Vol 13, Issue 2, 254-263, February 2003
LETTER
Transcriptional Interactions Between Yeast tRNA Genes, Flanking Genes and Ty Elements: A Genomic Point of View
Eric C. Bolton and
Jef D. Boeke1
Department of Molecular Biology and Genetics, The Johns Hopkins
University School of Medicine, Baltimore, Maryland 21205, USA
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ABSTRACT
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Retroelement insertion can alter the expression of nearby genes. The
Saccharomyces cerevisiae retrotransposons Ty1–Ty4 are
transcribed by RNA polymerase II (pol II) and target their integration
upstream of genes transcribed by RNA polymerase III (pol III), mainly
tRNA genes. Because tRNA genes can repress nearby pol II-transcribed
genes, we hypothesized that transcriptional interference may exist
between Ty1 insertions and pol III-transcribed genes, the preferred
targets for Ty1 integration. Ty1s upstream of two pol III-transcribed
genes (SNR6 and SUP2) were recovered and analyzed by
RNA blot analysis. Ty1 insertions were found to exert a neutral or
modest stimulatory effect on the expression of these genes. Further RNA
analysis indicated a modest tRNA position effect on Ty1 transcription.
To investigate the possible genomic relevance of these expression
effects, we compiled a comprehensive tRNA gene database. This database
allowed us to analyze a genome's worth of tRNA genes and Ty elements.
It also enabled the prediction and experimental confirmation of tRNA
gene position effects at native chromosomal loci. We provide evidence
supporting the hypothesis that tRNA genes exert a modest inhibitory
effect on adjacent pol II promoters. Direct analysis of PTR3
transcription, promoted by sequences very close to a tRNA gene, shows
that this tRNA position effect can operate on a native chromosomal
gene.
[The following individuals kindly provided reagents,
samples, or unpublished information as indicated in the paper: S.
Sandmeyer and J. McCusker.]
It is well known that the integration of retroelements
can interfere with the expression of nearby genes
(Roeder et al. 1980 ; Harrison et al. 1989; Kobayashi et al. 1998;
Gaisne et al. 1999; Morgan et al. 1999). The five Ty retrotransposons
found in the budding yeast Saccharomyces cerevisiae apparently
target their integration to nonessential regions or "safe havens."
Whereas Ty5 was found to preferentially insert in vivo at telomeric and
silenced mating regions (Zou et al. 1996), Ty1–4 seem to target their
integration upstream of tRNA genes. Moreover, Ty1 (Devine and Boeke
1996) and Ty3 (Chalker and Sandmeyer 1992) were found to target their
integration just upstream of genes transcribed by RNA polymerase III
(pol III), including SNR6, 5S rDNA, and tRNA genes. Because
Ty1 and Ty2 insertions near genes transcribed by RNA polymerase II (pol
II) can activate, vary, or inactivate their transcription (Boeke and
Sandmeyer 1991) and Ty3 insertions in either orientation moderately
increase steady-state levels of SUP2 pre-tRNA (Kinsey and
Sandmeyer 1991), we hypothesized that Ty1 insertions might affect the
expression of target pol III-transcribed genes as well.
In S. cerevisiae, actively transcribed tRNA genes have been
shown to transcriptionally repress the expression of adjacent pol
II-transcribed genes (Kinsey and Sandmeyer 1991; Hull et al. 1994;
Kendall et al. 2000). This relatively ill-defined phenomenon, which we
refer to as tRNA position effect, has also been called tRNA-mediated
gene silencing (Kendall et al. 2000), and reportedly operates over a
few hundred base pairs (bp) on both sides of the tRNA gene. The tRNA
gene is only inhibitory when it is transcriptionally active (Hull et
al. 1994; Kendall et al. 2000). The severity with which tRNA position
effect down-regulates nearby pol II-transcribed genes, however, depends
on the pol II promoter used in the reporter.  -factor-induced
transcripts corresponding to Ty3  elements and full-length Ty3
elements were inhibited to varying degrees. elements were
reportedly transcriptionally inhibited two- to 60-fold, whereas Ty3
elements were inhibited two- to 14-fold (Kinsey and Sandmeyer 1991;
Hull et al. 1994). On the other hand, several artificially-juxtaposed
pol II-transcribed reporter genes were strongly inhibited by a
neighboring tRNA gene (Hull et al. 1994). We hypothesized that
appropriately oriented Ty1 insertions near tRNA genes might suffer
similar transcriptional inhibition. Alternatively, because Ty1 is
probably evolutionarily adapted to survival in a tRNA-proximal
environment, it may be relatively resistant to this form of inhibition.
Presently, the mechanism by which tRNA position effect occurs remains
unclear. Kendall et al. (2000) have proposed, however, that subnuclear
localization of tRNA genes to the nucleolus antagonizes transcription
of nearby pol II-transcribed genes. Despite the evidence for tRNA
position effect, little has been done to directly examine whether or
not it operates on any of the 274 native chromosomal tRNA loci. One
possibility is that pol II-transcribed genes found bordering these loci
may be exempt, or conditionally resistant to this form of
transcriptional inhibition. Alternatively, selective pressure might
maintain the close association of such pol II-transcribed genes with
tRNA loci as a general regulatory mechanism designed to maintain low
levels of expression.
To test whether there is transcriptional interference between tRNA
genes and Ty1 elements, we analyzed several in vivo Ty1 element
insertions into target plasmids, containing either a marked
SNR6 gene (U6mg) or a marked SUP2 tRNA gene
(sup2+b) (Chalker and Sandmeyer 1992, 1993). We found
Ty1 insertions to have a neutral, or in one case a stimulatory effect,
on the expression of adjacent pol III-transcribed genes. Additionally,
tRNA position effects on Ty1 insertions were measurable at considerable
distances. The inhibition was partial, however, and quite modest.
Finally, we present direct evidence that modest levels of tRNA position
effects can actually operate at a native chromosomal locus,
PTR3.
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RESULTS
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Integration of Ty1 Into Two pol III-Transcribed Target Regions
When a Ty1 element inserts upstream of a tRNA gene in divergent
transcriptional orientation, the Ty1 pol II promoter and the tRNA pol
III promoter can be separated by as little as 100–500 bp. To determine
whether the neighboring pol II and pol III promoters were interfering
with each other, Ty1-neo insertions were captured in vivo to
generate a set of systematically structured constructs with a
Ty1-neo element next to a pol III-transcribed reporter gene.
The pol III-transcribed reporter genes U6mg (Chalker and
Sandmeyer 1993) and sup2+b tRNA (Kinsey and Sandmeyer
1991) produce products distinguishable by size or hybridization
properties, respectively, from their native counterparts, allowing
effects on that target gene to be determined specifically without
altering the functions of the chromosomal counterparts. The reporter
genes are contained within the two 2 micron (2µ) HIS3 target
plasmids, pDLC605 and pDLC356, respectively, and have been
characterized previously. Figure 1 depicts
all of the Ty1-neo insertions that were captured in each
target plasmid. Twenty-two of 23 Ty1-neo integration events
into pDLC605 occurred within the region upstream of the U6mg
target gene, and one outlier lay downstream of U6mg (Fig. 1A).
Similarly, most (20 of 29) of the Ty1-neo elements integrated
upstream of the sup2+b target gene (Fig. 1B). The
other nine insertions mapped just downstream of
sup2+b. Clearly, the preferred sites of integration,
as indicated by multiple insertions at the same position, lie within
the region (200 to 83 bp) just upstream of either target gene. Although
Ty1 targeting in these plasmids is qualitatively similar to that
reported previously (Ji et al. 1993; Devine and Boeke 1996), the
presence of a relatively small upstream genomic window combined
with the presence of 2µ sequences, which are required for plasmid
propagation, may have limited the Ty1 target area used in this study.
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Figure 1. Integration of Ty1-neo into SNR6- and
SUP2-containing target plasmids. (A) U6mg
represents the marked SNR6 gene, and (B)
sup2+b is the marked SUP2 gene. The term
genomic signifies that the sequence is the native sequence flanking the
SNR6 or SUP2 loci, respectively, whereas
2µ and HIS3 are vector sequences. Ty1-neo
insertions into the target plasmids are depicted as small black
arrowheads. Arrowheads pointing downward represent Ty1-neo
insertions in the same transcriptional direction as the target gene,
whereas those pointing upward are transcribed divergently from the
target gene. The distance in base pairs from the major pol III
transcription start sites (+1) is indicated (Kinsey and Sandmeyer 1991;
Chalker and Sandmeyer 1993). The lowercase letters indicate the
individual Ty1-neo insertion constructs that were used for
expression studies.The Ty1-neo integration positions are
–273, –147, –90, +318, –608, –289, –96, and –83 for a–h and
–166, –105, –179, and –103 for t, u, w, and x, respectively.
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Ty1 Insertions Exert Neutral or Mildly Positive Effects on Adjacent pol III-Transcription
To examine the effect that Ty1 insertions have on the expression of
the genes they target for integration, total RNA isolated from cells
carrying plasmid constructs with a single Ty1-neo insertion
was subjected to quantitative RNA blot analysis, using U14
(control) and either U6- or sup2+b-specific
probes. Figure 2 shows the effects that
various Ty1-neo insertions had on the relative amount of
U6mg transcripts that were produced. The level of
U6mg RNA in cells containing a Ty1-neo insertion is
expressed relative to the level in cells containing the pDLC605 control
plasmid (Fig. 2B). The steady-state level of U6mg RNA was
unaffected by any of the Ty1-neo insertions at the tested
positions, lanes a–h in Figure 2, with the possible exceptions of
insertions b and h, which show a 30% reduction in expression.
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Figure 2. Effect of Ty1-neo insertions on U6mg target gene
expression. (A) RNA blot analysis of U6mg and
U14 transcripts containing the target plasmid with a single
integrated Ty1-neo element. Multiple yeast transformants
containing the same Ty1-neo target plasmid were analyzed. RNA
was also isolated from cells lacking the U6mg target plasmid
(-target). The endogenous U6 snRNA (U6) present in all cells
is indicated. The ratio of U6mg to U14 RNA was
determined for each sample. These ratios were further normalized for
plasmid copy number in each transformant. (B) Normalized RNA
levels are relative to that of a target plasmid lacking a
Ty1-neo insertion (-Ty1).
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Only one Ty1 insertion had a significant effect on the production of
sup2+b transcripts (Fig.
3). The level of sup2+b
pre-tRNA in cells containing a Ty1-neo insertion is expressed
relative to the level in cells containing the pDLC356 control plasmid
(Fig. 3B). A Ty1-neo insertion at position x in Figure 3B,
oriented such that the Ty1-neo element and the tRNA gene are
transcribed divergently, increased the steady-state level of
sup2+b pre-tRNA nearly fourfold. The data for the
Ty1-neo insertion at position x shows a fair bit of scatter,
but the effect is clearly significant (Fig. 3B). In most instances,
however, especially for those positions of multiple independent
insertions, the steady-state level of sup2+b pre-tRNA
was only modestly affected by the Ty1-neo insertions
(positions t and w in Fig. 3B). For unknown reasons, the scatter among
independent transformants was greater for the sup2+b
plasmids than for the U6mg constructs. All observed expression
was tRNA promoter-dependent because control experiments done on
promoter mutant constructs showed no expression (Fig. 3B; t*, u*, x*
and w*).
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Figure 3. Effect of Ty1-neo insertions on pre-sup2+b
target gene expression. (A) Representative RNA blots of
pre-sup2+b and U14 transcripts from cells
harboring plasmids with a Ty1-neo inserted at position t or x
(* indicates the presence of a G56 mutation in box B of the
sup2+b gene, rendering it transcriptionally
inactive). Multiple yeast transformants containing the same
Ty1-neo target plasmid were analyzed. RNA was also isolated
from cells lacking the sup2+b target plasmid
(-target). RNA blots are not shown for the target plasmids containing
Ty1-neo insertions at positions u and w. The ratio of
sup2+b to U14 RNA was determined for each
sample, and normalized for plasmid copy number. As multiple species of
pre-sup2+b transcript were present, indicated by the
bracketed region, the sum of all bands in this region was measured.
(B) RNA levels are relative to that of a target plasmid
lacking a Ty1-neo insertion (-Ty1).
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tRNA Position Effects on Ty1 Transcription
Because pol II promoters are reportedly regulated by tRNA position
effect, we examined whether the Ty1 promoter was affected by its
proximity to the SUP2 pol III promoter. To address the
question of interference of a pol III promoter on a neighboring pol II
promoter, we used the juxtaposed sup2+b tRNA gene and
several integrated Ty1 elements, respectively. Additionally,
sup2+b tRNA variants with a previously characterized
C to G substitution in the box B internal promoter element
(Chalker and Sandmeyer 1992, 1993) were constructed as controls.
Mutation of this absolutely conserved C to a G at position 56 of the
mature tRNA has been shown to decrease in vitro expression of the
SUP4 tRNATyr gene  20-fold (Allison et al. 1983).
Total RNA isolated from cells containing the mutant
sup2+b(G56) tRNA constructs, indicated by an
asterisk, was subjected to RNA blot analysis (Fig. 3B). As
expected, no sup2+b-specific pre-tRNA was detected in
these samples.
Further RNA blot analysis of total RNA from cells containing the
sup2+b tRNA constructs and cells containing the
promoter mutant sup2+b(G56) tRNA gene constructs
(Fig. 4), indicated a modest difference in
Ty1-neo expression consistent with tRNA position effect. Only
those Ty1-neo insertions whose orientation placed the Ty1
promoter close to the tRNA promoter were affected (Fig. 4B, w and x).
For these constructs, the steady-state level of Ty1-neo RNA in
the presence of the transcriptionally inactive tRNA gene,
sup2+b(G56), was approximately threefold higher (Fig.
4B, w* and x*) relative to the level of Ty1-neo RNA observed
in the presence of the transcriptionally active tRNA gene,
sup2+b. In Ty1 insertions at positions x and w, the
transcription start site of the Ty1 element lies 330 bp and 400 bp,
respectively, from the transcription start site of the
sup2+b tRNA gene (Fig. 4B). Clearly, the
transcriptionally active sup2+b tRNA gene interferes
with expression from the upstream Ty1 promoter. These results, along
with similar results observed for and Ty3 (Kinsey and Sandmeyer
1991; Hull et al. 1994) and other pol II-transcribed promoters fused to
various reporter genes (Hull et al. 1994), indicate that tRNA genes
might repress surrounding genes at their native chromosomal locations.
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Figure 4. Effect of sup2+b tRNA gene expression on
Ty1-neo expression. (A) Representative RNA blots of
Ty1-neo and ACT1 transcripts from the cells used in
the previous figure. RNA blot is not shown for the target plasmid
containing Ty1-neo insertion at position x. The ratio of
Ty1-neo to ACT1 RNA was determined for each sample.
Ratios, or levels, were adjusted for relative plasmid copy number.
(B) Ty1-neo RNA levels in the absence of an actively
transcribed tRNA gene (* indicates the presence of the G56 mutation in
sup2+b) are relative to those in the presence of an
actively transcribed tRNA gene.
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Assembly and Survey of a Database of tRNA Gene Loci
We generated a tRNA- and Ty-centered database for several reasons:
(1) to analyze the intergenic windows flanking tRNA genes to address
the hypothesis that regions upstream of tRNA genes are "safe
havens" for Ty element insertion; (2) to probe the hypothesis that
windows exist as buffer regions to relieve potential tRNA position
effects on adjacent pol II promoters; (3) to identify exceptions to the
general trend of larger windows as buffer regions to examine whether
tRNA position effects operate on flanking non-Ty sequences in the
genome; and (4) to generate a public resource to foster further studies
of tRNA loci and their associated Ty sequences.
The database covers the locations and orientations of the 274 tRNA
genes in the yeast genome and their neighboring open reading frames
(ORFs) as well as Ty sequences (SGD data as of July 2, 2002). The
database was used to analyze the features surrounding tRNA genes (Fig.
5A). From an initial survey of the
database, we show that the intergenic windows upstream of tRNA genes
are larger that the downstream windows even after the removal of all
annotated Ty sequence (Fig. 5B). The total mean upstream window is
2,330 bp long and the downstream window is 560 bp long. For comparison,
the average genome-wide inter-ORF distance excluding telomeric regions
(genome) was only 480 bp long. The upstream window value, however, is
significantly inflated by inclusion of full-length Ty elements. Removal
of these (Fig. 5B; -Ty elements) reduced the length of the upstream
window to 1,310 bp and the downstream window to 510 bp. Even when all
annotated Ty sequences including LTR fragments were removed from the
windows (Fig. 5B; -Ty sequence) the upstream window was still 1,010 bp
long, whereas those downstream were 490 bp long. Moreover, the
noncoding pol II-transcribed genes, such as the small nuclear RNA genes
except SNR6, are neither under- nor over-represented at tRNA
loci.
If there is no transcriptional interference between tRNA genes and
adjacent pol II-transcribed genes, there should be no orientation bias
in the genome. Analysis of the promoter orientations, expression
levels, and transcriptional initiation rates at tRNA loci, however,
provides additional evidence supporting the inhibition of upstream pol
II promoters by adjacent pol III promoters. The generic genomic tRNA
locus (Fig. 6A) contains the tRNA gene,
upstream and downstream non-Ty ORFs, and may or may not contain Ty
sequence. Sixty-seven percent of tRNA genes have Ty sequence between
them and the nearest upstream or downstream non-Ty ORFs. We analyzed
the transcriptional orientations of the adjacent upstream and
downstream non-Ty ORFs relative to their associated tRNA genes
genome-wide (Fig. 6B,C). The upstream and downstream non-Ty ORFs may be
transcribed in either the same direction as the tRNA gene or the
opposite transcriptional direction as the tRNA gene. The latter results
in divergent transcription for upstream non-Ty ORFs and convergent
transcription for downstream non-Ty ORFs. For all 274 tRNA loci, 55%
of upstream non-Ty ORFs are divergently transcribed with the tRNA gene,
whereas 42% of downstream non-Ty ORFs are transcribed in the same
direction as the tRNA gene. These two transcriptional orientation
combinations are relevant to the issue of tRNA position effect because
they place the adjacent non-Ty gene promoter closest to the tRNA gene
promoter, generating the conformations that could lead to tRNA position
effects. To identify tRNA loci possessing maximal tRNA position effect
conformations (i.e., genes that are actually immediately adjacent to
and within a few hundred base pairs of a tRNA gene), we examined the
distance distribution (includes all Ty sequences) of the adjacent
non-Ty ORFs with the above promoter orientations for the upstream (Fig.
6B) and downstream (Fig. 6C) genes.
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Figure 6. Genomic distribution of the distances from tRNA genes to their nearest
neighbor genes. (A) Cartoon of the generic tRNA locus in the
yeast genome. The gray double-headed arrows represent the distances
from tRNA genes to their nearest (B) upstream and (C)
downstream genes. Solid bars represent tRNA genes that are transcribed
in the opposite direction as the neighboring gene, and open bars
represent tRNA genes that are transcribed in the same direction as the
nearby gene. The minor peaks in the distributions for distances >6,000
and 12,000 bp indicate the presence of one or two full-length Ty
elements, respectively.
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For upstream genes within 400 bp of a tRNA gene there is a modest
transcriptional bias toward the inhibitory conformation; 67% are
transcribed divergently relative to the tRNA gene, thereby generating
the conformation favoring tRNA position effect. Supporting the notion
of tRNA position effect in yeast chromosomes, the subset of divergently
transcribed genes within 400 bp of a tRNA gene has a lower mean
expression level (0.6 copies/cell, Fig. 7A)
and transcription initiation rate (data not shown). This subset of
genes is transcribed on average 3.5-fold less (P < 0.0001)
than the mean of all genes upstream of tRNA genes (2.1 copies/cell), as
determined by examination of a transcription database (Holstege et al.
1998). These bioinformatic data indicate the presence of tRNA position
effects at the subset of loci very close to tRNA genes; all of these
lack interposed Ty sequences. In contrast, the mean expression level
for all upstream genes is unaffected by whether or not the flanking
genes are separated from the tRNA gene by Ty sequences. Interestingly,
the subset of genes within 400 bp upstream and convergently transcribed
relative to tRNA genes also has a lower mean expression level (0.6
copies/cell, Fig. 7B) than the mean of all genes upstream of tRNA
genes. The latter result should be interpreted with caution because of
the small sample number (n = 8 convergently transcribed
upstream genes within 400 bp of the tRNA gene).
For downstream genes within 200 bp of a tRNA gene, there is again a
transcriptional bias. The bias, however, is away from the inhibitory
conformation; only 38% of downstream genes within 200 bp of a tRNA
gene are transcribed in the same transcriptional orientation as the
tRNA gene. Also, this subset of genes in the same transcriptional
orientation is transcribed on average 1.5-fold less
(P = 0.04) than the average of all genes downstream of tRNA
genes (data not shown) as determined by examination of a gene
expression database (Holstege et al. 1998). Taken together, these
findings indicated that tRNA position effects exist at a number of
genomic tRNA loci in yeast chromosomes. Transcriptional inhibition of
the adjacent upstream genes, however, appears to be more dramatic.
tRNA Position Effects on a Genomic pol II-Transcribed Gene
Examination of the tRNA database allowed us to identify those rare
cases in which non-Ty ORFs were located very close to pol
III-transcribed genes. One such case is represented by the tY(GUA)F2
locus (Fig. 8A) in which the distance
between the 5'-end of the mature tRNATyr and the ATG of the
upstream PTR3 gene is merely 230 bp. Whereas PTR3
encodes a plasma membrane sensor of extracellular amino acids (Island
et al. 1991; Klasson et al. 1999; Forsberg and Ljungdahl 2001), it has
not been reported to function in either pol III transcription or tRNA
position effects. To determine whether pol III-transcription of this
tRNA gene interferes with the expression of the adjacent PTR3
gene in vivo, we engineered several congenic strains containing various
alleles at the tY(GUA)F2 locus. The wild-type
tY(GUA)F2 gene (wt) was replaced using a homologous
recombination strategy with one of three alleles: (1) a complete gene
substitution tY(GUA)F2 ::CaURA3MX4 allele
(URA3MX4); (2) the tY(GUA)F2-sup2+b allele
(sup2+b); or (3) the transcriptionally inactive
tY(GUA)F2-sup2+b(G56) allele
(sup2+b*). Total RNA was isolated from several
isolates for each of the strains containing the above chromosomal
tY(GUA)F2 alleles and subjected to quantitative RNA blot
analysis using PTR3- and ACT1-specific probes (Fig.
8B). In cells containing the URA3MX4 allele, the steady-state
level of PTR3 RNA was found to be more than fourfold higher
relative to the level of PTR3 RNA observed in the presence of
the wild-type or the sup2+b alleles. Moreover, the
presence of a transcriptionally inactive tRNA gene, the
sup2+b* allele, stimulated the steady-state level of
PTR3 gene expression >2.5 fold (P < 0.0001)
relative to that measured in the presence of a transcriptionally active
tRNA gene, the wild-type, or the sup2+b alleles.
PTR3 expression in the strain containing the URA3MX4
allele is possibly the highest because the presence of the pol
II-transcribed URA3MX4 cassette might physically recruit the
RNA pol II to the locus. These results directly demonstrate that tRNA
position effects occur in at least one native yeast locus.
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Figure 8. Effect of a native chromosomal tRNA gene on PTR3 expression.
(A) Cartoon of the tY(GUA)F2 locus on chromosome VI. Open
arrowheads indicate the transcriptional orientation, and the distance
between the tRNA gene and the flanking non-Ty ORFs is given in base
pairs. (B) RNA blot analysis of cells containing various
chromosomal alleles of the tY(GUA)F2 tRNA gene. The level of
PTR3 to ACT1 RNA was determined for each sample.
(C) PTR3 levels in the presence of no tRNA gene, an
actively transcribed tRNA gene and a transcriptionally inactive tRNA
gene are relative to those in the presence of the wild-type tRNA gene,
URA3MX4, sup2+b and
sup2+b* relative to wt. (* indicates the presence of
the G56 mutation in sup2+b).
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DISCUSSION
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Effect of Ty1 insertions on Adjacent pol III Transcription
Because Ty elements target their integration to noncoding regions,
it is reasonable to suggest that the reason for this is to minimize
deleterious effects on the host cell (Boeke and Devine 1998). One way
of doing so is to target integration upstream of pol III-transcribed
genes or to telomeres and silent mating loci; these regions are known
to contain relatively few genes and for their repressive (silencing)
effects. Ty1 elements are commonly found inserted upstream of tRNA
genes and other genes transcribed by RNA polymerase III. Surprisingly,
however, their effect on tRNA gene transcriptional activity has never
been directly evaluated. Possibly, this is because tRNA promoter
elements are internal, and it is widely assumed that sequences external
to the tRNA gene will have little impact on transcriptional activity,
in spite of evidence to the contrary (Kinsey and Sandmeyer 1991; Hull
et al. 1994; Kendall et al. 2000). Also, it is technically difficult to
measure the transcriptional activity of individual tRNA genes because
most are repeated. The latter problem can be overcome by carefully
marking the pol III-transcribed gene of interest, U6 and
SUP2 (Kinsey and Sandmeyer 1991; Chalker and Sandmeyer 1993).
Twelve different insertions, reflecting the common insertion sites
upstream of two pol III-transcribed genes were studied, including both
orientations of the Ty1 relative to the target gene. This revealed that
the U6 gene was relatively unaffected by the presence of a
nearby Ty1 element, perhaps because the native U6 gene already
contains a Ty1 LTR within its 5' flanking region (Brow and Guthrie
1990). Two of 4 insertions evaluated, however, showed a significant
(P < 0.05) increase in sup2+b tRNA
transcript abundance, in one case four times that observed in the
absence of the Ty1 element. It is formally possible that the increased
transcription results from insertion of DNA and not from the Ty1
sequence itself. Significant inhibitory effects on transcription were
not observed for any insertion. Analysis of the tRNA gene database
shows that 33 tRNA genes in strain S288C are as closely linked to Ty1
elements as are the insertions that stimulate tRNA transcription.
Therefore it is possible that a significant fraction of total tRNA gene
expression is attributable to adjacent Ty1 elements. Furthermore,
because Ty1 LTRs (delta elements) are found upstream of 67% of tRNA
genes, and these solo LTRs can recombine with full-length elements, it
is possible that Ty1 to solo LTR recombination could be a significant
force in modulating tRNA expression levels as cellular needs for tRNAs
change in evolution.
Effect of tRNA Genes on Ty1 Transcript Abundance
Just as Ty1 elements affect tRNA transcription, active tRNA genes
can affect Ty1 promoters. We show here that the Ty1 promoter is subject
to such effects, but that they affect only divergently transcribed
elements, and the maximal effect on transcript levels is a threefold
reduction. Our current data for tRNA position effects on Ty1 insertions
agree well with those reported for and Ty3 (Kinsey and Sandmeyer
1991; Hull et al. 1994). Whereas the initial isolation of
Ty1-neo insertions depended on selection for G418-resistant
yeast, we cannot rule out the possibility that the data for the Ty1
elements analyzed in this study may under-represent the extent of tRNA
position effects on Ty1 elements. Examination of the tRNA database
shows that 36% of the Ty1s in the genome are suitably oriented and
close enough to a tRNA gene to have their transcription affected in
this way. An interesting corollary to this type of regulation, which
depends on the transcriptional activity of the tRNA gene, is that any
cellular state, such as stationary phase, which interferes with tRNA
transcription (Tower and Sollner Webb 1988), might well have
the opposite effect on the transcription of suitably positioned Ty1
elements. Along these lines, we have searched for temporal and
functional commonalities among the upstream genes within 400 bp of tRNA
genes. By our analysis, this subset of genes does not appear to cluster
by any recognizably similar function, pathway, or expression profile.
tRNA Gene Database Allows a Genome-Wide Look at Ty1 Integration Windows
We have described a database of tRNA genes that can be used to study
the relationships between those genes, Ty1 elements, and neighboring
non-Ty ORFs. Using the database, we have calculated the mean distance
between the tRNA gene boundaries and their neighboring genes. At the
upstream boundary of tRNA genes, there is a remarkably large distance
(2,330 bp) to the nearest non-Ty1 genes, relative to the distance from
the downstream boundary (560 bp) or to the genome-wide inter-ORF
distance (480 bp). To determine whether this long 5'-intergenic
interval exists in the absence of Ty elements, we examined this
distance after deleting all full-length Ty elements (1,310 bp) or all
vestiges of annotated Ty sequences (1,010 bp) and found it was still
modestly longer than the control distances. These facts indicate that
the native yeast genome structure has evolved to put more DNA between
tRNA genes and ORFs, possibly to buffer the consequences of tRNA
position effect.
Transfer RNA Position Effect: Extent of the Effect and Possible Functions
We have described a database of tRNA genes that can be used to study
the relationships between those genes, Ty1 elements, and neighboring
pol II-transcribed genes. Examination of this database allowed us to
examine the relative orientations of the pol II-transcribed genes
flanking tRNA genes to seek evidence for or against position effects of
tRNA genes on the genes flanking them. The null hypothesis that there
are no position effects on adjacent genes would predict no bias in the
orientation of flanking pol II-transcribed genes relative to tRNA
genes, when one focuses on the subset of very closely spaced genes. We
found a strong deviation from randomness in the intergenic window
upstream of tRNA genes (Figs. 5B and 6B). Unexpectedly, we observed an
over-representation of genes divergently transcribed relative to the
tRNA gene. Consistent with the action of a tRNA position effect, we
observed that the mean expression level and transcription initiation
rate for the divergently transcribed genes was 3.5-fold less than
control genes. Why might such a position be beneficial to this subset
of genes? This subset of genes might represent genes that function best
at very low levels, or perhaps these genes are derepressed specifically
when tRNA genes are at their lowest expression level.
Finally, in this paper we present the first direct evidence that tRNA
position effect can influence the expression of a chromosomal gene, the
PTR3 gene. Although the effect is modest, it is reproducible.
Extrapolation of this result to the entire genome indicates that as
many as 12 genes in the yeast genome are down-regulated by tRNA
position effects by 3.5-fold or more, and there may be many more
affected to a lesser extent.
|
METHODS
|
|---|
Strains and Media
All strains used in this study are described below. Media were
prepared as described (Sherman et al. 1986).
Targeting Assay and Plasmids
The Ty1 donor plasmid, pSD530, consists of a neo-marked
Gal–Ty1 element in a pRS316 (URA3 CEN6) plasmid backbone
lacking the bla gene (Devine and Boeke 1996). Growth of the
yeast carrying similar Gal–Ty1 constructs on galactose has been used
to induce Ty1 transposition 20- to 100-fold (Boeke et al. 1985, 1988).
The neo marker within the Ty1 element confers G418 resistance
to yeast cells harboring newly transposed Ty1-neo elements
(Boeke et al. 1988). Therefore, after induction and subsequent loss of
the donor plasmid, cells that are auxotrophic for uracil and
G418-resistant have undergone transposition.
The target plasmids used here, pDLC356 and pDLC605, respectively,
consist of the sup2+b or U6mg target genes
and flanking genomic sequence in a high copy, bla- and
HIS3-marked, shuttle vector (Chalker and Sandmeyer 1992,
1993). These targets contain pol III-transcribed genes that were
modified in subtle ways to allow their products to be distinguished
from their endogenous wild-type counterparts. Because the donor plasmid
contains Ty1-neo (KanR) and the target plasmid
contains the bla gene (AmpR),
KanR/AmpR clones arise only by transposition of
Ty1-neo into the target plasmid. Therefore, transposition
events into either of the target plasmids can be rescued by
transformation into Escherichia coli and identified by
selection on medium containing ampicillin and kanamycin.
The donor strain, ySD10, was described previously (Devine and Boeke
1996) as YPH499 (MATa ura3-52
trp163 his3200 lys2-801
ade2-101 leu21) (Sikorski and Hieter 1989)
containing pSD530 (Devine and Boeke 1996). Each of the target plasmids
was then transformed into ySD10 by the lithium acetate method (Gietz et
al. 1992) and carried through the transposition assay detailed below.
Approximately 30 independent transformants for each target plasmid were
patched onto SC medium lacking histidine and uracil (SC-His-Ura) with
2% glucose (selection for both plasmids) and incubated at 30°C for 2
d. Yeast patches were then replica-plated onto SC-His-Ura with 2%
galactose (induces Gal–Ty1 expression and transposition) and incubated
at 22°C for 4 d. Patches were replica-plated onto SC-His-Ura with 2%
glucose (represses Gal–Ty1 transposition) and incubated at 30°C for
2 d. The donor plasmid was shuffled out by growth on YPD medium at
30°C overnight followed by growth on SC-His containing 1 g/L of
5-fluoro-orotic acid (5-FOA) (Boeke et al. 1984) with 2% glucose at
30°C for 2 d. Finally, patches were replica-plated onto YPD medium
containing 0.5 mg/L of G418 (selects for Ty1-neo transposition
events) and incubated at 30°C for 1–2 d.
Recombinant target plasmids containing T1-neo insertions were
recovered into E. coli by isolating total DNA from yeast
patches using the glass bead-phenol method (Kaiser et al. 1994)
followed by electroporation of DH10 cells using a Bio-Rad (Hercules,
CA) electroporation apparatus. Individual Ty1 insertions were mapped by
sequencing and labeled a–h (U6mg) and t–x
(sup2+b).
The sup2+b(G56) box B mutations were constructed as
follows in the pDLC356 target plasmid derivatives containing individual
Ty1-neo transposition events. A 1.9-kb BstE
II–MluI fragment from pDLC565, containing the mutated
box B promoter element of the sup2+b tRNA
gene, (Chalker and Sandmeyer 1992, 1993) was ligated to both the 4.9-kb
SalI –BstE II fragment and the 7.1-kb
MluI–SalI fragment of Ty1-neo insertion t
to yield the t* mutant. After digestion with MluI and partial
digestion with BstE II, the 1.9-kb BstE II
–MluI fragments of insertions w and x, were replaced with
that of pDLC565 (Chalker and Sandmeyer 1992) to generate the w* and x*
mutants, respectively.
Generation of Strains With Various tY(GUA)F2 Alleles
In the parent strain, BY4741 (MATahis31 leu20 met150
ura30) (Brachmann et al. 1998), the tY(GUA)F2
gene was first replaced with the CaURA3MX4 cassette
(Goldstein and McCusker 1999) by homologous recombination generating
a tY(GUA)F2::CaURA3MX4 allele. In the resulting
strain, the CaURA3MX4 cassette was then replaced with the
sup2+b and the sup2+b(G56) alleles
of the tRNATyr by homologous recombination to generate the
tY(GUA)F2-sup2+b and the
tY(GUA)F2-sup2+b(G56) alleles, respectively. All gene
replacements in the resulting strains were verified by PCR and by
sequencing across all of the recombination junctions.
RNA Isolation and Blot Analysis
Total RNA was isolated from several independent yeast
transformants. Yeast cultures were grown at 30°C to an
OD600 of 0.7–1.0 in SC-His with 2% glucose. Thirty
milliliters of culture was used for total RNA isolation, and 30 ml was
used for total DNA isolation. Total RNA was extracted by hot acid
phenol (Collart and Oliviero 1993), denatured by formaldehyde and/or
formamide, and fractionated by denaturing gel electrophoresis as
described below.
For quantification of low molecular weight RNAs, U6mg,
U14, and sup2+b total RNA (10 µg) was
boiled in sample buffer (85% deionized formamide, 9 mM EDTA, and 0.1%
xylene cyanol) and iced before loading onto 10% polyacrylamide, 8 M
urea denaturing gels in 1X TTE (90 mM Tris base, 29 mM
Taurine and 0.5 mM EDTA) running buffer. The
denaturing gels were run at 23 V/cm for 4.5 h. RNA was then stained
with ethidium bromide to examine its integrity and to position the rRNA
molecular weight markers before being electrophoretically transferred
to Gene Screen Plus filters as described by the manufacturer (NEN Life
Science Products, Boston, MA). After transfer, the RNA was fixed by UV
crosslinking. Membranes were prehybridized at 42°C with hybridization
solution (5X SSPE, 5X Denhardt's solution, 5% dextran
sulfate-Na+ (500 kD), 0.1% SDS, and 50% deionized
formamide). U6mg-specific (5'-CCTTATGCAGGGGAACTG-3'),
U14-specific (5'-CCGAGAGTACTAACGATGGGTTCGTAAGCGTACTCC-3'), and
sup2+b-specific
(5'-GATTTCGTAGGTTACCTGATAAAT TACAG-3') oligonucleotides were 5'-end
labeled with T4 polynucleotide kinase and purified over G25 Sephadex
spin columns. Filter-bound RNAs were hybridized at 25°C in
hybridization solution for at least 12 h to 30 pmoles.
32P-labeled oligonucleotide probes mixed with 2.5 mg of
boiled sheared herring sperm DNA. After hybridization, filters were
washed twice for 20 min at 25°C with 1X SSPE, 1X SSPE + 1% SDS
and 0.1X SSPE + 0.1% SDS and exposed to a Molecular Dynamics
phosphoimager screen.
For quantification of higher molecular weight RNAs, ACT1,
PTR3, and Ty1-neo, 20 µg total RNA was boiled in
sample buffer (55% deionized formamide, 1X MOPS buffer, pH 7.0, 5%
formaldehyde, 8 mM EDTA) and 0.1% bromophenol blue) and iced before
loading onto 1% agarose, 1X MOPS buffer, pH 7.0 (40 mM MOPS, 10 mM
sodium acetate, and 1 mM EDTA) and 2% formaldehyde gels. The gels were
run in 1X MOPS buffer at 6 V/cm for 4 h. RNA was stained with
ethidium bromide before being transferred by capillary action to Gene
Screen Plus filters as described by the manufacturer (NEN Life Science
Products, Boston, MA). Following transfer, the RNA was fixed by UV
crosslinking. Membranes were then prehybridized as indicated previously
and then hybridized at 42°C in hybridization solution for at least 12
h to 32P-labeled DNA probes mixed with 2.5 mg boiled sheared
herring sperm DNA. ACT1-specific (1.2 kb
BamHI–HindIII fragment of p10-AHX3 [Chapman and
Boeke 1991]), PTR3-specific (1.7 kb intra-ORF PCR product),
and neo-specific (1.0 kb BamHI fragment (Joyce et al.
1993) of pGH54) DNA probes were internally labeled and purified over
G25 Sephadex spin columns. Filters were washed twice for 20 min at
25°C with 2X SSPE, twice at 60°C with 2X SSPE + 2% SDS, and
twice at 25°C with 0.2X SSPE and then exposed to a Molecular
Dynamics phosphoimager screen for at least 24 h. Quantitation of the
relative steady-state transcripts was done using ImageQuant v1.11
(Molecular Dynamics) and Microsoft Excel software.
Relative Plasmid Copy Number
The relative copy number of the various U6mg- and
sup2+b-containing plasmids was determined by DNA blot
analysis with bla- and ACT1-specific probes used to
detect plasmid and yeast genomic DNA, respectively. Total yeast DNA was
isolated as described (Boeke et al. 1985) from 30-mL aliquots of the
same cultures used for RNA isolation. The genomic DNA samples were
digested with BamHI and XbaI and fractionated by
agarose gel electrophoresis. The DNA fragments were transferred by
capillary action to Gene Screen Plus membranes as described by the
manufacturer (NEN Life Science Products, Boston, MA). Hybridization was
analyzed using a Molecular Dynamics phosphoimager. The ratio of
bla- to ACT1-specific hybridization of different
samples provided a measure of the relative plasmid copy number present
in transformed populations. These ratios were used to normalize the
relative measurements of U6mg, sup2+b, and
Ty1-neo transcripts for gene dosage.
Sequencing
The insertion sites of Ty1-neo recombinants were sequenced
by extending primers homologous to the subterminal regions of the
Ty1-neo outward into the adjacent sequences. A single
recombinant clone was selected from each patch to ensure that each
recombinant sequenced represented an independent transposition event.
Ty1-neo sequencing primers were JB939
(5'-CCTTAGAAGTAACCGAAGCAC-3') and JB940 (5'-GATCTATTACATTATGGGTG-3').
PCR products spanning the homologous recombination junctions of at the
various tY(GUA)F2-allele loci were sequenced using primer
JB3286 (5'-GTGATTGTTGTTCTAGTCGCTTGC-3').
tRNA Gene Loci Database
Data pertaining to all annotated tRNA loci were extracted from the
Saccharomyces Genome Database (SGD) at
http://genome-www.stanford.edu/Saccharomyces/ and the yeast
Transcriptome database at http://web.wi.mit.edu/young/expression/
(Holstege et al. 1998) and reassembled into a flat-file tRNA- and
Ty-centered database using Microsoft Access and Excel. The tRNA gene,
flanking gene, and Ty database is accessible at
http://www.bs.jhmi.edu/MBG/BoekeLab/Boeke_Lab_Homepage, Supplements to
Publications. The coordinates and transcriptional orientation of each
tRNA gene are given as well as the coordinates, orientations, and
available expression levels of the non-Ty ORFs that flank the tRNA
gene. Moreover, the coordinates and orientations of annotated Ty
sequences, LTRs and full-length Tys (Kim et al. 1998) are listed with
their associated tRNA gene. Distance measurements were made using the
annotated gene coordinates, which are actually the ORF coordinates.
Therefore, the actual distance between transcription start sites and
therefore, promoters, is modestly overestimated for divergently and
convergently transcribed genes.
Several (86) ORFs annotated as hypothetical ORFs were disallowed during
the construction of this database because they lack homologs in yeast,
worms, or humans according to BLAST searches conducted through the SGD
website or any evidence supporting their identity as genes. Forty-five
ORFs were disallowed because they were in fact discovered to consist
entirely of Ty sequence. Of the remaining 41 ORFs, 20 were disallowed
because they encoded polypeptides less than 200 codons long and lacked
serial analysis of gene expression (SAGE) tags (Velculescu et al. 1995)
and were absent from the Transcriptome data set (Holstege et al. 1998).
Seventeen were disallowed because they encoded polypeptides <200
codons long. Four were disallowed because they contained internal tRNA
genes, and one was disallowed because it lacked a SAGE tag and was
absent from the Transcriptome data set.
Calculations
The mean genome-wide inter-ORF distance excluding telomeric regions
(genome) was calculated to be 480 bp long as follows. Using the
annotated gene coordinates from SGD
(http://genome-www.stanford.edu/Saccharomyces/), we designated the
first and last named genes as the ends of each chromosome to minimize
the bias imposed by the long, gene-poor telomeric regions. This crude
method, however, by no means designates precise telomere boundaries.
The total base pairs in these "telomereless" segments for the 16
yeast chromosomes was then summed. The number of bp for all of the ORFs
in these telomereless segments was summed as well. Subtracting the
number of base pairs for the ORFs from the total number of base pairs
in the genome gives the number of base pairs for the inter-ORF sequence
in the telomere-free genome. The number of intergenic regions for the
telomere-free genome corresponds to the number of ORFs (ORFs disallowed
in our data set were excluded here as well) less 16 (one for each
chromosome because the chromosomes begin and end with ORFs). Therefore,
dividing the number of base pairs for the inter-ORF sequence by the
number of inter-ORF regions yields the mean length of the intergenic
regions in the telomere-free genome. Note that this estimate of
inter-ORF length is actually inflated by the fact that tRNA genes and
their flanking regions were not deleted before this analysis.
Probability (P) values were determined by two-tailed
t-test analysis using Microsoft Excel.
|
WEB SITE REFERENCES
|
|---|
http://genome-www.stanford.edu/Saccharomyces/;
Saccharomyces Genome Database (SGD).
http://web.wi.mit.edu/young/expression/; Young Lab Home Page
(Transcriptome database).
http://www.bs.jhmi.edu/MBG/BoekeLab/Boeke_Lab_Homepage; this manuscript
(tRNA gene, flanking gene and Ty database).
|
Acknowledgements
|
|---|
We thank the members of the Boeke laboratory for helpful
discussions and reagents. We also thank Suzanne Sandmeyer and John
McCusker for generous gifts of plasmids and Brendan Cormack for
comments on the manuscript. This work was supported by National
Institutes of Health grant GM36481 to J.D.B.
The
publication costs of this article were defrayed in part by payment of
page charges. This article must therefore be hereby marked
"advertisement" in accordance with 18 USC section 1734 solely to
indicate this fact.
|
Footnotes
|
|---|
1 Corresponding author. 
E-MAIL jboeke{at}jhmi.edu; FAX (410) 614-2987.
Article and publication are at
http://www.genome.org/cgi/doi/10.1101/gr.612203.
|
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Received July 10, 2002;
accepted in revised format December 5, 2002.
13:254-263 © by 2003 Cold Spring Harbor Laboratory Press ISSN 1088-9051/03 $5.00
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ABSTRACT
RESULTS
