ABSTRACT
Sequences similar to mitochondrial large and small subunit rRNAs are found as
small scattered fragments on a tandemly reiterated 6 kb element in the human
malaria parasite
Plasmodium falciparum
. The rDNA sequences previously identified include strongly conserved portions
of rRNA, suggesting that fragmented rRNAs derived from them are able to
associate into functional ribosomes. However, sequences corresponding to other
expected rRNA regions were not found. We here report that 10 of the 13
previously described rDNA regions have abundant small transcripts. An
additional 10 transcripts were found from regions not previously known to
contain genes. Five of the latter have been identified as rRNA fragments,
including those corresponding to the 5
'
end and 790 loop sequences of small subunit rRNA and the sarcin/ ricin loop of
large subunit rRNA. Demonstration that most of the previously described rDNA
regions have abundant transcripts and the identification of new transcripts
with other portions of conventional rRNAs provide support for the hypothesis
that these small transcripts comprise functional rRNAs.
Mitochondrial protein coding genes are present on tandemly reiterated copies of
a 6 kb sequence element in
Plasmodium
species (
1
-
3
), thus identifying this element as the malaria parasite's mitochondrial genome.
Ribosomal RNAs are also invariably encoded by mitochondrial genomes (
4
) and the 6 kb element has small regions of sequence similar to rRNAs (
2
,
3
,
5
,
6
). Unexpectedly, these small rDNA regions are not contiguous: they are
interspersed with each other, are scrambled in order, and are encoded on both
strands of the DNA. However, the nucleotide sequences are highly conserved
between
Plasmodium
species, they correspond to highly conserved regions of rRNA, and at least some
have relatively abundant small transcripts (
5
). These characteristics suggest that rRNA fragments corresponding to these
regions may assemble into functional ribosomes.
Fragmented rRNAs, while uncommon, have been described from other eukaryotes (
7
,
8
), prokaryotes (
9
,
10
), and organelle genomes (
11
-
15
). In some cases the fragmentation is minor: the
Tetrahymena pyriformis
mitochondrial rRNAs each have two fragments (
11
,
16
). More complex fragmentation is seen for the nucleo-cytoplasmic large subunit rRNAs of trypanosomatid protozoa, which consist
of seven fragments (
7
,
17
,
18
), and that of
Euglena
gracilis
, which has 14 fragments (
8
,
19
). Many examples of fragmented rRNAs share the characteristic that the rRNA fragments are encoded
in the expected linear order. The fragmented mitochondrial rRNAs of
T.pyriformis
(
11
,
16
) and of
Chlamydomonas
species (
12
,
20
), on the other hand, are encoded out of expected order. These examples of
fragmented rRNAs all differ significantly from those of
Plasmodium
in their aggregate size. Thirteen rDNA regions, from 28 to 178 nucleotides (nt), have been identified from the
Plasmodium falciparum
6 kb element, based on significant similarity in nucleotide sequence and
potential secondary structure to highly conserved regions of other rRNAs (
5
). The combined size of regions corresponding to large subunit (LSU) rRNA is 822
nt and for the small subunit (SSU) rRNA, the predicted size total is 429 nt (
5
). This is much smaller than other fragmented rRNAs, which generally approximate the size of their non-fragmented counterparts. As a comparison, the LSU and SSU rRNAs of
E.coli
rRNAs are 2904 and 1542 nt, respectively, and the corresponding fragmented
mitochondrial rRNAs of
Chlamydomonas reinhardtii
sum to 2419 and 1200 nt (
12
).
Comparison of the
P.falciparum
rRNA fragments to the sequence and secondary structures expected for organelle
rRNAs has shown that they correspond to a number of highly conserved regions of
rRNA (
5
). However, sequences corresponding to some expected regions were not found in
initial analyses. These include the sarcin/ricin loop of the LSU rRNA and
portions at the 5' end and the 790 loop of the SSU rRNA. It seems unlikely that a ribosome
would function without at least some of these rRNA sequences. In addition,
although a variety of small transcripts have been shown to hybridize to 6 kb
element probes (
3
,
6
,
21
), only a few (
5
,
22
) have been shown to correspond to specific rDNA regions. We have employed transcript mapping approaches to verify transcription of the previously described
P.falciparum
rDNA regions and to search for potential rRNA fragments from regions of the 6
kb element not known to encode genes. Our analyses have shown that 10 of the 13
originally proposed rDNA regions encode small, abundant transcripts while
similarly abundant transcripts could not be detected for three rDNA regions. We have also detected an additional 10 small, abundant transcripts, five of which have sequence similarity to regions of rRNAs from other organisms
and/or contain sequences complementary to the other rRNA fragments.
The C10 line of
P.falciparum
was employed for these studies. Parasites were cultivated by the method of
Trager and Jensen (
23
) and prepared for RNA extraction by lysing infected erythrocytes with saponin,
followed by washes with phosphate-buffered saline. Isolated parasites were quick-frozen in liquid N
2
and stored at -80oC for later use.
Total RNA was prepared from mixed populations of parasites, generally 50% or
more trophozoites, by lysis in guanidinium thiocyanate and extraction with
acidic phenol:chloroform (
24
), as previously described (
25
). For RNA blots, total RNA was electrophoresed on 12% acrylamide, 7 M urea gels
in TBE (0.1M Tris-borate, 0.9 mM EDTA) and electrophoretically transferred to nylon membrane
in TAE (40 mM Tris-acetate, 1 mM EDTA). Probing with radiolabeled
in vitro
transcripts or oligonucleotides was carried out as described previously (
25
), with modification to the temperatures for oligonucleotide probings as
dictated by their characteristics. Probe locations are shown in Table
1
.
Table 1
Sequence data for the
P.falciparum
6 kb element is found in Genbank entry M76611. The additional rRNAs described
here were identified by searching, using the Generunner program (Hastings
Software, Inc.), for short highly conserved motifs in the regions known to
encode transcripts and then manually assessing the potential for longer alignments. Proposed secondary structures were modeled after consensus structures (
26
,
27
) which include rRNAs from three phylogenetic domains (archaea, bacteria, and eukarya) and two organelle types (chloroplasts and mitochondria).
The
P.falciparum
6 kb element is densely populated with genes, having three protein coding genes
and 13 predicted rDNA fragments (Fig.
1
). To verify that transcripts existed for each rDNA region and to determine
whether other potential rRNA fragments were encoded by other portions of the 6
kb element, we embarked on a systematic transcript mapping project. Radiolabeled
in vitro
transcripts were made from short clones, among them covering the entire length
of the 6 kb element separately for each strand, and used to probe RNA blots
prepared from denaturing acrylamide gels. When necessary, oligonucleotide
probes were used to resolve whether multiple transcripts detected by a single
in vitro
transcript probe were derived from the same or adjacent sequences. Abundant
small transcripts were found for 10 of the originally proposed rDNA regions
(Fig.
2
, Table
1
) but not for the SSUC, LSUB and LSUC regions, discussed below. Two of the rDNA
regions, SSUF and LSUF, have abundant transcripts of two different sizes, as confirmed by hybridization with oligonucleotide probes.
Comparison of rRNA sequences from a wide variety of organisms provides an
insight into those regions most strongly conserved. Two types of conservation
should be considered. One is conservation of nucleotide sequence and the other
is positional conservation, meaning the maintenance of particular structures,
such as the size and location of loops and length of helices. Mitochondrial
rRNA sequences and potential structures often tend to be unusual. Adding mitochondrial sequences to comparisons of rRNAs from the three phylogenetic domains (archaea, bacteria and eukarya)
and chloroplasts produces a noticeable decrease in both sequence and positional conservation (
26
,
27
). Consideration of the fragmented
P.falciparum
mitochondrial rRNAs indicates that many correspond to regions with significant
sequence and positional conservation (Fig.
4
) and have proposed secondary structures consistent with other rRNAs (
5
). In attempting to identify additional rRNA fragments from the 6 kb element, we
first searched for conserved sequence motifs or for sequences complementary to
appropriate portions of previously identified rRNA fragments. This was followed by consideration of the potential secondary structure, and its similarity to consensus structures.
Two of the new rRNAs correspond to regions missing from the originally proposed
SSU rDNA fragments. One of these, the 70 nt RNA4, was detected with a probe
that contains sequence similar to the most conserved portion of the 790 loop of
the SSU rRNA. An oligonucleotide complementary to the conserved sequence also
hybridizes to a 70 nt transcript (data not shown). The consensus sequence at the terminal loop is GAUNAGAUACC (where N is less highly conserved than the other sites). Ten of the sites in
the terminal loop are conserved in >90% of SSU rRNAs in the three domain, two
organelle consensus (Fig.
4
). Of these, RNA4 matches eight, with its loop sequence being GAUAAGAUGAC (Fig.
5
). The GA at positions 26 and 27 (Fig.
5
) replaces the normally conserved AC. The consensus stem below the loop has only
a few scattered 90+% conserved nucleotides but a specific structure, with
conserved side bulges (Fig.
4
). The proposed RNA4 structure conforms very well to the consensus, with
similarly placed and sized side bulges (except for the bulged G at position 35,
which is missing in the consensus structure) and similar numbers of paired
nucleotides in helices.
In addition to the RNAs similar to SSU rRNA, two of the newly described RNAs
have been identified as probable components of the fragmented LSU rRNA. One of
these corresponds to the sarcin/ricin loop. This sequence is found near the 3' end of the LSU rRNA. Its central 12 nt are almost universally conserved
and it is sensitive to site-specific endonucleolytic cleavage by [alpha]-sarcin and to depurination by ricin. Treatment with either of
these toxins abrogates the elongation step of protein synthesis and thus
signifies a crucial role for this region of rRNA (
33
). The structure of the sarcin/ricin loop has been extensively studied and has a
GAGA tetraloop, which contains the site of ricin and [alpha]-sarcin action, at the tip of the stem (
34
). In mitochondrial LSU rRNAs, the tetraloop sequence is often altered (Fig.
8
A) but still fits a motif very common in rRNAs: GNRA, where N is any nucleotide
and R is a purine (
35
). The 6 kb element contains a sequence which matches the terminal consensus
sequence exactly except for the tetraloop region, which bears the sequence GUAA
(Fig.
8
A) and is thus consistent with the common tetraloop motif. An oligonucleotide
complementary to the putative sarcin/ricin loop sequence described here detects
the 100 nt RNA10 (Fig.
3
, panel 10). The sequences flanking the sarcin/ricin loop region form an
extended helix (Fig.
8
B) which corresponds to a positionally conserved helix in the consensus
structure (Fig.
4
). Detailed studies of the conformation of a synthetic oligonucleotide based on the rat sarcin/ricin loop suggest that the G at position 12 is bulged
and the As at positions 11 and 23 form a non-canonical pair (
34
). This structure is also possible with the
P.falciparum
sequence (Fig.
8
C).
The RNA blotting experiments presented here have demonstrated abundant
transcripts for 10 of the originally proposed rDNA regions of the
P.falciparum
6 kb element. This set of RNAs corresponds to regions critical for ribosome
function but is missing important sequences expected in an organelle rRNA.
Transcript mapping has now detected 10 additional small transcripts. For five
of these, specific locations in secondary structure can be predicted, filling
in major gaps from the previously proposed structures. With the exception of
the GTPase center, the earlier identification of which is now in question (see
below), by far the majority of regions with highly conserved nucleotide
sequence expected in rRNAs are now represented. Identification of rRNAs
corresponding to structures that are predominantly positionally conserved is more difficult and remains an issue for future study. The identification of these new rRNA fragments further supports the potential for the small transcripts from the
6 kb element to participate in functional ribosomes.
The newly identified rRNA fragments generally include shorter contiguous regions
of strong similarity than the originally identified rDNA sequences, explaining
the difficulty in finding them. Based on the functions and/or conservation of corresponding regions of other rRNAs, however, they constitute important portions of the rRNA and presumably the mitochondrial ribosomes. The 790 loop (RNA4) is implicated in the interaction of the large and small
subunits during protein synthesis and alteration of its sequence or ability to hydrogen bond decreases subunit association (reviewed in
37
). The abrogation of translation by the action of the ribotoxins [alpha]-sarcin and ricin (
33
) attest to the importance of the sarcin/ricin loop (RNA10). The identities of
RNA8 and RNA9 rest to a significant degree on their ability to appropriately
complement sequences of other rRNA fragments, and are bolstered by occurrence
of a few highly conserved nucleotides and positional conservation of their
potential structure. These rRNA regions have less clearly defined roles than
the 790 loop and sarcin/ricin loop but correspond to generally well-conserved regions. RNA1 has maintained sufficient similarity to
E.coli
rRNA for its identification, has half a dozen highly conserved nucleotides, and
has maintained two of three predicted tertiary interactions with LSUE.
Experiments to verify the association of these and other rRNA fragments into
ribosomes are in progress.
The combination of the discontinuous and scrambled nature of the 6 kb element rDNA sequences and the unconventional secondary structures often found for small mitochondrial rRNAs makes it particularly
difficult to determine the identity of each of the small transcripts. No rRNA
similarities have yet been found for five of these although their size and
abundance make them excellent candidates for rRNA fragments. While further
sequence analysis may reveal hints to their identities, it is also possible
that they correspond to positionally conserved regions that are not well-conserved in sequence. Alternately, they may exhibit unique structures
called for by the fragmented nature of the
P.falciparum
mitochondrial rRNAs. They may, for example, interact with other fragments to
maintain a particular secondary structure and bring important nucleotides into
the proper orientation and proximity. Presumably such interactions between RNAs and/or with proteins in ribosomes allow the various rRNA fragments to maintain the correct spatial
arrangement for function. In such a situation, there may be important
structural roles for (some of) the five as yet unidentified small RNAs which do
not require extensive sequence or positional conservation but only the ability
to interact with other rRNAs at selected sites. It is also possible that some
of them have undiscovered functions unrelated to rRNA.
Abundant, appropriately sized transcripts were not detected for three of the
originally proposed rDNA regions, SSUC, LSUB and LSUC, by RNA blotting or RNase protection assays. These regions are transcribed,
as demonstrated by data showing extensive polycistronic transcription of the
P.falciparum
6 kb element (
38
). The polycistronic transcripts are not very abundant, suggesting rapid
processing to the smaller RNAs, and those portions of transcript which
correspond to the `missing' RNAs may be relatively rapidly turned over. The
lack of easily detectable, abundant transcripts suggests that the earlier
identification of the
P.falciparum
SSUC, LSUB and LSUC rDNA regions was in error.
The absence of a transcript corresponding to SSUC is not critical since the
abundant small transcript designated RNA9 has the potential to fill the same
position in the secondary structure, and presumably the same function in the
ribosome. The absence of LSUB and LSUC RNAs is more troubling since they were
predicted to comprise the GTPase center, which is required for protein
synthesis. Three possibilities may explain this apparent absence. The GTPase
center functions may be provided by one or more of the remaining unidentified
small transcripts from the 6 kb element, RNAs with the GTPase sequences may be
imported from outside the mitochondrion, or the LSUB and LSUC sequences may be
transcribed but then modified in such a way as to render them unable to
hybridize to the probes we employed. The first of these possibilities may
require a novel sequence or structure to provide the GTPase function, since no
significant similarity has yet been detected between the unidentified RNAs and
conserved GTPase sequences. As to the other possibilities, small RNAs are known
to be imported into mitochondria (
39
,
40
), and post-transcriptional modification of rRNA sequences has also been reported (
41
-
43
). Sorting out these possibilities will probably require analysis of the RNA components in the mitochondrial ribosomes.
As with the GTPase center, the deviations of SSUA RNA sequence from the generally conserved characteristics of the 530 loop provoke questions. The 530 loop has been extensively studied and specific sites
in and around it have been implicated in interactions with EF-Tu (
44
), polysome formation and/or stability (
45
), and interactions with ribosomal proteins S12 and S4, which are associated
with the fidelity of translation (
46
,
47
). Some of the critical sites are maintained in SSUA RNA, notably a G in the
site corresponding to
E.coli
position 530 (position 29 in SSUA, Fig.
6
A). In contrast, studies with
E.coli
mutations suggest that substitution of the G at position 517 with a C increases misreading (
48
); the corresponding site in the
P.falciparum
SSUA RNA sequence (position 16 in SSUA, Fig.
6
A) is a C. Perhaps most importantly, potential for the pseudoknot expected
between the bulged loop and terminal loop appears decreased, with only one of
the set of three basepairs able to form a canonical pair. Mutational analysis
has shown that the absence of basepairing potential between these sites results
in decreased cell viability and even substitution of wobble basepairing for
Watson-Crick interactions has negative functional consequences (
30
). However, there are a few examples of mitochondrial rRNAs which do not exhibit strong basepairing potential, such as those from kinetoplastid protozoa (
49
-
51
). The lack of expected basepairing for the 530 loop pseudoknot may thus still
allow functioning ribosomes.
The characteristics of the small abundant RNAs are, for the most part, consistent with a functional protein synthetic apparatus, albeit probably a most unusual one. The loss of sequence, compared with
eubacterial rRNAs, may reflect adaptation to the very small size of the
mitochondrial genome in
Plasmodium
, with less critical sequences being evolutionarily jettisoned.
Theileria parva
, a related parasite, also contains a very small mitochondrial genome with highly
fragmented rRNAs (
36
), and these unusual rRNAs may be characteristic of other members of phylum
Apicomplexa as well (reviewed in
52
). Evidence for the existence of the protein products of the mitochondrial mRNAs
(reviewed in
52
) suggests that the
Plasmodium
mitochondrion has developed mechanisms for overcoming the challenges posed by the extreme fragmentation,
small fragment size, and apparent absence of some sequences and expected
interactions. Deciphering them may provide insights into the role of RNA
structure in ribosome function.
We thank Mark Drew, Holli Banister and Julie Anderson for technical assistance,
Shaofeng Yan for work performed during a graduate student rotation, and Martha
Thorning for invaluable assistance with figure preparation. We further thank Dr Ira G. Wool for helpful discussions about the sarcin/ricin loop, Drs Robin Gutell, Michael
Gray and Murray Schnare for suggestions on secondary structures, and Drs Gerard
Cangelosi and Marilyn Parsons for comments on the manuscript. This work was
supported by NSF grant MCB 9205809 to J.E.F., who is also a Burroughs Wellcome New Investigator in Molecular Parasitology.
*To whom correspondence should be addressed at: Seattle Biomedical Research
Institute, 4 Nickerson Street, Seattle, WA 98109-1651,
USA. Tel: +1 206 284 8846; Fax: +1 206 284 0313; Email: feagin@u.washington.edu
Gene
Probe type
Probe location
a
Transcript size (nt)
SSUA
oligonucleotide
1919-1943
140
SSUB
oligonucleotide
431-452
110
SSUD
oligonucleotide
5395-5417
65
SSUE
oligonucleotide
1656-1675 rc
40
SSUF
oligonucleotide
5468-5489
74, 58
LSUA
in vitro
transcript
4957-5163
175
LSUD
in vitro
transcript
5525-5967
78
LSUE
in vitro
transcript
5525-5967
190
LSUF
in vitro
transcript
1411-1657 rc
125, 110
LSUG
oligonucleotide
332-353
115
RNA1
in vitro
transcript
544-664
95
RNA2
in vitro
transcript
1658-1851 rc
75
RNA3
in vitro
transcript
1852-2159 rc
85
RNA4
in vitro
transcript
4526-4748 rc
70
RNA5
in vitro
transcript
4749-4956 rc
92
RNA6
in vitro
transcript
4790-4956 rc
58
RNA7
oligonucleotide
5226-5252
94
RNA8
in vitro
transcript
5894-5967
115
RNA9
in vitro
transcript
2-130 rc
53
RNA10
oligonucleotide
644-665
100
REFERENCES
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