype (24). Thus, the sequence of this domain
may not be important except to serve as spacer that separates the 5'-
and 3'-domains of the U4 RNA.
It has long been debated whether trypanosome snRNAs carry a bona fide
Sm binding site. The L. collosoma U4 Sm-like site (positions 87-93) deviates from the canonical sequence, because the U stretch is
disrupted by an A. Many trypanosomatid SL and snRNA Sm-like sites
deviate from the canonical consensus. In L. collosoma U2 snRNA, the U stretch is disrupted by a C (27), whereas the U6 Sm site
is very divergent with only a single U (18). The only two RNAs that
carry canonical Sm sites are the SL RNA (28) and the U5 snRNA (5).
However, this property is not shared with other trypanosomatids, for
instance the T. brucei SL RNA and U5 Sm sites (4, 29, 30).
By compiling all the Sm-like sites a consensus trypanosome Sm site can
be derived: AAAN4G (where in 71% cases N is a U). Based on the strong
deviation of these sites from the consensus Sm-like sequence and the
finding that trypanosome proteins are not recognized by anti-Sm sera,
it was proposed that trypanosome core proteins and their corresponding binding site may differ significantly from those in mammals. Affinity selection using antisense biotinylated oligonucleotides to T. brucei snRNP and SL RNP RNAs suggests that core proteins are
shared among all these particles (31). Moreover, antibodies raised against these proteins immunoprecipitate all the spliceosomal RNAs
including U6 and SL RNA (32). The recent finding of an SmE homologue in
trypanosomes, which carries both Sm motifs 1 and 2, suggests that Sm
proteins do exist in trypanosomes (7). Yet, the lack of recognition of
these proteins by anti-Sm sera is intriguing. Changes in both the
Sm-like site and the core proteins may have co-evolved. These
deviations may stem from the need of these snRNAs to interact with
different capping enzymes, because SL RNA has a unique cap structure
and U5 unlike U2 and U4 does not possess a trimethylguanosine cap (5),
whereas the U6 most probably possesses an inverted cap, like in all
other eukaryotes (2).
Extragenic Sequence Elements Controlling the Expression of the U4
Gene--
To investigate the elements that control expression of the
U4 gene, the coding region was tagged in the internal loop III by
inserting 6 bp between position 82 and 83 (Figs. 5 and
7). The marked gene carrying 210 bp of
upstream sequence carrying the tRNA-like coding region and 91 bp of
downstream sequence was cloned into the pX expression vector (Fig.
7A). RNA prepared from transfected cell lines was analyzed
by primer extension using oligonucleotide (25000) complementary to the
3'-end of the U4 snRNA. The results indicate that the tagged gene was
expressed efficiently (Fig. 7B, lane 2),
suggesting that the tRNA-like element, but not the tRNAPro
and tRNAGly, is important for the U4 expression. To further
explore elements essential for the expression, we mutated the A and B
boxes separately and established cell lines. Analysis of U4 expression
(Fig. 7B) demonstrated that mutation of the A box had no
effect, whereas mutation of the B box completely inactivated the gene.
To control for gene dosage (plasmid copy number), the level of the
neo mRNA encoded by the pX vector was examined by primer
extension and found to be almost identical in all cell lines (Fig.
7B, bottom). This pattern of dependence on the
tRNA-like extragenic A and B box elements is identical to that obtained
for the U2 gene, except that mutation in the T. brucei box A
also had a slight effect on expression (10). The T. brucei
U2 B box was identified as a binding site for a nuclear protein (33).
The protein might be involved in chromatin organization. Indeed, a B
box located downstream to the yeast U6 coding region was shown to
affect chromatin organization (34).
Mapping 2'-O-Methyl Modifications on the L. collosoma U4, U5, and
U6 snRNAs--
An important characteristic of U snRNAs is their
specific modification in regions highly conserved in evolution. In the
vertebrate U4 and U6, modifications are clustered in the 5'-terminal
region of U4 and the central region of U6 (35). Modifications are less numerous in yeast (36). We therefore examined the modifications of
snRNAs from trypanosomes, which diverged much earlier than yeast from
the eukaryotic lineage. The position of the 2'-O-methyl groups was determined by primer extension in the presence of low dNTP
concentration (14), which causes the reverse transcriptase to pause one
nt before the methylated site (13). The presence of specific stops in
low (0.02 mM) but not high (5 mM) dNTP
concentrations (Fig. 8) suggests
2'-O-methylation at positions A63 and A72 of U4. The A63
position is conserved in vertebrates and is the nearest nt to the
potential stem I of the U4/U6 duplex. A72 is in the vicinity of U4/U6
stem III (see Fig. 6), but this position is not modified in
verterbrates (35). In U6 snRNA 2'-O-methyls can be assigned
to positions G42, G47, U50, C53, and U56, located in stem I and stem II
of the U4/U6 duplex (Figs. 6 and 8). The modified nt of trypanosome U6
RNA agree well with their positions in vertebrate U6 (35), where they
appear in regions involved in intramolecular base pairing and are
expected to be at or near the spliceosome catalytic center.
Interestingly, U5 is modified (Fig. 8) in position A19 of the invariant
loop, which is the one position that deviates from the canonical U5
invariant loop sequence (5). We have suggested that this position
interacts with the SL RNA +2 position by base pairing (4, 5). Another
U5 modification appears in position A28, which seems to be specific to
trypanosome, because no modified nt were found in other eukaryotes
downstream of the invariant loop.
Recent study has shown that 2'-O-methylation of vertebrate
U6 snRNA is guided by box C+D snoRNAs (37), suggesting that
modification may take place in nucleolar or coiled bodies that have a
close relationship with the nucleoli. Indeed the modification of U6 position U47 was shown to be guided by a guide RNA that also guides the
methylation of 28 S rRNA. So far, no snoRNAs that have potential to
guide modification of trypanosome U4 or U6 snRNAs have been discovered,
although a guide that directs modification of 5.8 S rRNA has been
described (19).
We thank Joan A. Steitz for her interest in
the study and critical comments on the manuscript. We acknowledge Maile
Ray for performing inverse PCR to initially identify and characterize the L. collosoma U4 snRNA gene and Albrecht Bindereif for
communicating unpublished results.
*
This work was supported by German-Israeli Foundation Grant
I-353-078.03/94 and by National Institutes of Health Grant GM26154 (to
Joan A. Steitz).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) AF204671.
¶
To correspondence should be addressed. Tel.: 972-3-531-8068;
Fax: 972-3-535-1824; E-mail: michaes@biu.mail.ac.il.
The abbreviations used are:
SL, spliced leader;
sn, small nuclear;
RNP, ribonucleoprotein;
nt, nucleotide(s);
sno, small nucleolar;
PCR, polymerase chain reaction;
bp, base pair(s).
The trans-Spliceosomal U4 RNA from the Monogenetic
Trypanosomatid Leptomonas collosoma
CLONING AND IDENTIFICATION OF A TRANSCRIBED tRNA-LIKE ELEMENT
THAT CONTROLS ITS EXPRESSION*
,
Faculty of Life Sciences, Bar-Ilan
University, Ramat-Gan 52900, Israel the § Department of
Molecular Biophysics and Biochemistry, Howard Hughes Medical Institute,
Yale University School of Medicine, New Haven,
Connecticut 06536-0812, and the ¶ Department of Biological
Chemistry, The Weizmann Institute of Science,
Rehovot 76100, Israel
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
410
to 430 within the tRNAGly; 25001, 5'-CATCTCCCTGCGCAAGGCTT-3' antisense to positions 1-20 of U4; 26264, 5'-GGCCGTCTGCCGCCTGCCCT-3' antisense complementary to positions 120
to 140 in the tRNA-like RNA; 24772, 5'-CAGTACTCCTATCCATGGACGGGAATATT-TGCA-3' carrying insertion
(underlined) of NcoI site in position 82; 24422, 5'-AGGGTTACGGCTGGACTCCCAAGGCGGG-3' sense complementary to
positions 144 to 171 carrying substitutions (underlined)
in the B box positions 153 to 162; 24427, 5'-TTCCCTCGCGCTCATAGCAGGGCAGGCG-3' sense
complementary to positions 102 to 129 carrying substitutions (underlined) in the A box positions 112 to
117; 24424, 5'-CGGGATCCCCGGGAATCGAACCCGGG-3' sense to
positions 415 to 432 including a BamHI site for PCR mutagenesis; 24423, 5'-CGGGATCCCTCATATTTCTTGCTCCGCG-3'
sense to positions 191 to 210 including a BamHI site
used for deleting the two upstream tRNA genes; 24425, 5'-CGGGATCCCCCGCGGCGTTCCTTTCC-3' antisense to
positions 188-205 including a BamHI site, the 3'-primer for mutagenesis.
EMBL3 library was screened with an RNA probe
synthesized with T7 polymerase (Promega) using the U4-1 plasmid as a
template. Three positive plaques were obtained and digested with
AluI. A fragment of 1.2 kilobase was cloned into pBluescript
and sequenced with T3, T7, and the internal primer 25001.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
223
and 372, respectively. The tRNAPro is 90% identical to
the tRNAPro of humans, mice, and Caenorhabditis
elegans. Positions 4-53 of tRNAGly are identical to
the tRNAGly of humans, C. elegans, and mice. The
tRNAPro is transcribed in the same direction as the U4
gene, whereas the tRNAGly is divergently transcribed. The A
boxes of tRNAPro and tRNAGly (5'-TAGTCTAGTGG-3'
and 5'-TGGTCTAGTGG-3', respectively) conform to the consensus box A
sequence 5'-TRRYNNAGTGG-3' (the
most conserved positions are underlined). The B boxes of
these tRNAs (5'-GTTCAATTCC-3' and 5'-GTTCGATTCC-3', respectively) are
consistent with the box B consensus
5'-GTTCRANNCC-3'. Both tRNAs can
fold into the canonical tRNA cloverleaf structure.
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Fig. 1.
DNA sequence of the U4 gene locus. The
sequence of a 1.2-kilobase AluI fragment is presented. The
coding region of U4 snRNA is underlined, bold,
and indicated by uppercase lettering with its +1 position
indicated; tRNAPro, tRNAGly and tRNA-like genes
in the upstream region of U4 are underlined in bold
letters. The direction of transcription of the U4 and tRNA genes
is marked with arrows. The conserved boxes A and B of the
tRNA and tRNA-like genes are indicated by boxes and
uppercase letters.
104 whereas the U4 A box is located at position 107. The
U2 A box sequence (5'-TGGCCCGGGTGT-3') deviates more from the consensus
sequence than the U4 A box. The U4 box A contains all the highly
conserved positions (underlined), whereas the U2 A box
contains two deviations at positions 8 and 12 with G and T instead of A
and G. The B box in the U2 locus (5'-GGTTCGAGCCT-3') is located at
position 155 and that of U4 is located at position 153; both the U2
and U4 B boxes agree well with the consensus. Thus, the tRNA-like boxes
of U2 and U4 are positioned similarly with respect to the snRNA coding
regions.
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Fig. 2.
Comparison of the clusters of tRNA or
tRNA-like genes found upstream of L. collosoma and
T. brucei snRNAs (9). The coding regions of the
snRNAs are marked by frames with inside arrows indicating
the direction of transcription. The tRNAs or tRNA-like sequences are
represented by black arrows. The distance between the RNAs
is indicated.
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Fig. 3.
Secondary structure of the tRNA-like
transcript from the L. collosoma U4 locus.
A, comparison of the tRNA-like sequence (right)
with tRNAPro (left). The anticodon sequence is
framed and the positions of Box A and Box B are circled.
B, primer extension analysis with the antisense
oligonucleotide 26264 specific to the 3'-end of the tRNA-like coding
region. The 5'-end of the tRNA-like is indicated with an
arrow. DNA sequencing with the same oligonucleotide is shown
on the right.
C stem-loops. The acceptor stem is
extended at the 5'-end and the anti-codon loop is composed of 5 instead
of 7 nt. However, the RNA can still be folded into the cloverleaf
structure, and the A and B boxes are located in their conserved positions.
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Fig. 4.
A, Southern blot analysis of the U4
gene. L. collosoma genomic DNA (10 µg) was digested with
different restriction enzymes, transferred to membrane, and hybridized
with a 600-bp random primer-labeled U4-1 DNA. Sizes of fragments in a
1-kilobase DNA ladder (Life Technologies, Inc.) are indicated.
B, Northern blot analysis of U4 snRNA. Total L. collosoma RNA (3 µg) was analyzed on a 10% polyacrylamide-7
M urea gel and electroblotted onto a Nytran membrane. The
membrane was hybridized with labeled U4 antisense oligonucleotide
25000. The U4 transcript is indicated with an arrow. The
marker was end-labeled with pBR322 HpaII digest.
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Fig. 5.
The proposed secondary structure of L. collosoma U4 RNA and the comparison with U4 snRNAs from
L. ternatolae (GenBankTM/EBI
Data Bank Accession no. X97621) and T. brucei (2).
Squares and circles indicate nt differences
between L. collosoma and T. brucei and L. ternatolae, respectively. Bars indicate gaps in the
alignment. The Sm-like site is indicated.
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Fig. 6.
The proposed secondary structure of the
L. collosoma U4/U6 complex. The model is based on
yeast and T. brucei U4-U6 interactions (2, 23). The
asterisks indicate positions of the
2'-O-methylated nt (determined in Fig. 8).
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Fig. 7.
Expression of U4 snRNA. A,
schematic representation of U4 expression constructs. Lane
1, genomic locus of the U4 gene; lane 2,
construct carrying the tag, CCATGG, between positions 82 and 83 (black square) with only the upstream tRNA-like gene.
lane 3, construct carrying the U4-tagged gene,
upstream tRNA and tRNA-like genes, and a box A mutation,
TCGCGCTCATAG instead of TCGCGGAGTGGG; lane
4, the same as lane 3, but with a box B mutation,
GGCTGGACTCCC instead of GGTTCGATCCC. The arrows
indicate the direction of transcription. B, primer extension
analysis of U4 expression. Primer extension was performed using a
labeled oligonucleotide, 25000, antisense to the 3'-end of U4. The
positions of the U4 and tagged U4 are indicated with arrows.
Lanes 1, 2, 3, and 4 are as
in A. DNA sequencing with the same oligonucleotide is shown
on the right. Primer extension with a neo antisense
oligonucleotide was the control. The primer extension products are
indicated. WT, wild type; PE, primer
extension.
View larger version (56K):
[in a new window]
Fig. 8.
Mapping of the ribose methylated sites in
L. collosoma U4, U5 (5), and U6 (18) snRNAs.
Primer extension was performed in the presence of 5 or 0.02 mM dNTPs (indicated) with specific oligonucleotides
antisense to the 3'-end for each snRNA. The 5'-ends of the snRNAs are
indicated by arrows. The position of the reverse
transcriptase stops (one nt before the methylated site) are indicated
with the number of the nt.
ACKNOWLEDGEMENTS
FOOTNOTES
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
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