Department of Biology, Massachusetts
Institute of Technology, Cambridge, Massachusetts 02139
Received 18 June 1998/Returned for modification 17 August
1998/Accepted 29 September 1998
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INTRODUCTION |
Telomeres in Drosophila
melanogaster are composed of multiple copies of two non-long
terminal repeat (LTR) retrotransposons, HeT-A and
TART, instead of the short DNA repeats generated by telomerase on the chromosome ends of most eukaryotes (13,
24). Successive transpositions of HeT-A and
TART yield arrays of repeats that are larger and more
irregular than the repeats produced by telomerase. Nevertheless, these
transpositions are, in some sense, equivalent to the
telomere-generating action of telomerase; both telomerase and the
transposition of HeT-A and TART extend chromosome ends by RNA-templated additions of specific sequences.
HeT-A and TART share two features that
distinguish them from other known retrotransposable elements. Both
transpose only to the ends of chromosomes (apparently to any chromosome
end in D. melanogaster), and each contains a large segment
of untranslated sequence (Fig. 1). We
have recently shown that, for HeT-A, each of these
distinguishing features is also conserved in related species
(10), even when phylogenetic separation is great enough for
the HeT-A sequences to have diverged by nearly 50%.
HeT-A and TART also have some significant
differences. TART encodes its own reverse transcriptase
(RT); HeT-A does not. However, HeT-A seems
efficient in utilizing RT from some other source because most of the
documented transpositions onto broken ends have been HeT-A
rather than TART (1, 2, 33). A second difference is seen in the large 3' untranslated regions (UTRs). HeT-A
elements have a distinctive pattern of A-rich segments in this region
(9). Although the sequence of the HeT-A 3' UTR
diverges even faster than that of the coding region, the specific
pattern of A-rich segments is conserved in other species, suggesting
that it has a function (10). The 3' UTRs of TART
elements are more heterogeneous; D. melanogaster has at
least two subfamilies of TART elements, A and B, whose 3'
UTRs do not cross-hybridize (33). TART 3' ends
also show much less evidence of sequence patterning than do
HeT-A UTRs. However, as discussed below, TART has
its own distinctive sequence features.
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FIG. 1.
Diagrams of the two telomeric retrotransposons. The bars
under each diagram indicate the sequences used as probes for the RNA
hybridization experiments reported here. Each sequence was transcribed
in vitro to yield both sense and antisense probes. TART
elements can be divided into several families on the basis of their 3'
UTR sequences. The two probes marked A and B identify different
subfamilies of TART and do not cross-hybridize under the
conditions used here. HeT-A elements are ~6 kb, and the
TART element shown is >10 kb. (A)n, site of the
poly(A) tail on RNA transposition intermediate.
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What is now known about the two telomeric retrotransposons raises
several questions about function and, ultimately, about evolution. Are
the features shared by HeT-A and TART sufficient for either element to fulfill its telomeric function(s)? Are the differences important, so that the two elements must cooperate to form
a telomere? Are the two elements derived from a single ancestor, or are
they products of convergent evolution? These questions are difficult to
answer directly. Every D. melanogaster stock that we have
examined has multiple copies of both HeT-A and
TART. We detect no pattern in the distribution of the two elements that would suggest either a functional difference or a simple
way of breeding a fly with no copies of one of the elements.
To learn more about the relationship between HeT-A and
TART, we have extended our studies of the transcription of
these elements. We found earlier that the predominant transcript of
HeT-A was, as expected for a non-LTR retrotransposon, a
sense-strand copy of the entire element (6). We detected no
antisense copies of this RNA. Surprisingly, we now find both sense and
antisense TART RNAs. Furthermore, the antisense transcripts
are more than 10-fold more abundant than sense-strand RNAs. Our
analyses indicate that transcriptionally active TART
elements are more variable in size and sequence than HeT-A
elements; however, the distribution of variants differs among fly
stocks, suggesting that these variants are functionally equivalent. The
major differences in the sets of transcripts found for HeT-A
and TART are not aberrations of the genetic background of a
particular stock. We have surveyed several stocks and a distantly
related Drosophila species and found the same general patterns.
Another unexpected finding of our studies is an RNA with similarity to
the TART RT coding sequence but not to any other part of
TART DNA. All the eukaryotic RTs which have been studied,
with the notable exception of telomerase, have been encoded by
retroelements and transcriptionally linked to the gag coding
region. Thus, this new RNA is a novel RT mRNA. HeT-A is an
unusual non-LTR retrotransposon in that it does not encode its own RT.
It is intriguing that the RT mRNA is found in cells that are expressing
HeT-A.
We have found that both subfamilies of TART for which
sequence is available have one pair of remarkably conserved repeats. These repeats resemble those found in a small subgroup of non-LTR retrotransposons that have been described as having unequal, or nonidentical, terminal repeats (30). HeT-A does
not have repeats of this type. This difference in sequence
organization, in conjunction with the differences in transcription
patterns, suggests that an ancestral gag sequence has become
associated with two different subclasses of non-LTR elements to form
HeT-A and TART.
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MATERIALS AND METHODS |
Drosophila stocks and cell lines.
Of the
D. melanogaster stocks used in this work, 2057 (an isogenic
y; cn bw sp stock used for the Drosophila Genome
Project P1 phage library [35]) was the generous gift
of E. Lozovskaya, and the stocks shown in Fig. 5 were the generous gift
of A. A. Hoffmann (20). The other D. melanogaster and Drosophila yakuba stocks have been in
our collection for many years. D. melanogaster cultured
cells, Schneider line 2, were grown in Dulbecco's modified Eagle's
medium with lactalbumin hydrolysate, 10% fetal bovine serum, and 10 mM
nonessential amino acids. Schneider line 3 cells were grown in
Schneider's medium (Gibco BRL, Gaithersburg, Md.) plus 10% fetal
bovine serum.
Sequences for RNA probes (Fig. 1).
The following
HeT-A probes were from element 23Zn-1 (GenBank accession no.
U06920): 5' UTR probe, nucleotides (nt) 982 to 1746; open reading frame
(ORF) probe, nt 1746 to 4421; and 3' UTR probe, nt 4851 to 6481. The
TART ORF probes were ORF1 (accession no. U14101), nt 1801 to
3336, and ORF2 (accession no. U02279), nt 434 to 2683. The 3' UTR
sequence for TART subfamily A was amplified from genomic DNA
of stock 2057, and that of subfamily B was amplified from P1 phage
13-14, using PCR primers derived from accession no. U02279 (A probe)
and U14101 (B probe). The amplified sequences were cloned in Bluescript
II SK (Stratagene, La Jolla, Calif.). For each probe used in this
study, sense and antisense strands were transcribed from DNA fragments
of identical length.
RNA extraction.
One hundred flies were homogenized in a
Dounce homogenizer or 2 × 108 to 3 × 108 cultured cells were resuspended in 2 ml of buffer (100 mM NaCl, 50 mM Tris-HCl [pH 7.0], 20 mM EDTA). The solution was
brought to 1% sodium dodecyl sulfate (SDS) and 50 µg of Proteinase
K/ml. The homogenate was incubated at 37°C for 20 min and then
extracted with phenol-chloroform and chloroform. Nucleic acids were
precipitated with 3 volumes of ethanol plus 0.2 M LiCl. The pellet was
resuspended in 200 µl of H2O and precipitated with an
equal volume of 4 M LiCl at 4°C overnight. Finally, the pellet was
resuspended in 200 µL of H2O and precipitated with 3 volumes of ethanol plus 0.3 M Na acetate.
Northern hybridization.
RNA samples (20 µg per lane) were
treated with glyoxal, separated on an 0.8% agarose gel, and
transferred to Hybond-N nylon membrane according to the method of
Sambrook et al. (27). 32P-labeled riboprobes
were transcribed in vitro from DNA fragments inserted into Bluescript
II SK with T7 or T3 RNA polymerases, according to the Promega (Madison,
Wis.) protocol. Hybridization was performed at 65°C in 4× SET (1×
SET is 0.15 M NaCl, 0.03 M Tris-Cl [pH 7.0], and 2 mM EDTA), 5×
Denhardt's solution, 0.5% SDS, and 50 µl of salmon sperm DNA/ml
(27). The filters were washed three times with 1× SSC (0.15 M NaCl plus 0.015 M sodium citrate) plus 0.5% SDS at 65°C and then
treated with 100 U of RNase T1 (Boehringer Mannheim,
Indianapolis, Ind.)/ml in buffer (10 mM Tris-Cl [pH 7.5], 300 mM
NaCl, 5 mM EDTA) for 1 h at 37°C, rinsed with 1×
SSC-0.5% SDS, and exposed for autoradiography.
Cell fractionation.
Schneider 2 cells (2 × 108) were washed once in phosphate-buffered saline,
resuspended in 8 ml of homogenization buffer (10 mM Tris HCl [pH 8],
1 mM EDTA, 10 mM NaCl, 10 mM MgCl2), and placed on ice for
10 min. The cells were disrupted in a Dounce homogenizer and
centrifuged for 10 min at 800 × g. The pellet was used
as the nuclear fraction. The supernatant was centrifuged again at 800 × g, and the supernatant from this spin was used
as the cytoplasmic fraction. RNA was isolated from both the nuclear
pellet and the second supernatant, as described above.
Nucleotide sequence accession numbers.
The sequences of the
clones discussed in this study have been deposited in the GenBank
database under accession no. AF072856 (A subfamily, 990-nt probe) and
AF072857 (B subfamily, 950-nt probe).
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RESULTS |
Technical details of hybridization.
It is important to note
that both HeT-A and TART are present in the
genome in multiple copies with various degrees of divergence. In
addition, parts of the noncoding regions of these elements are closely
related to sequences in other types of repeats (5). Thus,
there are many potential sources for sequences hybridizing with the
probes used here, and it is important to eliminate hybridization to
similar, but not identical, sequences. For this reason, all hybrids in
these experiments were treated with RNase T1 before autoradiographic exposure. This treatment, adapted from RNase protection experiments, assures that the transcripts detected have
strong sequence similarity to the probe, although not necessarily over
their complete lengths.
HeT-A elements produce only sense-strand
transcripts.
When RNA from either flies or cultured cell lines is
probed with sequence from any part of HeT-A, the major
species of RNA detected is a sense-strand transcript of ~6 kb (Fig.
2). This is the size expected for a
full-length transcript of the element. HeT-A elements which
have been sequenced differ by multiple insertions and/or deletions
(9, 25), but the net result is that the elements are
approximately the same size and should be expected to migrate as the
slightly broad band seen in our gels (Fig. 2; see also Fig. 7). Two
minor bands of apparently larger RNAs detected by these probes are
thought to be readthrough transcripts of tandem elements.
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FIG. 2.
HeT-A elements produce only sense-strand
transcripts. Autoradiograph of a Northern blot of total RNA from adult
D. melanogaster females (lanes F) and males (lanes M) probed
with HeT-A sequences. Single-strand probes detecting
sense-strand RNA were used for lanes marked "sense," while
"antisense" lanes were probed with the opposite strand. See Fig. 1
for the extent of sequence in each probe. Each probe detects a
prominent sense-strand RNA of ~6 kb (arrow). Less abundant RNAs
(asterisks) of approximately twice this size and greater may represent
readthrough transcripts of tandem elements. (The label at ~2 kb is
due to RNA trapped by rRNA.) Each of the probes for sense-strand
transcripts also detects one or more small (<2-kb) RNAs. Some of these
RNAs appear to be sex specific. No antisense transcripts are detected
with any of the probes. The lanes probed for antisense ORF sequences
are shown. Similar results were obtained when blots were probed for 5'
and 3' UTR antisense sequences. The marker sizes are shown in
kilobases.
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In addition to the large sense-strand RNAs, each of these probes detect
one or more much smaller (<2-kb) RNAs. These smaller RNAs are
different for each probe and also show sex-specific differences in
expression. We do not yet know the source of these RNAs. They may be
transcribed from full-sized HeT-A elements or from truncated elements.
Although the sequence of HeT-A is transcribed into several
sense-strand RNAs, no part of the sequence has been found in antisense RNA. The complete absence of hybridization seen when a Northern blot is
probed for antisense RNA containing the ORF sequence (Fig. 2) is
typical of the result obtained when probes from any part of the
HeT-A sequence are used.
TART produces both sense and antisense transcripts of a
heterogeneous array of full-length elements.
In striking contrast
to HeT-A, TART produces transcripts of both
strands in both flies and cultured cells. We have concentrated our
study on the RNAs, both sense and antisense, that run as several distinct bands with estimated sizes between 7.5 and 12 kb (Fig. 3). The numbers, sizes, and relative
intensities of these bands differ from stock to stock (compare Fig. 3
through 8). Our studies suggest that all of the RNAs in the 7.5- to
12-kb range represent full-length elements. As discussed below,
TART appears to be a more heterogeneous family than
HeT-A. TART elements whose 3' UTRs differ by nearly 2 kb in
length have been sequenced. The 5' end of TART has not yet
been defined. The longest published TART sequence is 10,636 nt (33), and it does not have a complete 5' end. We do not
yet have probes for the 5' UTR, but all of the probes that we do have
(ORF1, ORF2, and 3' UTR [Fig. 3 and 4])
hybridize to the same bands in this region of the gel; thus, each
species of RNA seems to have the entire sequence, even though some are
shorter than 10 kb.
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FIG. 3.
TART produces multiple full-length sequences,
both sense and antisense. An autoradiograph is shown of a Northern blot
of total RNA from adult Oregon R flies probed for transcripts carrying
sequence of TART ORF1 (lanes 1) and ORF2 (lanes 2). Both
ORFs are found in the same set of transcripts. Both sense and antisense
probes detect sets of transcripts migrating between 7.5 and 12 kb;
however, the sense and antisense RNAs do not exactly comigrate. The
transcripts in the 7.5- to 12-kb region in Oregon R flies differ (in
number and size) from those in other fly stocks and in the two cultured
cell lines studied (compare Fig. 3 through 8). Note that the
autoradiographic exposures shown are chosen to best display the bands
produced by each probe and therefore vary from probe to probe. The
detection of sense-strand RNAs requires significantly more exposure
than detection of antisense transcripts. The label at ~2 kb is due to
RNA trapped by rRNA.
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FIG. 4.
TART A- and B-subfamily elements yield
full-length transcripts of several sizes. An autoradiograph of a
Northern blot of total RNA from adult 2057 flies probed with
TART sequences is shown. In this stock ORF probes detect two
bands of sense-strand RNA migrating above the 9.5-kb marker. (Both ORF1
and ORF2 probes give this result; ORF2 is shown.) The A-subfamily 3'
UTR probe identifies the top band as TART A RNA, while the
B-subfamily 3' UTR probe labels a band corunning with the lower band
but seen only in RNA from males (indicated by the arrow in the B 3' UTR
lane). Because females in this stock lack full-length B elements, we
conclude that the band just above 9.5 kb hybridizing with ORF probes in
females represents a third subfamily of TART found both in
males and females. This third-subfamily RNA comigrates with the B
transcript in males. Both 3' UTR probes detect very abundant short
sense RNAs (<2 kb). Probes for antisense ORF sequences detect two
transcripts whose gel mobilities differ slightly from that of the large
sense RNAs. The larger antisense transcript may comigrate with the
smaller sense RNA. Both of the large antisense RNAs hybridize with
A-subfamily 3' UTR probes. The B-subfamily 3' UTR detects a smaller and
less abundant antisense RNA seen only in males with either ORF or B 3'
UTR probes (indicated by the arrow in the B 3' UTR lane). The label at
~2 kb is due to RNA trapped by rRNA.
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Although both sense and antisense transcripts appear to contain the
complete TART sequence, they do not exactly comigrate in the
gel. This suggests that they are transcribed from different copies of
TART. The failure to comigrate may be partly explained by
their biased base compositions. Sense-strand transcripts are twice as A
rich as the antisense strands, and this difference in base composition
might affect gel mobility. However, if this were the entire explanation
we would expect that in each stock the two sets of RNAs would have the
same numbers and relative intensities of hybridizing bands, with one
set somewhat offset from the other in the gel. This is not what is observed.
Like HeT-A, TART probes also hybridize to a
heterogeneous set of small (<2-kb) RNAs. TART probes detect
unusually large amounts of small sense-strand transcripts complementary
to the A and B 3' UTR sequences but not to other parts of the element
(Fig. 4). As with the small HeT-A RNAs, it is not clear
whether these small RNAs are transcribed from full-length or partial elements.
TART elements form a heterogeneous family and yield a
complex set of transcripts.
Both TART (33)
and HeT-A (9) can be divided into subfamilies on
the basis of their 3' UTR sequences. The intrafamily divergence of
TART subfamilies is greater than that of HeT-A
subfamilies. The 3' UTRs of different TART subfamilies are
unable to cross-hybridize under the conditions used in this work, while
different HeT-A subfamily 3' UTRs do cross-hybridize.
TART 3' UTRs also have greater length differences than
HeT-A elements. The small number of nearly full-length
elements which have been sequenced show TART 3' UTRs of 5.1 and 3.3 kb, while HeT-A 3' UTRs are 2.6 to 2.3 kb. These limited data are supported by the sizes of the apparent full-length transcripts of these elements. The TART sense transcripts
migrate on gels as though they were 7.5 to 12 kb, while
HeT-A transcripts run in a single broad band of ~6.0 kb.
The transcription of TART subfamilies is complex and differs
among fly stocks. The 3'-most sequences of A and B elements do not
cross-hybridize and can be used as probes to identify transcripts of
the subfamilies. Each probe hybridizes to a subset of the presumed full-length transcripts. Other transcripts hybridize with neither probe
and presumably indicate the presence of additional subfamilies. A
typical example of these studies is shown in Fig. 4, taken from our
analysis of stock 2057. In this stock, the A probe hybridizes with the
larger sense transcript, consistent with the longer 3' UTR in the
published TART A sequence. Also consistent with the size of
the published TART B 3' UTR, the B probe hybridizes with a
smaller sense-strand transcript found only in males. The limitation to
males is understandable because full-sized B elements are found only in
DNA from males (data not shown) and must be on the Y chromosome. We
assume that 2057 has transcripts of a third TART subfamily because TART ORF probes detect a sense RNA in females which
is the same size as the B subfamily in males (running with the 9.5-kb marker). This RNA does not hybridize with either A or B probes in
females and must comigrate with the B-subfamily transcripts in males.
The relation between TART subfamily and transcript size is
not always simple. Both of the major antisense transcripts in 2057 hybridize with the A probe. The B probe binds a smaller antisense
transcript, again seen only in males. In Oregon R, sense and antisense
B probes hybridize to RNAs of at least two different sizes (data not shown).
The sharply defined bands in the 7.5 to 12 kb region suggest that
TART elements have a limited number of variant forms. To see
whether this limitation was due to the small breeding populations in
the laboratory, we examined TART elements in a series of
lines (Fig. 5) derived from a wild-caught
population, collected in Australia in 1994 and therefore geographically
distant from any other stocks we have studied (20). We
examined these lines after 3 years of segregation (see the legend to
Fig. 5). The TART bands in these new lines show transcripts
of many different sizes. The sources of these transcripts appear to be
in the process of being segregated in the different lines. We conclude
that there are many variants of TART in wild populations. In
the same populations, HeT-A transcripts migrate in a single
broad band, as do those of laboratory stocks.
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FIG. 5.
Elements yielding different-size TART
transcripts can be segregated by breeding. An autoradiograph of a
Northern blot of total RNA probed for TART antisense ORF2
RNA is shown. Lanes 1 to 6 contain RNA from lines selected from a
mass-bred population of D. melanogaster initiated from
females collected in Australia in February 1994 (20). Lines
1 to 3 were then selected on the basis of heat shock resistance, and
lines 4 to 6 were parallel control lines. (Our samples, a gift of A. Hoffmann, were taken after 3 years of selection.) For our purposes the
six lines illustrate the variation in TART expression
patterns that can be selected from a large population of flies. Each
line shows a somewhat different pattern of TART RNA. Probes
for ORF1 show a similar picture. The different patterns seen clearly in
the antisense strands are reflected in the sense strands (not shown),
although the two strands do not give identical patterns.
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In spite of their differences, the TART subfamilies may be
functionally interchangeable. In stock 2057 we detect full-length B-subfamily elements only on the Y chromosome, and these elements make
only a minor contribution to the transcripts in the male. In contrast,
in the Oregon R stock, B-subfamily elements are abundant in both males
and females and contribute strongly to the pool of transcripts in both
sexes. No transcripts of A elements are detected in Oregon R flies
(data not shown).
TART antisense transcripts are more than 10 times as
abundant as sense transcripts.
Blots probed for sense-strand RNAs
require significantly longer autoradiographic exposures than do blots
probed for antisense RNA. To obtain a more quantitative estimate of the
relative amounts of the two sets of transcripts, we have used equal
amounts of each probe and varied autoradiographic exposures until they
yielded bands of nearly equal densities for sense and antisense
transcripts. These comparisons show clearly that there is a marked
excess of antisense RNAs. Figure 6 shows
a typical example of these comparisons. The exposure for the lanes of
sense-strand RNAs is six times that for lanes of antisense RNAs. One
more factor must be taken into account in estimating the relative
amounts of transcripts of the two strands. The sense strands are twice
as A rich as the antisense strands. The probes used for these
experiments are labeled with [32P]UTP by in vitro
transcription. Therefore, the specific activity of the probe depends on
the specific activity of the nucleotide mix and the sequence
transcribed. We have used the same nucleotide mix for both sense and
antisense probes. The fragment transcribed to give the probe for the
sense strand has approximately twice (1.94 times) as many A residues as
does its complement, and therefore it will be labeled 1.94 times as
heavily as probes for the other strand. Taken together, the probe
strength and the exposure time used for Fig. 6 lead to the conclusion
that antisense strands are approximately 12 times as abundant as sense
strands.
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FIG. 6.
TART elements produce many more antisense-
than sense-strand transcripts. An autoradiograph of a Northern blot of
total RNA (replicate preparations) from the D. melanogaster
Schneider 2 cell line probed with sequence from TART ORF2 is
shown. Autoradiographic exposures were chosen so that probes for sense-
and antisense-strand RNA produced bands of approximately equal
intensity. The exposure used for sense-strand transcripts (18 h) is
approximately six times that for antisense RNA (3 h). Because of the
strong strand bias in A residues, the probe used to detect sense-strand
RNA has incorporated nearly twice as much 32P as the
complementary probe, indicating that antisense transcripts are more
than 10-fold more abundant than sense RNA. ORF1 and 3' UTR probes from
TART produce similar blots, with the exception that only the
ORF2 probe (containing RT sequences) detects a sense-strand transcript
of 5.5 kb, which we call RTx (arrow).
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TART probes detect an unusual RT mRNA.
The RT of
retroviruses and retrotransposons is typically encoded by their
full-length sense-strand transcripts. The enzyme is translated as part
of a polyprotein linked to the product of the gag gene
either by a frameshift or by readthrough of a leaky stop codon
(14). Full-length TART transcripts partially fit the general pattern. They contain both ORF1, the gag gene,
and ORF2, which has the RT coding sequence; however, this ORF2 appears to require an internal initiation for translation, unlike many retrotransposons (33). It was surprising, therefore, to find a sense-strand RNA that hybridized with TART ORF2 probes
(Fig. 6) but not with TART sequence either 5' or 3' of the
RT sequence (data not shown). Thus, this RNA appears to be an mRNA for
RT, and we refer to it as RTx RNA. Because it hybridizes with
TART at very high stringency, it might be a processed
product. However, if so, it must have undergone removal of both 5' and
3' sequences, something that has not been reported for other
retrotransposons. RTx RNA might also be the product of a
"free-standing" RT gene, perhaps related to the RT of
TART in much the same way that viral oncogenes are related
to their cellular counterparts. No antisense copy of RTx RNA has been detected.
RTx RNA is present in both lines of cultured Drosophila
cells which we maintain (Schneider 2 and Schneider 3). We have not yet
detected RTx RNA in intact flies; however, all of our studies thus far
have used RNA from whole flies. If RTx RNA is restricted to a subset of
tissues or developmental periods, it might well have been missed in
analyses of bulk RNA. The presence of RTx RNA in the two immortal cell
lines studied is intriguing. The two lines had separate origins
(29), and they also have different patterns of full-size
TART RNA (data not shown). In spite of these differences
both lines now express RTx RNA.
The intracellular distribution of TART transcripts
differs from that of HeT-A transcripts.
Full-length
sense-strand transcripts of typical retrotransposons serve as mRNA for
the products of the gag and pol genes. The same
transcripts can also serve as templates for the reverse transcription
required for transposition to a new chromosomal site. For non-LTR
retrotransposons, such as HeT-A and TART, this reverse transcription occurs in the nucleus, primed by chromosomal DNA
at the site of integration (19). Therefore, the dual roles of full-length transcripts suggest that these transcripts will be found
in both the nucleus and the cytoplasm. There is no reason to expect the
intracellular distribution of HeT-A transcripts to be
different from that of TART transcripts, yet the
distributions of these transcripts differ markedly (Fig.
7). When we assay RNA from cell
fractions, the sets of presumed full-length TART
transcripts, both sense and antisense, are found almost entirely in the
nuclear fraction. Therefore, these TART RNAs must be either
inside the nucleus or tightly associated with it. In contrast, a
significant fraction of full-length HeT-A RNA (which is
sense strand only) is present in the cytoplasmic fraction. RTx RNA is
also detected in the cytoplasmic fraction, consistent with the
supposition that it is an mRNA.
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FIG. 7.
The nucleocytoplasmic distribution of TART
transcripts differs from that of HeT-A transcripts. An
autoradiograph of a Northern blot of RNA from cultured cells after
nucleocytoplasmic fractionation is shown. N, nuclear fractions; C,
cytoplasmic fractions. TART sequences were detected with
ORF2 probes. HeT-A sequences were detected with a 3' UTR
probe. Full-length HeT-A RNA (right arrow) and
TART RTx RNA (left arrow) are found in nuclear and
cytoplasmic fractions, while the large TART transcripts
(both sense and antisense) are almost entirely limited to the nuclear
fraction. The 6-kb HeT-A RNA is seen with all
HeT-A probes. The ~4-kb HeT-A RNA is detected
only with 3' UTR probes and only in cultured cells; its origin is not
known. The label at ~2 kb is due to RNA trapped by rRNA.
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Transcription patterns of the telomeric transposons are conserved
across species.
We have begun our studies of telomeric elements in
other species with those of D. yakuba, thought to be
separated from D. melanogaster by 5 to 15 million years
(15). Previously, we cloned HeT-A elements from
D. yakuba (HeT-Ayak) and found that,
although the overall sequence identity between HeT-Ayak and HeT-Amel is
only 55%, the D. yakuba HeT-A elements show all the unusual features of HeT-Amel: they transpose only to
telomeres, where they form long arrays; they have the unusual, long 3'
UTR; and they lack RT coding sequences (9). Our studies now
show that, like HeT-Amel,
HeT-Ayak produces only sense-strand RNA and its
full-length transcripts are relatively homogeneous in size, migrating
as a single rather broad band in our gels (data not shown).
TART elements have not yet been cloned from D. yakuba; however preliminary evidence suggests that D. yakuba has its own TART elements. Southern blots of
D. yakuba DNA probed with TART coding sequences
from D. melanogaster show multiple faint bands of
hybridizing restriction fragments (data not shown). This result would
be expected if D. yakuba has a family of TART
elements whose sequence has diverged from the D. melanogaster
TART sequences, much as the HeT-A elements have
diverged. Because D. melanogaster TART probes detect
TART sequences in D. yakuba DNA, we have used
these D. melanogaster probes to study D. yakuba
TART transcripts. These probes detect multiple transcripts
migrating approximately with full-length TART transcripts
from D. melanogaster (Fig. 8).
ORF1 and ORF2 probes hybridize to all of the bands in this set, as expected from our studies of D. melanogaster. Significantly,
TART antisense transcripts are much more abundant than sense
transcripts in D. yakuba, as they are in D. melanogaster. Northern blots of D. yakuba RNA require
much longer autoradiographic exposures than blots of D. melanogaster RNA, presumably reflecting the lower level of
hybridization with the D. melanogaster probe. Although we
can obtain reasonable pictures of the blots showing antisense transcripts, the lower levels of sense-strand RNA yield pictures that
are very difficult to reproduce and therefore are not shown here.
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|
FIG. 8.
The abundant antisense transcription of TART
is conserved in related Drosophila species. An
autoradiograph of a Northern blot comparing TART transcripts
in D. melanogaster (m) with those in D. yakuba (y) is shown. Both samples have been probed with
sequences from D. melanogaster TART ORF1 and ORF2. The
D. melanogaster lanes were exposed for 20 h, and the
D. yakuba lanes were exposed for 44 h. Only lanes with
antisense transcripts are shown. In D. yakuba, as in
D. melanogaster, TART antisense transcripts are
much more abundant than TART sense RNAs.
|
|
Comparisons of the HeT-A and TART gag
coding sequences give evidence of a common ancestor.
The coding
region of HeT-A appears to be closely related to ORF1 of
TART. Both sequences are clearly related to the ORF1
sequences of several insect non-LTR elements, but HeT-A and
TART are most closely related to each other (25).
Dot matrix analyses show that the 3' half of the D. melanogaster
TARTmel ORF1 has a high level of nucleotide identity
with the equivalent part of the coding regions of HeT-A
elements from both D. melanogaster and D. yakuba,
even though the coding regions of the two HeT-A elements are
only ~65% identical to each other at the nucleotide level (Fig.
9).
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|
FIG. 9.
TART ORF1 has significant sequence similarity
with the HeT-A coding region. (A and B) Dot matrix
comparisons of the nucleotide sequence of TART ORF1 from
D. melanogaster with sequences of the entire coding regions
of HeT-A elements from D. melanogaster (A) and
D. yakuba (B). (C) The two HeT-A elements have
65% sequence identity distributed evenly over the coding region.
HeT-Amel has 38% identity with TART,
and HeT-Ayak has 41% identity with
TART. For both HeT-A elements, the regions of
identity with TART are strongest in the C-terminal end of
the coding region (boxed and marked by stars).
|
|
When comparisons are based on amino acid sequences,
TARTmel ORF1 has similarities throughout the
HeT-A coding region (25) and is as similar to the
D. yakuba HeT-A as to the D. melanogaster HeT-A.
HeT-A elements of D. melanogaster and D. yakuba show 64% amino acid sequence similarity (identities plus
conservative replacements). A 707-amino-acid stretch of the
TARTmel ORF1 product shows 50% amino acid
sequence similarity with HeT-Amel and 52%
similarity with HeT-Ayak. Furthermore,
two-thirds of the similar amino acids in each comparison between
TART and HeT-A are positions that are also
conserved when the two HeT-A products are compared. (We note
that HeT-A elements within D. melanogaster can
vary both in sequence and in length [3, 25].
Therefore, these percentages vary with the particular element used in
each comparison. The numbers given are representative.) Thus, the
sequences that are conserved in the HeT-A ORF show a strong
tendency to be conserved in TART ORF1, a situation that suggests that they are evolving from a common ancestor.
HeT-A and TART belong to different
subfamilies of non-LTR retrotransposons.
Our sequence analyses
reveal that TART displays several features which it does not
share with Het-A; instead, these features characterize a
small subgroup of non-LTR transposons, elements with unusual terminal
repeats (see Fig. 10). A striking feature of non-LTR retrotransposons
is their tendency to be truncated at the 5' end. This tendency led to
the suggestion that these elements are reverse transcribed into their
site of transposition, with variable failure of reverse transcription
to complete the copy of the 5' end (31). This basic
mechanism of transposition has been elegantly confirmed for the R2Bm
element (19). HeT-A and TART are also
frequently truncated at the 5' end, although the extent to which this
is due to failure of reverse transcription as opposed to erosion of the
chromosome end is unclear. The complete 5' end of HeT-A has
been defined by the analysis of several elements that had identical
junctions with an upstream element, a 5' UTR, and a complete coding
region (7).
No junctions between apparently complete TART elements and
other sequences have been found. The longest available TART
sequence is that of a B element extending 962 bp 5' of the start of
ORF1. Sheen and Levis (33) noted that this element had a
perfectly identical repeat of 1,046 bp (Fig.
10). The 5' copy of this repeat extended from the start of the sequence to bp 84 of ORF1. The 3' copy
of this repeat was in the 3' UTR and ended 560 bp from the 3' end of
the element. We have recently cloned a junction between two
TART A elements. In this junction, the poly(A) tail of the
distal element is joined to the proximal element 33 bp before the start
of ORF1 (26a). This sequence showed that the A subfamily
also has two perfect repeats, which differ in sequence from the B
repeats but have similar positions in the element. The 5' repeat of the
A element extends from the junction 117 bp into ORF1, and the 3' repeat
ends 374 bp from the poly(A) end of the element. Because of the 5'
truncation of the cloned element, the full size of the repeat is
unknown. In view of the absolute sequence conservation between repeats
within each element, the divergence between elements is striking, the
more so because most of the sequence that we have to compare is within
ORF1. The 84 bp of B repeat that lie in ORF1 have only 71% identity
with the first 84 bp of the A repeat lying in ORF1. The amino acid
sequence encoded by these nucleotides has only 70% identity.
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|
FIG. 10.
Diagrams of TART A and TART B
showing locations of perfect repeat sequences (indicated by the black
bars under the elements). Both elements are truncated at the 5' end.
The TART B diagram is based on data from reference
33;0. (A)n, site of the poly(A) tail on
RNA transposition intermediate.
|
|
The complete sequence identity of the repeats within TART
elements contrasts sharply with the divergence between TART
elements. Similar conserved repeats have been reported for other
non-LTR retrotransposons, most notably DER from
Dictyostelium discoideum (30) and TOC1
from Chlamydomonas reinhardtii (11). In these elements a 5' repeat is completely identical with a 3' repeat that,
like the TART repeats, is some distance from the 3' end of
the element. These repeats have been called unequal terminal repeats
because the terminal 3' sequences have no match at the 5' end; the
region of identity is in the subterminal sequence. The identity of the
repeats suggests that they are replicated from the same template, as
are retroviral LTRs (12). Because of this resemblance, Day
et al. (11) have proposed that these elements use a
variation of the retroviral integration mechanism.
| |
DISCUSSION |
The Drosophila telomeric transposons present intriguing
evolutionary questions.
Drosophila telomeres are a striking
exception to the almost-universal eukaryotic telomere, which is
maintained by the enzyme telomerase. Drosophila must have
shared ancestors with organisms that now have telomerase. Therefore, it
is interesting to speculate on the evolution of the
Drosophila retrotransposon-type telomere. We have suggested
that telomere retrotransposons have evolved from components of
telomerase (24, 26). This hypothesis has recently received
support from evidence that the catalytic subunit of telomerase
resembles the RTs of non-LTR retrotransposons (18, 21, 22).
This finding is particularly intriguing, since both HeT-A
and TART are non-LTR retrotransposons. Nevertheless, there is no compelling evidence to eliminate other possible explanations for
the Drosophila telomere, such as the "domestication" of
existing, parasitic retrotransposons to replace telomerase in
Drosophila or the evolution of telomerase from
retrotransposons, with Drosophila demonstrating the
ancestral mechanism.
Regardless of the explanation for the evolutionary origin of
Drosophila telomeres, the question of the relationship
between HeT-A and TART is important. Are these
two elements simply variants, fulfilling a function that is so simple
that it places few constraints on the form of the element? For example,
if the only role of telomere sequences is to protect genes by providing
extra DNA on the end of the chromosome, any sequence that can be added
to the chromosome should be adequate. If so, the differences between
HeT-A and TART would be entirely irrelevant,
reflecting only chance differences. On the other hand, these
differences might be evidence that these two elements have evolved to
fulfill different roles in the cell. For example, both sequences might
be required to form an effective telomere. One obvious possibility is
that TART provides the RT for HeT-A, which does
not encode the enzyme. This raises the question of why HeT-A
exists at all, if TART owns the RT. The question is even
more puzzling because HeT-A is significantly more abundant than TART in the genome. If TART supplies the RT,
it would seem that HeT-A must also have something to
contribute. An alternative explanation for the two elements is that one
or both of them, or an encoded protein, might have a second task in the
cell, a task not necessarily related to forming the telomere. This
possibility is raised by studies of Saccharomyces
cerevisiae, where multiple copies of genes adaptive for certain
environments are associated with chromosome ends, suggesting that the
association with telomeres facilitates their amplification
(23). The functional importance of the yeast genes is easy
to see because we understand the gene products. The roles of the
transposon products are more cryptic, but they too might be exploiting
the ease with which gene amplification can occur at telomeres.
HeT-A and TART belong to different
subgroups of non-LTR retrotransposons.
The studies reported here
offer a new insight into the relationship between HeT-A and
TART. TART displays several features which it does not share
with HeT-A but which are found in a different subgroup of
non-LTR retrotransposons. This evidence that the two elements belong to
different subgroups suggests that the evolutions of HeT-A
and TART converged by acquiring related gag
proteins, which may be responsible for their telomere localization.
The features that TART shares with the subgroup of non-LTR
retrotransposons are not limited to the 5' and subterminal 3' perfect repeats. In addition, the 5' repeat of TART elements extends
some distance into ORF1, another unusual characteristic of this group (30). Our preliminary studies indicate that the placement of TART promoters differs from that of HeT-A
promoters and instead may resemble that of the promoters described for
the imperfect repeat element, DER (30). In
earlier work we showed that the promoter of HeT-A is in an
unusual position (8): it is at the 3' end of the element and
serves to promote transcription of its downstream neighbor. We have
begun a similar study on TART promoter activity
(13a) and have found that the region of TART
equivalent to the HeT-A promoter, the 3'-most 900 bp, of
both TART A and TART B has little, if any,
sense-strand promoter activity. However, this region of both A and B
does have good promoter activity for antisense RNA and therefore
resembles the antisense promoter of DER. DER has been shown
to have a promoter for sense-strand transcription in the 5' UTR, just
upstream of the 5' repeat region (30). If the similarity
between TART and DER is complete, the
TART sense-strand promoter should be at the 5' end. We are
attempting to identify this 5' end to test this possibility.
Antisense transcripts have been reported for very few non-LTR
retrotransposons.
From their structure, it would appear that a
sense transcript of non-LTR retrotransposons should contain all of the
information necessary both for translation of element-specific proteins
and for templating of the DNA copy when the element transposes.
HeT-A conforms to this expectation. We have studied
transcripts in both males and females of several stocks of D. melanogaster, in two immortal cell lines, and in one stock of
D. yakuba. In every case we find only sense-strand
transcripts of HeT-A. In contrast, parallel studies of
TART show, in every case, a large excess of antisense transcripts. Since these studies detect steady-state RNA, the excess
could be due to more active promoters for the antisense RNA, to a
slower turnover of the RNA, or both.
It is not obvious why TART produces antisense RNA when
HeT-A does not. One possible use for antisense RNA could be
to allow an element to encode additional proteins; however, for several reasons, it seems unlikely that TART antisense RNA provides
extra coding capacity. First, the antisense strand has only very small ORFs, no larger than those on the antisense strand of HeT-A,
which is not transcribed. Second, most, if not all, TART RNA
remained with the nucleus in our cell fractionation experiments. Thus, it is not likely to be serving as a template for protein synthesis; however, we cannot yet rule out the possibility that TART
RNA is on polysomes that preferentially fractionate with the nucleus.
Several reports of antisense RNAs for retrotransposons have suggested
that these RNAs were accidental products of readthrough transcription.
Mammalian LINE-1 elements transpose to many chromosomal sites and thus
can come under the control of irrelevant promoters. These irrelevant
promoters probably produce many of the heterogeneous transcripts
detected by LINE-1 hybridization, while transcripts capable of
transposition are produced by a small subset of the elements (28,
34). Although irrelevant promoters could produce the
TART antisense transcripts, this explanation seems very
unlikely. TART elements are intermingled with
HeT-A elements in long head-to-tail arrays on telomeres. The
only promoters for which we have any evidence are the TART
antisense promoters mentioned earlier, which would yield bona fide
TART RNA, and HeT-A promoters, which in principle
could drive TART transcription but should yield only sense transcripts.
Other retrotransposons which produce defined sets of antisense
transcripts (16, 30) suggest additional roles for
TART antisense RNA. The Drosophila
retrotransposon, micropia, produces an antisense copy of its
RT and RNase H coding sequence in the testis but not in other tissues.
It has been suggested that the testis-specific transcript blocks
transposition of micropia in the male germ line
(16). This seems an unlikely model for TART antisense RNA. If antisense RNA acts as an inhibitor, it should block
expression from TART in all tissues, because antisense RNA appears to be present wherever TART sense RNA is found.
Antisense depression of transcription might explain why TART
is much less abundant than HeT-A in the genome.
Another example of a role for antisense RNA is offered by the
Dictyostelium element, DRE, mentioned earlier.
The DRE sense strand is not a complete copy of the element;
it lacks the 3'-most sequence. This 3'-most information is found on an
antisense RNA, which is also an incomplete copy of the element but
lacks sequence from the 5' end of the element. It has been proposed
that, by pairing of the overlapping regions, the two strands form a
structure in which both strands can be extended by RT to produce a
mosaic RNA-DNA hybrid with the complete DRE sequence. This
hybrid could then insert into the new chromosomal site (30).
A similar role in transposition is an intriguing possibility for the
TART antisense RNA. However, the invariant orientation of
TART elements on chromosome ends is more easily explained by
a transposition mechanism thought to be more typical of non-LTR
retrotransposons. The mechanism has been demonstrated for the R2Bm
retrotransposon: RNA is reverse transcribed at the site of integration,
primed on the 3' OH of the chromosomal DNA (19). The polar
orientation seen for TART is consistent with its reverse
transcription being primed off the chromosome end. Thus, any model for
integration of a double-stranded TART requires a mechanism
for maintaining orientation on the chromosome. Clearly, many questions
about the abundant antisense TART transcripts remain.
A novel RNA encoding RT.
Our Northern blots show a
sense-strand RNA that hybridizes at very high stringency with the probe
for TART ORF2 (the RT coding sequence) but does not
hybridize with TART sequences either 5' or 3' of the RT
gene. Because this RNA appears to encode RT, we have named it RTx RNA.
Our data are consistent with three possible explanations for the origin
of RTx RNA: (i) it could be transcribed from TART with
posttranscriptional processing or altered transcriptional initiation
and termination; (ii) it could be the transcript of a new
retrotransposon; or (iii) it could be the mRNA product of a cellular
gene, as opposed to a retroelement gene. (We refer to this as the
"free-standing gene" alternative.)
Each of the possible origins for RTx is interesting. Alternative
transcripts of non-LTR retrotransposons are rare. The
Neurospora non-LTR retrotransposon, Tad, produces
an alternative transcript that would encode RT activity
(32), although it does not require the 3' processing needed
to derive RTx from TART. If RTx represents a new
retrotransposon, that retrotransposon must be a telomere-specific element, like HeT-A and TART, because the
TART ORF2 probe hybridized in situ only to telomeres
(reference 17 and our unpublished observations).
None of the analyses of cloned telomere DNA in our laboratory or others
have identified an additional telomere transposon.
The third alternative, mRNA from a free-standing gene, is consistent
with our preferred hypothesis for the evolutionary origin of
HeT-A (see above). This hypothesis suggests that the
Drosophila gene for the catalytic subunit of telomerase is
still present in the genome and that it produces the RT for
HeT-A (24, 26). Thus, Drosophila may
have a cellular gene capable of providing the RT for the non-LTR
element, HeT-A. If RTx RNA proves to be mRNA from a cellular
gene, it will identify the first cellular RT gene. It may also provide
insight into the evolution of retrotransposon-type telomeres.
It is interesting that we have detected RTx RNA in the two immortal
cell lines that we work with. These two lines have separate origins and
express different sets of TART RNAs. Therefore, the expression of RTx RNA must have occurred independently in the two
lines. Bodnar et al. (4) have expressed the catalytic
subunit of human telomerase in normal human cells and have shown that the life span is extended (at this time, apparently indefinitely) while
the cells still maintain the normal karyotype. Thus it is possible that
the expression of RTx RNA is associated with the immortality of the
Drosophila cell lines.
This work was supported by grants from the National Institutes of
Health (GM 50315 and GM57006).
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Abstract
Introduction
