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Mechanism. The
transcripts of 12 (the precise number varies with the species) of
the maxicircle protein-coding genes are edited posttranscriptionally
by the insertion and occasional deletion of uridine (U) residues
mostly within coding regions, thereby correcting frameshifts and
producing translatable mRNAs. The minicircles encode gRNAs, which
are complementary to mature edited sequences if G:U as well as
canonical base pairs are allowed . The gRNAs have 3′ nonencoded
oligo(U) tails that may be involved in stabilizing the initial
interaction of the gRNA and the mRNA by either RNA–RNA or
RNA–protein interactions . Fifteen gRNAs are encoded in intergenic regions of the
maxicircle DNA of Leishmania tarentolae. This division of the
mitochondrial genome into two physically separate genomes, with the
RNA transcripts of one interacting with the incomplete mRNA
transcripts of the other to produce translatable mRNAs is
unprecedented and is suggestive of an unique evolutionary origin.
The mechanism of U-insertion/deletion editing
involves a series of enzymatic cleavage-ligation steps, with the
precise cleavages determined by base pairing with the cognate gRNAs
. A single gRNA mediates the editing of a “block” of approximately
1–10 sites. Multiple overlapping gRNAs mediate the editing of a
“domain” . The overall 3′ to 5′ polarity of editing site selection
within a domain is determined by the creation of upstream mRNA
“anchor” sequences by downstream editing . Editing usually also
proceeds 3′ to 5′ within a single block. A variable extent of
“misediting” at the junction regions between fully edited and
unedited sequences also has been observed, and this varies from gene
to gene and from species to species . Misediting, which appears to
be a consequence both of correct guiding by an incorrect gRNA and by
stochastic errors in the editing process, is not deleterious, as
misedited sequences appear to be re-edited correctly, in a 3′ to 5′
polarity.
Comparative Analysis of
Editing in Different Kinetoplastid Species. The
U-insertion/deletion type of RNA editing has been detected in
multiple trypanosomatid species. The extent of editing for several
genes varies in different species. For example, the ND7 gene in the
trypanosomes, T. brucei and T. cruzi, is pan-edited in
two domains, whereas in the Leishmania-Crithidia clade this
gene is edited only at the 5′ end of each domain. The A6 gene in the
trypanosomes is pan-edited, whereas in the Leishmania-Crithidia
clade, the editing of the A6 gene shows a gradient of restriction to
the 5′ end of the single domain, from L. tarentolae, to
Herpetomonas muscarum, to Phytomonas serpens, and to
Blastocrithidia culicis .
To date, the deepest lineage in which U-insertion
editing has been detected is the bodonid group. Because minicircles,
which presumably encode gRNAs, are observed in B. caudatus,
B. saltans, and C. helicis, this suggests that the
free noncatenated state is a primitive feature. Catenation of
minicircles to form the kDNA network probably arose in an ancestor
of the trypanosomatids as a molecular mechanism designed to avoid
minicircle losses by missegregation. Concatenation of minicircles in
the 180-kb megacircle as observed in T. borreli might have
independently arisen as another solution to the same problem.
However, additional analyses of the kDNA structure in bodonids are
required to shed more light on kDNA evolution.
The only mitochondrial gene isolated from the
deeper branching Diplonema
and Euglena gracilis is the COI gene, which is unedited. In addition, no
evidence was obtained for small gRNA-like molecules in E.
gracilis mitochondria by 5′ capping experiments .
This preliminary evidence does not, of course, eliminate the
possibility of editing in these cells, but the simplest hypothesis
is that this type of editing evolved in the mitochondrion of an
ancestral bodonid after the split from the euglenoid lineage.
Minicircle-Encoded gRNAs in
Two Strains of L. tarentolae. The only species
for which the entire complement of gRNAs is known
is the UC strain of L. tarentolae, which has been maintained
as the promastigote form in culture in various laboratories for more
than 60 years. There are 15 maxicircle-encoded gRNAs and 17
minicircle-encoded gRNAs in this strain. Five pan-edited genes
(G1–G5) show a complete absence of productive editing in this
strain, as evidenced by an inability to PCR-amplify mature edited
transcripts by standard methods. These genes are productively edited
in T. brucei. Two of the minicircle-encoded gRNAs in the
L. tarentolae UC strain, gLt19 (=gG4-III) and gB4 (=gND3-IX),
represent nonessential gRNAs for these nonfunctional editing
cascades. This was determined by analyzing the minicircle-encoded
gRNA complement of LEM125, a recently isolated strain of L.
tarentolae.
LEM125 has the same 15 maxicircle gRNA genes but also has an
estimated 80 total minicircle-encoded gRNAs, of which 30 have been
cloned and sequenced and the remainder inferred to be present
because of the existence of completely edited mRNA transcripts in
this strain. These additional gRNAs mediate the editing of three
components of complex I of the respiratory chain, ND3, ND8,
and ND9, and also two unidentified genes, which were termed
G-rich regions 3 and 4 (G3 and G4). It was proposed that multiple
gRNA-encoding minicircle sequence classes had been lost from the UC
strain probably because of a lack of a requirement for complex I
activity in culture. The presence of productively edited ND8, ND9,
G3 (=CR3), G4 (=CR4), and ND3 mRNAs in T. brucei and
the presence of productively edited G3 mRNA in P. serpens
(D.A.M., unpublished results) implies that the corresponding
minicircle-encoded gRNAs also exist in these species, and this
provides phylogenetic evidence for our hypothesis that the ancestral
cell had a complete complement of minicircle classes. In addition,
the presence of two minicircle-encoded gRNAs, gG4-III and gND3-IX,
in the UC strain, which are remnants of the complete editing cascade
of gRNAs for these two genes in LEM125, corroborates this evidence.
To propose a loss of multiple minicircle classes from the UC strain
is also more parsimonious than to propose a gain of multiple classes
in the LEM125 strain. And finally, the existence of a 5′ terminal
block of misedited sequence in the LEM125 ND3 mRNA
is indicative that this gene originally was completely edited and
has lost the 5′ terminal gRNA.
Minicircles from L. tarentolae and other
members of the Leishmania-Crithidia clade contain a single
gRNA gene situated at a constant distance from the origin of
replication. Minicircles from T. brucei, however, also have a
single origin of replication but contain three gRNA genes situated
between 18-mer inverted repeats ,
and minicircles from T. cruzi contain four gRNA genes
situated within each of the four variable regions between four
origins of replication .
The total number of different minicircle sequence classes in T.
brucei is estimated to be 200–300,
which would yield a total of 600–900 gRNAs. Although only 72 gRNAs
have been identified so far in T. brucei ,
it is clear that there are extensive redundant gRNAs, which are
gRNAs of different sequence but possessing the identical editing
information because of the allowed G:U base pairing .
In fact, 28 of the 72 identified gRNAs are redundant over the entire
length of the gRNA. Only a single redundant gRNA pair has been
observed in L. tarentolae.
T. brucei also contains gRNAs with several mismatches in the
anchor or guiding regions, which may be nonfunctional, but there is
no evidence for or against this suggestion.
Retroposition Model for Loss
of Editing in Evolution. Based on these observations and
on the known 3′-5′ polarity of editing, a retroposition model was
proposed to explain both the gradual restriction of editing to the
5′ end of domains and the complete loss of editing in some cases . We proposed that partially edited mRNAs were being
frequently converted to cDNAs by a postulated mitochondrial reverse
transcriptase activity, and those cells that had replaced the
original pan-edited cryptogene with a partially edited gene would
survive a loss of an entire minicircle sequence class encoding a
specific gRNA involved in that editing cascade. The retention of
editing at the 5′ end of a domain may allow regulation of
translation by creation of a methionine initiation codon and a
possible ribosome-binding site. This model is based on the
assumption that minicircles are distributed randomly to daughter
cells upon cell division.
Replication and Segregation
of Minicircles. One possible mechanism involved in the
random distribution of minicircles is the mode of replication and
segregation. The mitochondrial S phase is fairly synchronous with
the nuclear S phase, although the kinetoplast network physically
divides just before the nucleus . Closed minicircles are apparently
randomly removed from the side of the network facing the basal body
by a topoisomerase II activity and migrate by an unknown mechanism
to one of two replisomes that are located at the two antipodes
of the kDNA nucleoid body .
After replication, the daughter molecules remain nicked or gapped,
which may be a mechanism to ensure replication of each minicircle.
The daughter minicircles then are recatenated into the periphery of
the network. There is microscopic evidence that the networks in
Leishmania and Crithidia (and also T. cruzi) are
actually rotating, and this movement produces a complete peripheral
distribution of newly replicated minicircles . The networks in the
middle of S phase consist of an expanding ring of nicked circles and
a central core of closed circles, and at the end of S phase the
networks consist entirely of nicked circles. The minicircles then
become closed and then the network segregates into two daughter
networks as the single mitochondrion divides .
This mechanism of replication appears to introduce a certain amount
of randomness into minicircle segregation. In other words, sister
minicircles may not necessarily end up in different daughter cells.
A pulse–chase experiment performed with C. fasciculata cells
at the light microscope level previously showed that newly
replicated minicircles are spread throughout the network after one
cell cycle is completed .
In the case of T. brucei, the network
apparently does not rotate and two dumbbell-shaped masses of nicked
replicated minicircles accumulate at either end of the nucleoid
body, which then divides in half into the daughter cells . In this case there does not appear to be a mechanism for
randomization throughout the network, other than the possible random
selection and migration to the antipodal replisomes.
Plasticity of Minicircle
Sequence Class Copy Number in L. tarentolae in Culture. The
number of minicircles per network in L. tarentolae was
assayed by counting 4′,6-diamidino-2-phenylindole (DAPI)-stained
networks in a cell counting chamber using a fluorescent microscope
and measuring the DNA concentration spectrophotometrically.
Quantitative dot blot hybridization using an oligonucleotide probe
complementary to the conserved CSB-3 12-mer sequence yielded values
of 12,600 ± 300 and 12,700 ± 800 for the UC and LEM125 strains,
respectively. Similar dot blot hybridization analysis showed that
the copy number of maxicircle DNA molecules was very similar in the
UC and LEM125 strains (32 ± 2 and 25 ± 2 copies per network,
respectively).
Quantitation of the copy numbers of 17 specific
minicircle sequence classes in the UC strain was previously
performed by Southern blot analysis using specific oligonucleotide
probes for specific gRNAs. We have repeated these analyses with both
UC strain kDNA and LEM125 strain kDNA, by dot blot hybridization of
MspI-digested kDNA (all minicircles have at least one MspI
site), and a known amount of specific cloned minicircles using
primers specific to each gRNA. A primer to the conserved 12-mer
sequence was used as a loading control. The results in Table
1 show that homologous minicircle sequence class frequencies are
extremely variable, both between strains and between different kDNA
isolates from the same strain taken after several years of culture.
In general, the LEM125 strain kDNA exhibited lower copy numbers for
the sequence classes in common between the strains, which is
consistent with LEM125 possessing a more complex minicircle
repertoire.
In the UC strain kDNA as mentioned above, two
gRNAs, pLtl9 (= G4-III) and pB4 (= gND3-IX), are nonfunctional, in
that all of the other minicircle-encoded gRNAs in those editing
cascades are missing from this strain. It is of interest that these
nonfunctional minicircles showed the greatest plasticity in
frequency.
As was found previously ,
there was no correlation of minicircle copy number and gRNA relative
abundance (data not shown).
Computer Simulations of
Minicircle Sequence Class Plasticity. Using a population
dynamics model of minicircle segregation, Savill and Higgs
recently have shown that random segregation can indeed account for
much of the above experimental observations on minicircle
plasticity. The copy number of every minicircle class in every cell
in a population is tracked over many generations. In every
generation each cell replicates its minicircles, hence doubling the
copy number of all its classes. Then the cells divide and the
daughter cells receive a certain number of copies of each class. The
actual number of copies is randomly chosen according to a binomial
distribution that models a purely random segregation process. All
daughter cells that receive the full complement of minicircle
classes and have fewer than 12,000 minicircles in total are randomly
chosen to populate the next generation up to a maximum population
size. These two conditions model the reasonable assumptions that (i)
if a cell does not receive any copies of a particular class it is
therefore missing a gRNA and hence its mRNA cannot be correctly
edited, which is assumed to be lethal, and (ii) the network
is restricted in its maximum size because of physical constraints.
A typical simulation of a hypothetical species
with 17 minicircle classes is shown in Fig.
3. It clearly demonstrates that random segregation causes
fluctuations in the average minicircle class copy number from one
generation to the next. Moreover, it also leads to the
experimentally observed distribution of many classes having very low
copy number and a few having very high copy number. No two runs are
ever the same, thus explaining why homologous minicircle classes in
different strains have different copy numbers.
The loss of minicircle classes during the long
culture history of the UC strain also was modeled by starting with
70 classes, of which 15 are required and 55 are not. Fig.
4 shows the number of generations for each unnecessary class to
be lost, from the time when the UC strain was first cultured. Many
classes are lost fairly rapidly within the first few hundred
generations, but it takes successively longer for the remaining
classes to be lost, and the last few classes may take tens of
thousands of generations to be lost. Moreover, by averaging over
many simulations we found that the last remaining unnecessary class
was also the most abundant class in 27% of cases. Therefore, random
segregation can explain the observed long persistence time of
unnecessary classes and their high abundance. However, as shown in
Fig.
3, the highest frequency achieved by the most abundant class for
a hypothetical species with 17 classes (similar to the UC strain)
only reaches about 30% and never as large as the 67% observed in the
1,994 UC cells. This large abundance of one class is similar to the
situation in the CFC1 strain of C. fasciculata, in which one
minicircle sequence class shows over 90–95% abundance. It appears
that random segregation alone cannot explain the large abundance of
these classes, and therefore other selective forces must be present.
If the additional following assumptions are made,
simulations can explain the experimental results: (i) The
network has a minimum allowable size. If the network is too small,
it may not abut the replisomes. (ii) The number of copies of
each necessary minicircle class is regulated by an unknown
mechanism. (iii) The number of copies of each unnecessary
minicircle class is unregulated, i.e., once a minicircle becomes
unnecessary—by loss of other gRNAs in a cascade, its copy number is
not regulated and can vary freely. The model is modified so that if
the total number of minicircles in a daughter cell falls below a
predetermined threshold or if the copy number of each necessary
class exceeds a predetermined threshold, the cell does not survive
into the next generation. For simplicity, in the model this
threshold is set to the same value for all necessary classes, but in
reality it may vary between classes. The lower threshold for each
necessary class is one copy, as in the original model. Again, in
reality this may not be true. Fig.
5 shows a simulation where the minimum kinetoplast threshold
size is set to 10,000 minicircles and the upper threshold for
necessary classes is set to 200. Fifteen classes are required and 55
are not. Initially all classes have the same copy number of 170,
giving a total of 11,900 minicircles per cell, which lies between
the assumed lower and upper thresholds for the kinetoplast size
(i.e., 10,000 and 12,000 minicircles, respectively). The figure
shows the cumulative proportion of minicircles of all necessary
classes, all unnecessary classes, and the proportion of the most
abundant class. Initially the proportions are 21% (170 × 15/11,900),
79% (170 × 55/11,900), and 1.4% (170/11,900), respectively. Because
of assumption ii, the proportion of minicircles of necessary
classes cannot exceed 30% (15 × 200/10,000). Therefore, the
unnecessary classes must make up the difference for the kinetoplasts
to maintain their minimum sizes. However, as unnecessary classes are
lost because of random segregation over time, there are fewer
classes that can make up this difference. Finally, there will be
only one unnecessary class left to make up at least 70% of the
minicircles in the kinetoplast. This class is now necessary only to
maintain kinetoplast size, and the function of encoding gRNAs has
now been replaced by a buffering function. By adjusting the
parameters, it is even possible to obtain an unnecessary class with
over 90% abundance, as in the CFC1 strain.
This successful simulation of large frequencies for unnecessary
minicircle classes actually provides support for assumption ii.
The model of random segregation also makes
several interesting predictions. At every generation some daughter
cells become unviable and do not survive into the next generation
because they do not receive the full complement of minicircle
classes. Hence some fraction of the total population of daughter
cells is viable; we term this the daughter cell viability. We find
that cell viability increases with increasing kinetoplast size and
decreasing number of minicircle sequence classes. If the cells have some mechanism that more evenly segregates
sister minicircles between daughter cells, cell viability increases.
This implies that there could be some selection pressure for
trypanosomatids to segregate their minicircles more evenly, which
may have led to the development of the rotating network in the
Leishmania-Crithidia clade.
In the case of T. brucei, random
segregation of the 250+ sequence classes would lead to a predicted
cell viability in this model of less than 0.5, and hence population
extinction. However, incorporating the information that each
minicircle in this species encodes multiple gRNAs and that genetic
exchange occurs, it has been shown that the model can produce the
observed situation of evolutionary viability and multiple redundant
and nonfunctional gRNAs (N.J.S. and P. G. Higgs, unpublished
results). Mutation of the gRNA genes and drift in the minicircle
copy numbers lead to an ever-increasing number of necessary classes
encoding ever fewer functional gRNAs per minicircle. |
