Trans-splicing and RNA editing of LSU rRNA in Diplonema mitochondria.
ABSTRACT Mitochondrial ribosomal RNAs (rRNAs) often display reduced size and deviant secondary structure, and sometimes are fragmented, as are their corresponding genes. Here we report a mitochondrial large subunit rRNA (mt-LSU rRNA) with unprecedented features. In the protist Diplonema, the rnl gene is split into two pieces (modules 1 and 2, 534- and 352-nt long) that are encoded by distinct mitochondrial chromosomes, yet the rRNA is continuous. To reconstruct the post-transcriptional maturation pathway of this rRNA, we have catalogued transcript intermediates by deep RNA sequencing and RT-PCR. Gene modules are transcribed separately. Subsequently, transcripts are end-processed, the module-1 transcript is polyuridylated and the module-2 transcript is polyadenylated. The two modules are joined via trans-splicing that retains at the junction ∼26 uridines, resulting in an extent of insertion RNA editing not observed before in any system. The A-tail of trans-spliced molecules is shorter than that of mono-module 2, and completely absent from mitoribosome-associated mt-LSU rRNA. We also characterize putative antisense transcripts. Antisense-mono-modules corroborate bi-directional transcription of chromosomes. Antisense-mt-LSU rRNA, if functional, has the potential of guiding concomitantly trans-splicing and editing of this rRNA. Together, these findings open a window on the investigation of complex regulatory networks that orchestrate multiple and biochemically diverse post-transcriptional events.
- SourceAvailable from: David Farringdon Spencer[Show abstract] [Hide abstract]
ABSTRACT: In Crithidia fasciculata, a trypanosomatid protozoan, the nuclear-encoded ;28S' rRNA is multiply fragmented, comprising two large (c and d) and four small (e, f, g and j) RNA species. We have determined that the coding sequences for these RNAs (and that of the 5.8S rRNA, species i) are separated from one another by spacer sequences ranging in size from 31 to 416 bp. Coding and spacer sequences are presumably co-transcribed, with excision of the latter during post-transcriptional processing generating a highly fragmented large subunit (LSU) rRNA. Secondary structure modelling indicates that the C. fasciculata LSU rRNA complex (seven segments, including 5.8S rRNA) is held together in part by long-range intermolecular base pairing interactions that are characteristic of intramolecular interactions in the covalently continuous LSU (23S) rRNA of Escherichia coli. At least one functionally critical region (encompassing the alpha-sarcin cleavage site) is contained in a small RNA species (f) rather than in one of the two large RNAs. Within a proposed secondary structure model of C. fasciculata LSU rRNA, discontinuities between the different segments (created by spacer excision) map to regions that are highly variable in structure in covalently continuous LSU rRNAs. We suggest that ;rRNA genes in pieces' and discontinuous rRNAs may represent an evolutionarily ancient pattern.The EMBO Journal 05/1987; 6(4):1063-71. · 10.75 Impact Factor
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ABSTRACT: Mitochondria isolated from maize plants with S-type male-sterile cytoplasms are capable of synthesizing four species of RNA at concentrations of actinomycin D that eliminate all DNA-directed RNA synthesis. No RNA synthesis occurs under the same conditions with mitochondria from plants possessing normal (N) cytoplasm or with other subcellular fractions from plants with S cytoplasm. The actinomycin D-resistant RNA synthesis occurs within the mitochondria since the labeling of these species is unaffected by inclusion of RNase in the incubation medium and since they become completely sensitive to RNase upon lysis of the mitochondria with low concentrations of Triton X-100. Two of the actinomycin D-resistant products are double stranded. These are 2850 and 900 base pairs in length, whereas the remaining two are 2150 and 850 bases. The synthesis of all four RNAs occurs in at least five different accessions of S cytoplasm, suggesting it is a general feature of S mitochondria. The double-stranded RNAs show homology to single-stranded S mitochondrial RNA but not to N mitochondrial RNA. Our observations indicate that the replication of these RNAs occurs independently of mtDNA and that they thus represent a novel type of inheritable element in organelles, an RNA plasmid.Proceedings of the National Academy of Sciences 08/1986; 83(14):5175-9. · 9.81 Impact Factor
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ABSTRACT: Plasmodium falciparum contains two extrachromosomal DNAs, a 6 kb linear element and a 35 kb circular DNA; both encode rDNA sequences. The 6 kb element rDNAs comprise fragments of both large and small subunit rRNAs. Comparison of these with corresponding rDNA sequences from the 35 kb DNA and E. coli show that sequences conserved between the three are largely confined to highly conserved core regions; in fact, most of the 6 kb rDNA sequences correspond to core regions. Both the 6 kb element and 35 kb rDNAs show less conservation to each other than to E. coli sequences, suggesting that the two extrachromosomal DNAs of P. falciparum are not closely related. The characteristics of the fragmented rRNAs from the 6 kb element suggest they are functional, possibly in mitochondrial ribosomes.Nucleic Acids Research 03/1992; 20(4):879-87. · 8.81 Impact Factor
Trans-splicing and RNA editing of LSU rRNA in
Matus Valach*, Sandrine Moreira, Georgette N. Kiethega and Gertraud Burger*
Department of Biochemistry and Robert-Cedergren Centre for Bioinformatics and Genomics; Universite ´ de
Montre ´al, Montreal, H3C 3J7, Canada
Received July 26, 2013; Revised October 23, 2013; Accepted October 25, 2013
Mitochondrial ribosomal RNAs (rRNAs) often display
reduced size and deviant secondary structure, and
sometimes are fragmented, as are their correspond-
ing genes. Here we report a mitochondrial large
subunit rRNA (mt-LSU rRNA) with unprecedented
features. In the protist Diplonema, the rnl gene is
split into two pieces (modules 1 and 2, 534- and
352-nt long) that are encoded by distinct mitochon-
drial chromosomes, yet the rRNA is continuous.
To reconstruct the post-transcriptional maturation
pathway of this rRNA, we have catalogued transcript
intermediates by deep RNA sequencing and RT-PCR.
Subsequently, transcripts are end-processed, the
module-2 transcript is polyadenylated. The two
modules are joined via trans-splicing that retains at
the junction ?26 uridines, resulting in an extent of
insertion RNA editing not observed before in any
system. The A-tail of trans-spliced molecules is
shorter than that of mono-module 2, and completely
absent from mitoribosome-associated mt-LSU rRNA.
We also characterize putative antisense transcripts.
Antisense-mono-modules corroborate bi-directional
transcription of chromosomes. Antisense-mt-LSU
rRNA, if functional, has the potential of guiding
concomitantly trans-splicing and editing of this
rRNA. Together, these findings open a window on
the investigation of complex regulatory networks
that orchestrate multiple and biochemically diverse
the eukaryotic cell that contain not only a distinct
genome—typically a multicopy, single type of circular-
mapping chromosome—but also their own translation
mitoribosome are partly or completely encoded by the
nuclear genome, synthesized in the cytosol and imported
into mitochondria, the genes specifying the large subunit
(LSU) and small subunit (SSU) ribosomal RNAs always
Mitochondrial rRNAs (mt-rRNAs) are sometimes frag-
apicomplexans (2–4). In Plasmodium the ?20 gene pieces
are spread across the genome on both DNA strands, are
separately transcribed and then assembled into the
ribosome, without covalently joining of the rRNA pieces
(2). Further peculiarities observed in certain mt-rRNAs
are homo-nucleotide appendages at their 30end, e.g.
oligo(A) tails in Plasmodium (5) and short poly(U) tails
in kinetoplastids (6).
Identifying mt-rRNA genes and accurate termini
mapping in mitochondrial genome sequences can be
challenging, particularly in taxa that are not closely
related to model organisms and whose mtDNA has
diverged far away from its bacterial ancestor. This
applies in extremis to the unicellular protozoan (protist)
group diplonemids, the sistergroup of kinetoplastids.
Mitochondrial genes of Diplonema papillatum and its rela-
tives are not only highly divergent but also systematically
fragmented in a unique way. Genes consist of up to 11
pieces (modules) that are ?80–530-nt-long, and each is
encoded on a distinct circular chromosome of 6kb (class
A) or 7kb (class B). Modules are transcribed separately
and subsequently joined into continuous RNAs. With
sequence, the estimated genome size of Diplonema
mtDNA is unusually large [?600kb; (7)].
In contrast to the eccentric genome structure, the gene
complement of Diplonema mtDNA is rather conventional.
Mitochondrial genes encode components of the respira-
tory chain, oxidative phosphorylation and mitoribosome,
notably NADH dehydrogenase subunits 1, 4, 5, 7 and 8;
apocytochrome b, cytochrome oxidase subunits 1–3, ATP
components of the
*To whom correspondence should be addressed. Tel: +1 514 343 7936; Fax: +1 514 343 2210; Email: email@example.com
Correspondence may also be addressed to Matus Valach. Tel:+1 514 343 6111 (ext. 5172); Fax:+1 514 343 2210; Email: firstname.lastname@example.org
Nucleic Acids Research, 2014, Vol. 42, No. 4Published online 19 November 2013
? The Author(s) 2013. Published by Oxford University Press.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/
by-nc/3.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial
re-use, please contact email@example.com
synthase subunit 6 and LSU rRNA. The gene for mito-
chondrial SSU rRNA has not yet been identified (8). For
rnl (encoding LSU rRNA), we only found a 352-nt long
30-terminal portion that is otherwise well conserved.
Incidentally, this RNA piece is the most highly expressed
transcript in poly(A) libraries. However, the complete
sequence and overall organization of rnl has remained un-
recognized for many years, partly due to technical chal-
lenges in culturing sufficient cell material and isolating
mitochondria from Diplonema, but also, as we know
now, because of the intricate structure and biosynthesis
of mt-LSU rRNA. We succeeded to resolve the puzzle
by high-throughput RNA sequencing (RNA-Seq) and
show here that maturation of Diplonema mt-LSU rRNA
proceeds by multiple steps including extensive RNA
editing. We also identify antisense RNA molecules that
have the potential for guiding both trans-splicing and
RNA editing of mt-LSU rRNA, but their function has
yet to be demonstrated.
MATERIALS AND METHODS
Sequences deposited in public-domain databases
We have deposited in GenBank the genomic sequence of
rnl-module 1 plus adjacent chromosome regions (accession
no. KF633465) and the cDNA sequences of cytosolic 5.8S,
18S and 28S rRNA of D. papillatum (accession nos.
KF633466-KF633468). The sequence of rnl-module 2
was deposited previously under the accession number
JQ302963. A partial sequence of D. papillatum cytosolic
18S rRNA had been deposited before by others (GenBank
accession no. AF119811).
Strain, culture and extraction of mtRNA
D. papillatum (ATCC 50162) was obtained from the
American Type Culture Collection. The organism was
cultivated axenically at 16–20?C in artificial seawater
enriched with 1% fetal horse serum (Wisent) and 0.1%
bacto tryptone. For extended large-scale cultivations,
chloramphenicol (40mg/L) was added to prevent bacterial
contamination. To isolate mitochondria, cells were col-
lected by centrifugation at 3000g for 10min, washed
once with ice-cold ST buffer [0.65M sorbitol, 20mM
Tris (pH 7.5), 5mM EDTA] and disrupted by nitrogen
decompression at 600psi (Parr Instrument Company) in
the same buffer. Mitochondrial RNA and DNA were
extracted from an organelle-enriched fraction isolated by
differential and sucrose gradient centrifugation essentially
as devised earlier (9). More specifically, intact cells and
nuclei were removed by centrifugation at 3000g. The
mitochondria-enriched fraction was obtained after centri-
fugation at 30000g (20min) followed by two consecutive
separations on a discontinuous sucrose gradient [15, 25,
35, 45 and 60% sucrose supplemented with 20mM
Tris (pH 7.5) and 5mM EDTA] at 130000g (1h).
Mitochondria accumulated at the interface between the
sucrose layers of 35 and 45% (and/or 25 and 35%).
Mitoribosomes were enriched via separating a cell lysate
by two consecutive kinetic centrifugations, the first on a
step gradient (10–35% glycerol, in steps of 5%) at
250000?g for 2h and the second on a continuous
gradient (10–40% glycerol) at 250000g for 4h. Fractions
enriched in mt-LSU rRNA (as determined by agarose gel
electrophoresis) were pooled. RNA was extracted by a
home-made Trizol substitute (9). Residual DNA was
removed from RNA preparations by either RNeasy
(Qiagen) column purification or digestion with RNase-
(Invitrogen) followed by phenol-chloroform extraction.
Poly(A) RNA was enriched by a passage through
oligo(dT)-cellulose (Amersham), after denaturation of
the aqueous solution at 72?C for 2min and subsequent
chilling on ice.
DNase-treated RNA was separated electrophoretically in
a MOPS/formaldehyde denaturing gel (1.2% agarose, 3%
formaldehyde), side by side with the Riboruler High and
Low Range RNA ladders (0.2 – 6.0kb and 0.1 – 1.0kb,
Fermentas). As a size marker for smaller molecules, we
used single-stranded DNA, which was obtained from
denatured RT-PCR products of 130–440-nt-long rnl
segments. This marker was visualized by hybridization
to a radioactively labeled oligonucleotide (see later in
text). Primers used for RT-PCR (and product sizes)
dp72+dp208 (560nt). As size markers and positive
controls for mono-modules, we used RNAs synthesized
by in vitro transcription of PCR products amplified with
primer pairs dp230+dp216 (module 1) and dp232+dp168
(module 2). Oligonucleotides used as primers and hybrid-
ization probes are listed in Supplementary Table S1. The
electrophoretically separated nucleic acids were blotted on
a nylon membrane (Zeta-Probe, BioRad) and fixed by
baking the membrane at 80?C for 60min. As hybridiza-
tion probes, we used oligo-deoxynucleotides radio-labeled
by T4 polynucleotide kinase in the presence of [g-32P]ATP.
For the detection of antisense transcripts, we used an
oligoribonucleotide probe that was in vitro transcribed
from PCR amplicons that in turn were produced with
primer pairs dp225+dp210 (antisense targeting) and
dp226+dp211 (sense-targeting control); for each primer
pairs, one contained the T7 promoter in addition to
gene-specific sequence. In vitro transcription with T7
RNA polymerase [New England BioLabs (NEB)] was per-
formed in the presence of [a-32P]UTP, for internal
labeling. Membranes were hybridized overnight at 55?C
in either 5? saline sodium citrate (SSC) supplemented
with 5? Denhardt’s solution (0.1% polyvinylpyrrolidone,
0.1% BSA, 0.1% Ficoll 400) and 0.5% sodium dodecyl
sulfate (SDS) when oligonucleotide probes were used or
the ULTRAhyb buffer (Ambion) when RNA probes were
used. Subsequently, membranes were washed twice at
50?C in 2? SSC plus 0.1% SDS (oligonucleotide
probes), or twice at 68?C in 0.1? SSC plus 0.1% SDS
(RNA probes) and visualized using a phosphor-imaging
screen scanned by a Personal Molecular Imager (Bio-
Nucleic Acids Research,2014, Vol.42, No. 42661
intensities were conducted with the Image Lab 4.1
CircRT-PCR and RT-PCR
DNase-treated RNA was incubated with tobacco acid
phosphatase (TAP; Epicenter) and T4 polynucleotide
kinase (PNK; NEB). For circRT-PCR experiment, we
used an unmodified kinase that possesses 30-phosphatase
activity. RNA was diluted to 20ng/mL and circularized
using T4 RNA ligase (Roche). The first strand (cDNA)
was generated with Powerscript reverse transcriptase
of the Creator Smart cDNA library construction kit
(Clonetech) or avian myeloblastosis virus (AMV) reverse
transcriptase (Roche). PCR was performed with the
Takara PCR kit (Bio Inc.), typically for 35 cycles.
Generally, two gene-specific primers were used, but for
certain RT-PCR experiments, amplification was con-
ducted with only one gene-specific primer (for first-
strand synthesis) plus the Smart IV primer that anneals
with the overhanging G residues at the 50end extension of
the first-strand DNA (10). Primer sequences are given in
the Supplementary Table S1. For all RT-PCR experi-
ments, a negative control was performed where no
template RNA was added.
Cloning and sequencing of amplicons
Amplicon termini were rendered blunt with T7 DNA
polymerase and the Klenow fragment of DNA polymerase
I (NEB), agarose gel-purified, phosphorylated with T4
PNK (NEB) and ligated into the vector pBFL6cat,
which is an in-house constructed, small pBlueScript de-
rivative. Libraries of cDNA were cloned into pDNR-
LIB (Clonetech). After transformation into Escherichia
coli DH5a, plasmid DNA was extracted using the
Qiagen 96-well mini-prep kit. Sequencing reactions were
performed with the BigDye Terminator version 3.1 Cycle
Sequencing Kit from Applied Biosystems and sequenced
on an ABI 370 Analyzer.
High-throughput RNA sequencing
Total RNA and mitochondrial RNA-enriched samples
from D. papillatum were depleted of cytosolic 5, 5.8, 18
and 28S rRNA using a series of 50end biotinylated oligo-
nucleotides (IDT) complementary to these rRNAs. For
oligonucleotide design, we used the 5S rRNA sequence
published earlier by others (GenBank accession no.
AY007785) and the 5.8, 18 and 28S rRNA sequences
reported here. The amount of the overabundant mt-LSU
rRNA inmitochondrial RNA preparations wasreduced by
(for oligonucleotides, see Supplementary Table S1). After
streptavidin-coated magnetic beads (MyOne C1 and/or M-
270 Dynabeads; Invitrogen). The library PA was made
from cytosolic rRNA-depleted total RNA enriched for
poly(A) RNA (see earlier in text), and the libraries F1
and F2 from mitochondrial RNA, following the supplier-
recommended protocol devised for strand-specific RNA-
Seq libraries and using the ScriptSeqTM
Library Preparation Kit (Epicentre). The difference
between the F1 and F2 libraries is that for F2 the RNA
fragmentation step was omitted to minimize further frag-
mentation of short RNA molecules. The F1, F2 and PA
libraries were constructed and paired-end-sequenced
(2?101nt; Illumina HiSeq 2000) at the commercial tech-
nology platform Macrogen (Korea). According to the
service provider, spurious antisense reads are below 2%
and typically at 1% with the methodology used. For the
GG library, we used RNA extracted from a subcellular
fraction enriched in mitoribosomes. The library was con-
structed using the TruSeq Stranded Total RNA Sample
Prep kit (Illumina) following the suppliers instructions
and paired-end sequenced (2?250nt; Illumina MiSeq) at
the Genome Quebec Innovation Center in Montreal.
RNA-Seq data analysis
From the libraries F1, F2 and PA, we obtained between
50 and 70 Mio raw fastq reads of 101-nt length, and from
the library GG ?3 Mio raw reads of 250-nt length
(Supplementary Table S2). Reads corresponding to cyto-
solic rRNAs were filtered out using Geneious 5.6
(Biomatters, New Zealand) leaving 40% (F1), 33% (F2),
95% (PA) and 15% (GG) reads. Adapters were removed
from the 50and 30termini of reads with cutadapt version
article/view/200). As parameters, we used a sub-sequence
of 12nt at the 30end or 50end of the 50and 30adapters,
respectively, to allow for partial adapter sequence in the
reads. The error rate was set to 0.1. Cutadapt was also
used for quality clipping with a quality threshold of 20.
Reads <20nt were discarded. Statistics for the cleaning
steps of reads are compiled in Supplementary Table S2.
The data set used for further analysis was built from
paired reads; reads that lost their mate during filtering
were discarded using an in-house script. As a reference on
which to map the read pairs to, we constructed a set of
including the expected intermediary molecules from RNA
processing, trans-splicing and RNA editing. Paired reads
were mapped onto each of these reference transcripts
shtml). Bowtie was executed independently on each refer-
ence transcript and for each sense (forward and reverse)
using the corresponding –norc/–nofw option. Read pairs
where only one mate maps to the reference transcript or
which are discordant (i.e. not mapping to the same strand
or where the forward mate maps downstream of the reverse
mate) were discarded from the alignment. Finally, using in-
house scripts, pairs were removed that do not overlap with
any of the reference transcripts, have a mapping quality
<30, or a number of deletions ?3. From libraries F1 and
F2, we removed read pairs representing insert sizes ?165nt
that originate from spurious dp72-amplification products
primed by residual, contaminating dp72, an oligonucleotide
that was used during sample preparation for the removal of
cytosolic rRNA. Output files in sam format were subse-
quently transformed into ‘.bam’ files with SAMtools
version 1.4 (http://samtools.sourceforge.net/). Alignments
were visualized with tablet version 1.13.05.17 available at
URL http://bioinf.scri.ac.uk/tablet/ (11). The statistics for
2662Nucleic Acids Research, 2014,Vol.42, No. 4
the length distribution of the poly(A) tail and the poly(U)
tract were calculated using an in-house script, which filters
fastq files or the ‘sam‘ alignment file, respectively, for reads
that overlap the upstream and/or downstream modules by
a minimum number of nucleotides (typically 10–12nt) and
which contain a minimum number of homopolymeric nu-
cleotides (typically 4nt). The exact parameters are given in
the figure legends.
RNA secondary structure modeling
We searched for conserved primary sequence and second-
ary structure motifs of mitochondrial LSU rRNAs by
using the phylogeny-based consensus model available at
the Comparative RNA Web (http://www.rna.ccbb.utexas.
edu) (12). Thermodynamic folding was predicted by
RNAfold 2.0 (13). Identified conserved motifs served as
anchors for manual folding of the entire sequence to fit the
model. Conventional nomenclature for sequential num-
bering of secondary structure elements has been used
[e.g. (14)]. The secondary structure was drawn with
XRNA 1.1.12 (http://rna.ucsc.edu/rnacenter/xrna/xrna.
html) and finalized using CorelDRAW X4.
Identification of mt-LSU rRNA and its gene in Diplonema
The 352-nt-long 30-terminal portion of mt-LSU rRNA
from Diplonema was early on recognized as a top candi-
date for an unidentified rRNA, due to its extremely high
abundance (representing 1% of all ESTs) in cDNA
libraries constructed from total poly(A) RNA [(7);
GenBank record JQ302963]. This RNA species carries
an A tail of >25nt and as we show here, is a precursor
transcript of mt-LSU rRNA (see section later in text). For
identification of mt-LSU rRNA from Diplonema, neither
BLAST nor Rfam searches, nor comparison with mito-
chondrial rRNA sequences from other taxa was success-
ful. Counterparts from euglenozoan species (i.e. the
euglenid Euglena gracilis and kinetoplastids) not only
are as highly divergent as mt-LSU from Diplonema but
also display an extremely dissimilar nucleotide compos-
Euglena versus ?50% in Diplonema).
Mature Diplonema mt-LSU rRNA was first detected by
northern hybridization, using an oligonucleotide as a
probe that is specific for the 30-terminal rnl portion.
In total RNA, this probe lights up a major band of
(Figure 1A, right panel). The same band pattern is seen
when using the entire 30-terminal piece as a probe
(Supplementary Figure S1). The 0.9-kb band is most
likely the mature mt-LSU rRNA, whereas the smaller
one, present in >20-times lower steady-state concentra-
tion, corresponds to the polyadenylated 30-terminal
portion. A size of 0.9kb may appear small for mt-LSU
rRNA, but the kinetoplastid counterpart is not much
longer [1.1kb; GenBank acc. no. TRBKPGEN; (15)]. In
poly(A) RNA, the RNA species of 0.4kb is highly
enriched, whereas that of 0.9kb is nearly undetected
[Figure 1A, lane ‘poly(A)’; Supplementary Figure S1],
Figure 1. Mitochondrial LSU rRNA of Diplonema. (A) Northern blot
hybridization. Lane 1, in vitro transcription product of rnl module 1
(540nt); lane 4, in vitro transcription product of rnl module 2 (359nt;
synthetic RNAs are 6 and 7nt longer than the corresponding modules);
lanes 2, 3, 5 and 6, total RNA (?5mg); lanes 7 and 8, poly(A) RNA
(?0.5mg) extracted from whole cells. RNA in lanes 2 and 5 is from one
preparation; that in lanes 3 and 6 is from an independent preparation.
Blotted RNA was probed with radioactively labeled oligonucleotides
dp216 (lanes 1–3) and dp218 (lanes 4–8) that target module 1 and
module 2 of rnl, respectively. Bands represent the mature mt-LSU
rRNA (?900nt), mono-module 1 transcripts (?550nt; the weak band
in lane 3 is clearly visible on the original image), mono-module 2 tran-
scripts (?450nt) and presumptive end-processing intermediates of
single-module transcripts. The size markers are indicated on the left.
The signal ratio of mt-LSU rRNA versus mono-module 1 transcripts
varies noticeably from one preparation to another; it is 100:1 in lane 2
and 60:1 in lane 3. The signal ratio of mt-LSU rRNA versus mono-
module 2 transcripts (lanes 5 and 6; total RNA) is ?20:1. This ratio is
?1:5 to ?1:17 in poly(A)-enriched RNA (lanes 7 and 8), a variation
depending on the particular oligo(dT) pull-down experiment. Notably,
the steady-state of mono-module 1 transcript is lower than that of
mono-module 2. The same is seen in RNA-Seq experiments (see
Figure 4). (B) Upper part, schematic sequence of mtLSU rRNA. The
U-tract between modules 1 and 2 (black box) is not encoded by
northern hybridization probes dp216 and dp218 anneal are indicated.
Lower part, coding regions of mt-LSU rRNA on mitochondrial
chromosomes. Modules 1 and 2 are contained in cassettes of B-class
chromosomes, but oriented in opposite direction relative to the
chromosome’s constant region [indicated as B(+) and B(?), see text].
Non-coding regions within the cassettes (‘unique flanking regions’) are
shown in dark gray. The constant region of chromosomes (light gray) is
?95% identical across all B-class chromosomes (7). The black part of
the constant region is also present in A-class chromosomes (‘shared
Nucleic Acids Research,2014, Vol.42, No. 42663
which is in accordance with evidence from cDNA
sequencing. Apparently, mature mt-LSU rRNA has a
shorter A tail than the 352-nt RNA species, so that only
a small fraction of is pulled down during the poly(A) en-
The 50-terminal region of mt-LSU rRNA was identified
by RT-PCR applied to circularized RNA (circRT-PCR)
using a pair of ‘divergent’ primers annealing with the mol-
ecule’s 30end region (see ‘Methods’). Subsequent cloning
and sequencing revealed a 534-nt-long stretch upstream of
the 30end portion of rnl. As only two such clones were
obtained (in multiple experiments), we confirmed their au-
thenticity by northern hybridization. An oligonucleotide
specific to the presumed 50-terminal portion lights up the
0.9-kb product and in addition a faint 0.5-kb band that
corresponds to the 50-terminal portion alone (Figure 1A,
left panel). RNA-Seq data (later in text) provided the
ultimate confirmation for the 534-nt-long sequence being
the 50moiety of mt-LSU rRNA in Diplonema.
The most remarkable sequence feature of Diplonema
mt-LSU rRNA is a run of ?26 uridines (Us) immediately
upstream of its 30moiety (Figure 1B, upper part; Table 1
and Supplementary Figure S2). This homopolymer tract
was confirmed independently by RT-PCR using a primer
pair that anneals upstream and downstream of this tract
(Supplementary Table S5 and Supplementary Figure S3).
The observed U-tract length varies by about ±3, which is
apparently due to experimental rather than biological
variation (Supplementary Table S6); errors probably
occur during PCR amplification or the sequencing
reaction itself, as commercial RT-enzymes have high syn-
thesis fidelity. We posit that this long U-tract is the reason
why RT-PCR-based experiments yielded extremely low
numbers of reads. This bias is observed also in RNA-
Seq (see later in text).
The gene specifying Diplonema mt-LSU rRNA was pin-
pointed by mapping the rRNA sequence on the available
mtDNA sequence, revealing two previously unannotated
coding regions embedded in cassettes of separate B-class
chromosomes (for a definition of ‘cassette’, see legend of
Figure 1B). These coding regions are referred to as rnl
modules 1 and 2 (Figure 1B, lower panel). With 534bp
length, rnl module 1 is the longest among all known gene
modules in Diplonema mtDNA, whereas rnl module 2
(352bp) is of average size. Gene module 2 of rnl lacks a
30terminal A-homopolymer stretch, which is obviously
added by post-transcriptional polyadenylation. Also
absent from both the module 1 and module 2-coding
regions is a terminal T tract, otherwise present in the
center of the mt-LSU rRNA cDNA sequence. The
sequence of gene module 1 ends precisely upstream,
whereas that of gene module 2 starts exactly downstream
of the U tract in mt-LSU rRNA. Therefore, these non-
encoded nucleotides must be added post-transcriptionally,
resulting in U-insertion RNA editing. This is by far the
longest stretch of non-encoded Us seen in Diplonema
mitochondria and also the largest number of nucleotides
added at a single editing site ever observed.
2D structure modeling of mt-LSU rRNA
The secondary (2?) structure of the 30moiety from
Diplonema mt-LSU rRNA was modeled based on com-
parison with the mitochondrial consensus structure—the
homologs from kinetoplastids and E. gracilis are too di-
vergent for a meaningful comparison of covariant residues
(Figure 2A). Only domains IV to VI [as defined for E. coli
(Figure 2B)] are conventional, albeit reduced. Domain V
encompasses the peptidyl-transferase center (PTC) and is
the most conserved region of LSU rRNAs. As in many
other reduced mt-LSU rRNAs, the Diplonema molecule
lacks the helices H76-H79 that in E. coli bind the riboso-
mal protein L1 and H83-H86 that associate with 5S
rRNA. Domain VI lacks major parts, and the sequence
that connects H73 and H95 in most other mt-LSU rRNAs
(12,16) is unusually short. Just a few of the universally
conserved sequence motifs are readily recognizable in the
Diplonema molecule, namely, those corresponding to the
basis of helix H90 and its single-stranded junctions to H89
and H93, as well as the terminal loops of helices H80, H92
and H95 (the latter is also known as the a-sarcin/ricin
loop). Nonetheless, domain V of Diplonema mt-LSU
rRNA resembles bacterial 23S rRNA somewhat more
closely than that of kinetoplastids, the latter lacking for
example H97 (17–19).
Domain IV is most likely constituted by the 30third of
the module 1 sequence. We recognize the conserved helices
H69 and H71 with their surrounding single-stranded
regions that are involved in the majority of inter-subunit
contacts with ribosomal SSU and functionally important
interactions with ribosome-bound tRNAs (28). Two other
consensus helices of this domain lack a substantial periph-
eral portion in Diplonema as well as in kinetoplastids and
several other taxa. The structure model places the poly(U)
tract at the 30end of domain IV. Two 4-nt-long purine
stretches upstream in module 1 might base-pair with
poly(U) to form a helix analogous to H61. However,
this region could also remain single-stranded as in the 2?
Table 1. Non-encoded U-tract length of mt-LSU rRNA and its
Transcript structure Mean number of Us
am1, m2, rnl modules 1 and 2; [U]n, uridine-homopolymer of length n;
m1.[U]n, module 1 with 30-terminal U tract; m1.[U]n.m2, LSU-rRNA;
[U]n.m2, module 2 with 5’-terminal U tract; and ?, exact module
terminus not determined. n.d., not identified; /, not observed.
Supplementary Figure S2. Libraries F2, PA and GG display similar
U-tract length as F1 shown here.
cFour clones (dp11056, dp11060, dp11084, dp11088).
dThis type of transcript could not be identified unambiguously.
eSeven clones (dp9540, dp10594, dp11008, dp11009, dp11012, dp11017,
fNot-quality clipped individual reads from the F1 library.
2664Nucleic Acids Research, 2014,Vol.42, No. 4
model of kinetoplastid and nematode mt-LSU rRNAs
Although we were able to reconstruct a reasonable 2?
structure model of the 30half of mt-LSU rRNA from
Diplonema, folding the 50half of this molecule (domains
I-III) is challenging due to several reasons (but see
Supplementary Figure S7). First, this part of the
molecule is in general moderately conserved. In addition,
comparative modeling was not feasible due to low
sequence similarity between Diplonema mt-LSU rRNA
modeling based on thermodynamic folding leads to an
excessive number of alternatives because the G+C rich
(51%) sequence allows profuse base-pairing possibilities.
As to length and structure, the 50half of mt-LSU rRNA
from Diplonema is more reduced and shorter than
that from kinetoplastids, yet comparably deviant as
that from certain animals as detailed in the Discussion
Deep sequencing and RT-PCR analysis of the rnl
To capture rnl transcripts of Diplonema in a comprehen-
sive way, we performed massively parallel sequencing
(RNA-Seq) of three RNA samples (F1, F2, PA).
Samples F1 and F2 were extracted from a subcellular
fraction enriched in mitochondria; sample PA was
enriched for poly(A) RNA. The applied RNA-Seq
approach involved paired-end library construction by
RNA fragmentation for F1 and PA (but not F2),
random hexamer priming and strand-specific sequencing.
Average fragment (insert) length is 300nt, read length is
101nt and read depth is ?60 Mio reads per sample.
Primer and quality trimming resulted in ?100 Mio
paired reads of ?20-nt length for all three libraries
together (60%). Of these, 1.066 Mio paired reads (1%)
contain rnl sequences. A fourth small library (GG) was
constructed with an RNA sample that was extracted
Figure 2. Putative secondary structure of the mt-LSU rRNA (30moiety) from Diplonema. (A) The structure was modeled according to the
mitochondrial reference sequence and structure (http://www.rna.icmb.utexas.edu). Residues identical to the universal consensus sequence (12,16)
are shown in bold. Domain IV is composed of the 30portion of module 1 (dark gray shading) and the post-transcriptionally added U-tract (black
shading). Domains V and VI are encoded by module 2 (light gray shading). The thin dashed line marks helix 26a (see ‘Discussion’). Base pairing is
indicated as thin lines, thick lines, dots and open circles corresponding to A:U, G:C, G:U and other base pairs, respectively. Residues are
numbered according to nucleotide positions in rnl modules 1 (upstream of U-tract) and 2 (downstream of U-tract). The nucleotide pair
U305:A314 in the module 2 corresponds to a conserved trans Watson-Crick/Hoogsteen pair in the E. coli structure. (B) The 2?structure of
the 30moiety from Diplonema mt-LSU rRNA mapped onto the structure from E. coli LSU rRNA. Helices are numbered according to (14). H95,
a-sarcin/ricin loop. Thick gray and black lines indicate the structure elements present in the Diplonema model [same shading as in (A)]. Triangles
indicate breakpoints in the 30half of fragmented LSU rRNAs from apicomplexan (2,20,21) and dinoflagellate (3,4) mitochondria (light gray
triangles), several green algal mitochondria (gray triangles; 22–25), and the kinetoplastid (26) and euglenid (27) cytosol (black triangles). It is
noteworthy that among all known cases of discontinuous domain-IV LSU rRNA (apicomplexans and dinoflagellates), none is split in the 30half
Nucleic Acids Research,2014, Vol.42, No. 4 2665
from a mitoribosome-enriched subcellular fraction of
Diplonema. Reads of this library were used to characterize
the mitoribosome-associated LSU rRNA. Information on
RNA-Seq data are compiled in Supplementary Tables S2
First, we mapped read pairs to the sequence of mt-LSU
rRNA. Read coverage of the mitochondrial libraries is
depicted in Supplementary Figure S4A. Detailed inspec-
tion of coverage showed that only 14 (quality-clipped)
reads span completely the internal U-tract and include
?10nt of both adjacent modules, although ?150000
reads map to the module-1/module-2 junction region;
the majority of U-tract containing reads maps to either
the 30end of module 1 or the 50end of module 2
(Supplementary Table S4). This bias is due to low
sequence quality in homopolymer tracts. More than
99.9% reads have quality values <20 from the 13th
U-tract position on, so that all sequence beyond this
position is removed by quality clipping during the
read preprocessing step (Supplementary Figure S5).
Therefore, we used the inferred ‘inserts’ (i.e. the interval
inferred from paired-end reads) instead of reads for
mapping onto mt-LSU rRNA (Figure 3; for logarithmic
scale, see Supplementary Figure S4A) and most of the
other analyses described later in text.
For targeted detection of long transcripts and accurate
mapping of their termini, we conducted in addition RT-
PCR using specific primers that anneal within module 1 or
module 2 of rnl. In experiments with circularized RNA,
primers point in divergent direction, otherwise they are
oriented in convergent fashion.
Maturation intermediates of rnl transcripts
To characterize intermediates of mt-LSU rRNA, we
mapped RNA-Seq inserts from the mitochondrial libraries
F1 and F2 to three virtual reference transcript sequences,
which represent the primary transcript of each individual
module and a trans-spliced, edited and polyadenylated
transcript. LSU rRNA precursors were also characterized
by RT-PCR and circRT-PCR experiments.
End-processing intermediates are of two types, tran-
scripts including an rnl module plus either both adjacent
non-coding regions or a single adjacent region retained on
either end (Figure 4). Fully processed module transcripts
are seen as well (Table 2). Notably, not only fully pro-
cessed modules but also end-processing intermediates
engage in trans-splicing. For example, we detected a tran-
script with joined modules 1 and 2, whose 30end still has
non-coding sequence attached. Mapping of RNA-Seq
data to unprocessed reference sequences is shown in
Supplementary Figures S4B and C.
RNA editing almost certainly takes place before trans-
splicing, because neither RNA-Seq nor RT-PCR detected
reads where the 30end of rnl module 1 is immediately
upstream-adjacent to the 50end of module 2. Uridine
residues are most likely added 30to module 1 and not
50to module 2 according to circRT-PCR experiments
(Table 1). For Diplonema cox1, U-appendage editing of
the module upstream of the editing site has been validated
of RNAs did we observe upstream modules with Us
appended at the 30end, but under no condition was the
Figure 3. Coverage of Diplonema mt-LSU rRNA by RNA-Seq data. Mapping of inferred inserts from two mitochondrial libraries, F1 (dark gray)
and F2 (light gray). Vertical scales, counts of inserts. Cartoon in the center, schematic representation of the virtual reference transcript to which
inserts were mapped. Unfilled boxes labeled m1 and m2, rnl modules 1 and 2, respectively. Black box, poly(U) of ?26 length added by RNA editing;
dashed box upstream module 1, unique flanking region; gray line, transcribed constant region of B-class chromosomes (see Figure 1B). ‘A...A’,
A-tail. It should be noted that inserts (and reads) cannot be mapped unambiguously beyond ?80nt upstream and downstream of modules because
these regions are nearly identical in sequence with those from other modules residing on B-class chromosomes. Stacked-area chart on the right side,
coverage by sense (upper area) and antisense (lower area) inserts, respectively. The bar charts to the left represent the total number of reads covering
the corresponding area in the stacked-area chart. The scales for sense and antisense transcripts differ by a factor of 30. Sharp drop-off in antisense
read coverage ?100nt upstream of rnl module 1 (a zone corresponding to the constant region of B-class chromosomes) reflecting a discrete 30end of
antisense RNAs. Uneven read coverage along the sequence is probably due to sequence bias.
2666 Nucleic Acids Research, 2014,Vol.42, No. 4
downstream module found with Us attached to its
RNA editing intermediates of rnl that have excess or def-
icit Us cannot be determined reliably, because sequences
containing homopolymers are of low quality, especially
those where the U-tract is at the 50end of the read
(Supplementary Figure S5). At present, the two following
editing scenarios remain indistinguishable: (i) uncon-
trolled addition of numerous Us and subsequent precise
trimming as is the case for U-insertion editing of trypano-
some mitochondria (29) and (ii) controlled addition of the
exact number of nucleotides.
The A-tail of module 2-containing rnl transcripts
displays substantial differences in length (Table 3).
Mono-module 2 is polyadenylated by addition of up
to 90 As, whereas trans-spliced transcripts have predom-
incorporated in the mitoribosome has virtually no
A-tail. These differences are seen consistently in all three
experimental approaches used in this study. In northern
hybridization, we observe different signal ratios of mono-
module 2 versus mature rRNA. The ratio in total RNA is
?1:20, but nearly inverse in the poly(A) RNA-enriched
fraction (see Figure 1 and Supplementary Figure S1). In
circRT-PCR experiments, A-tails of rnl mono-module 2
transcripts are up to ?50-nt-long, whereas those of the
Finally, A-tail size distributions in RNA-Seq data from
total-cell poly(A) RNA exhibit a broad crest up to 80nt,
those from total mitochondrial RNA peak at ?20nt
dominant maximum at 0nt (Table 3 and Supplementary
Figure S6). The possible biological significance of this
variation in A-tail length will be examined in the
Antisense RNA covering module junction and editing
site of mt-LSU rRNA
We posited earlier that trans-splicing and RNA editing of
the mitochondrial protein-coding gene cox1 in Diplonema
might be instructed by antisense RNAs. Preliminary
evidence for antisense transcripts of a protein-coding
gene came from targeted RT-PCR experiments (8).
However, the yield of products was low and the inform-
ative sequence obtained (after subtraction of primer
sequences) was only a few nucleotides long. Here we
re-examine whether guiding antisense RNAs exist in
Figure 4. Maturation intermediates of rnl transcripts. Cartoons depict
schematically the regions where maturation processes take place. White,
hatched and black boxes indicate modules and the A tail, non-coding
regions and the U-tract at the module junction, respectively. Bar charts
beneath cartoons show the number of paired reads from the mitochon-
drial libraries F1 (medium gray) and F2 (light gray), and the
mitoribosome library GG (dark gray) that map to the designated
regions. The arrow below the bars specifies reads in sense (pointing
to the right) and antisense (pointing to the left) direction. Counted
reads suffice the following criteria: within a 100-nt-long region
around the maturation site, reads (forward or reverse read of
mapped read pairs) are required to cover at least 55 nt of this
window, i.e. overlap boundaries (between modules and other regions)
by at least 5nt. The proportion of immature rnl transcripts in the
library GG serves as a measure for mitoribosome enrichment.
Table 2. End processing intermediates of rnl modules transcriptsa
ModuleMethodologyNumber of clones/inserts
representing intermediate type
Module 1 (?534nt) circRT-PCR/
Module 2 (?352nt) circRT-PCR
aNumber of observed clones in RT-PCR experiments or inserts in
RNA-Seq libraries F1 and F2 (latter data taken from Figure 4).
Symbols and abbreviations used: —, non-coding adjacent region; m,
rnl-module 1 or 2; ^m^, module end-processed at both termini; ?m,
m?, nature of module’s 50end or 30end, respectively, is unknown (may
be unprocessed or processed); /, not observed.
bThree clones (dp11008, dp11034, dp11059); length of non-coding
regions is 324, 20 and 69nt, respectively.
cThree clones (dp9411, dp9613, dp11051l); length of non-coding regions
is 163, 22 and 3nt.
dLow probability of observation, because the libraries have an insert
size average of 300nt.
eThree clones (dp9408, dp10574b, dp10586).
fTwo clones (dp9411, dp9613).
gTwo clones (dp10439rb, dp10526a).
Nucleic Acids Research,2014, Vol.42, No. 42667
Diplonema mitochondria by focusing on one of the most
highly expressed mitochondrial genes, rnl, and by exploit-
ing strand-specific RNA-Seq data.
Strikingly, putative antisense transcripts of mt-LSU
RNA are detected at ?2.5%, which is significantly
above background (see ‘Materials and Methods’ section
and Figure 3A, lower panel). The existence of such
(Supplementary Figure S3 and Supplementary Table S5).
Remarkably, antisense read coverage drops off sharply
?100nt upstream of module 1, a zone corresponding to
the constant region of B-class chromosomes (Figure 3A).
This drop-off reflects a discrete 30end of rnl antisense
RNAs. The same phenomenon is seen in read mapping
shown), which is likewise a first module encoded on a
B-class (+) chromosome (see Figure 1B). Whether the
rnl-antisense 30terminus is generated by transcription
termination or processing remains to be investigated.
In contrast to their 30end, the 50terminus of rnl antisense
RNAs appears variable in the read coverage profile. We
attempted to determine the length of these transcripts by
northern experiments using either single-stranded oligo-
deoxynucleotides or in vitro transcribed RNAs as a
probe, but the signals were extremely weak (not shown).
Antisense transcripts might be a heterodisperse assemblage
of different length that do not form a homogenous band in
gel electrophoresis; an already weak signal spread out
instead of concentrated in a band would be difficult to
detect by northern hybridization. Neither could we find
the potential gene encoding the anti-mt-LSU RNA in the
available ?250kb mtDNA nor in the currently draft
assembly of nuclear DNA. It is possible that the gene was
not found because it is encoded in a yet unsequenced
genomic region, or alternatively, because there is no such
gene as elaborated in the ‘Discussion’.
Putative antisense transcripts of unprocessed modules
are also seen in RNA-Seq data. These RNAs apparently
originate from bi-directional transcription of rnl-module-
and C). Transcription in Diplonema mitochondria starts
in the shared, constant region of chromosomes located
opposite to modules (8). As modules are oriented in
either sense relative to the shared region (as for example
rnl modules 1 and 2 Figure 1B), the promoter(s) must be
able to drive transcription of both strands.
Regulation of rnl gene expression in Diplonema
Based on the observed types of rnl-transcript intermedi-
ates, two diametrically opposite maturation pathways of
mt-LSU rRNA can be postulated. One interpretation of
the results is that polyadenylation is a dead-end reaction,
tagging molecules that failed to be trans-spliced or
incorporated into the ribosome (Figure 5A). However,
this view does not explain why only module 2 but not
module 1 is polyadenylated. The other hypothesis, which
we favour, considers that polyadenylation is crucial for
mt-LSU maturation. We posit that module 2 is first
polyadenylated and then deadenylated in two subsequent
steps, with the particular A-tail length being the check-
points for trans-splicing of modules 1 and 2, and then
for assembly of the trans-splicing product into the
ribosome (Figure 5B). This view would explain the differ-
ence in predominant A-tail length of mt-LSU rRNA from
total mitochondrial RNA extractions (?20nt) versus
mitoribosome-extracted RNA (?0nt) as follows. The
former RNA preparation may contain mainly rRNA
that is not incorporated into the ribosome. Still, we
cannot fully exclude technical variation because different
protocols were used for constructing the two libraries.
The various biochemical reactions involved in the
expression of Diplonema mt-LSU rRNA, module-end pro-
cessing, adenylation, uridylation, trans-splicing and po-
tentially A-tail trimming of the molecule’s 30end, must
be catalyzed by an assortment of activities (ribonuclease,
polymerase and ligase), as well as trans-factors that guide
trans-splicing and editing. Traditionally, multi-step bio-
chemical pathways are pictured as a cascade of catalytic
steps, where the product of a given reaction is the sub-
strate for the subsequent step. However, in Diplonema
mitochondria, most transcript maturation-steps proceed
independently from one another in the sense that the
reaction at one extremity of the transcript is not influenced
by the nature of the other extremity. This excludes a
strictly linear, assembly line-like maturation pathway in
this system. Parallelization, thought to accelerate this
multi-step process, might be achieved by a molecular
machine that combines all activities in one (‘processo-
Table 3. Poly(A) tail length of rnl transcriptsa
Transcript (structure)Poly(A) tail
RNA-Seq: mean length
(major peak position)
rnl mono-module 2
46 (?60) (PA)d
33 (?20) (F1)f
0 (0) (GG)g
aSymbols used: m1, m2, rnl-modules 1 and 2; m1.Us.m2, mt-LSU
rRNA sequence including (from 50to 30) module 1, 20–30 Us, and
module 2; [A]n, adenine homopolymer of length n. Transcripts length
is ?900 and ?353nt for m1.Us.m2[A]n, and m2[A]n, respectively.
Supplementary Figure S6.
cFive clones (dp10594, dp11008, dp11009, dp11012, dp11017).
dLibrary PA was made from RNA that contains predominantly rnl
northern hybridization experiments; see Figure 1A, lane ‘poly(A)’].
eFifteen clones from the series dp104xx; e.g. dp10411r.
fLibrary F1 was made from RNA that contains predominantly mature
mt-LSU rRNA (trans-spliced rRNA:m2=?20:1 according to northern
hybridization experiments; see Figure 1A, lane ‘total’, probe m2). Two
mate pairs from this library span rnl modules 1 plus 2 (reads
1203:11003:25874 and 1216:19505:7846).
gLibrary GG was made from RNA that was extracted from a
subcellular fractions enriched in mitoribosomes. Contamination with
rnl transcripts not assembled in the ribosome is estimated at ?0.1%
(see Figure 4).
2668Nucleic Acids Research, 2014,Vol.42, No. 4
edito-spliceosome’), i.e. one that would properly position
guiding factors relative to its catalytic domains and allow
that the two extremities of a given transcript are sculpted
in an independent fashion and in no particular order. The
only steps where the nature of the ‘other’ end seems to
matter in mt-LSU rRNA maturation of Diplonema is
polyadenylation or deadenylation, which, according to
the two above pathway hypotheses, appear to be the
‘rubbish’ or ‘quality’ stamps of molecules.
In contrast to the here proposed integrated multi-
functional complex in Diplonema mitochondria, the
current view of kinetoplastids mitochondrial (m)RNA
maturation postulates the sequential action of two major
complexes, each having dedicated functions. The RNA
editing core complex conducts cleavage of pre-mRNA at
the editing site, removal or addition of Us and resealing of
the transcript, whereas the mitochondrial RNA-binding
complex 1 recruits guide RNAs and interfaces with
gRNA processing and mRNA tailing [reviewed in (30)].
We detected two types of antisense RNAs, anti-rnl-mono-
modules and anti-mt-LSU rRNA transcripts. Anti-mono-
module transcripts most likely arise by bidirectional
transcription of chromosomes, as the promoter(s) in the
shared region must accommodate modules encoded on
the plus and the minus strand [see Figure 1B and (8)].
The observed higher steady-state concentration of the rnl
sense transcript could be achieved by either an elevated
transcription rate in sense direction or faster degradation
controlled strand-dependent transcript regulation, whose
nature is yet to be unraveled.
The origin of anti-mt-LSU rRNA is less obvious, as a
corresponding gene has not been detected. Either the
gene is encoded in yet unsequenced portions of the mito-
chondrial or nuclear genomes or alternatively, no such
gene exists in Diplonema. The antisense RNA might be
transcribed from mature mt-LSU rRNA and inherited
epigenetically from generation to generation. Antisense
transcription templated by mt-LSU rRNA would require
an RNA-dependent RNA polymerase (RdRp). As this
activity has broad taxonomic distribution (31–35), the
Diplonema nuclear genome might well encode a mitochon-
drion-targeted enzyme. Epigenetic inheritance of RNAs
has precedents as well, for example, in ciliates (36) and
C. elegans (37), where RNAs transmitted to daughter
cells are involved in genome rearrangement and antiviral
Diplonema mt-LSU rRNA is extraordinarily short
With only ?910nt, mt-LSU rRNA of Diplonema is among
the smallest known, but still longer than that of certain
nematodes, bryophytes and rotifers [529–729nt; (38–41)].
It is the module-1 portion (534nt) that is substantially
shorter in Diplonema (and even more in the aforemen-
other euglenozoans and heteroloboseans [?730nt in
kinetoplastids (e.g. GenBank accession no. NC_000894),
>800nt in Euglena (42), and 1485nt in Naegleria
(GenBank accession no. AF288092)].
Diplonema rnl sequence into the consensus 50-half 2?
structure of mt-LSU rRNA
problems include low conservation, absence of compara-
tive data from close relatives and the possibility to build
is challenging. The
Figure 5. Maturation process of mt-LSU rRNA in Diplonema mitochondria. For clarity, the cartoon disregards end-processing of module 1 and
module 2 precursor transcripts. m1, m2, rnl module 1 and 2, respectively. U, post-transcriptionally-added U tract. AA, AAAA, poly(A) tails of
?20nt or ?40–90nt length, respectively. The gray-filled shape symbolizes the mitoribosome. (A) Hypothetical pathway where polyadenylated rnl-
transcripts represent dead ends instead of maturation intermediates. (B) Alternative pathway (preferred hypothesis) where the polyadenylation status
plays a key role in mt-LSU rRNA maturation: a poly(A)tail length of ?20 As signals a check point for trans-splicing, and absence of an A-tail from
the trans-spliced product is a requirement for incorporation of the transcript into the mitoribosome.
Nucleic Acids Research,2014, Vol.42, No. 4 2669
numerous alternative structures with this G+C-rich
sequence, making selection of the single most likely
model difficult. For illustration, one of the multiple
Supplementary Figure S7. With the availability of mt-
LSU rRNAsequences from other diplonemids,
portion of the molecule. Finally, it is conceivable albeit
not likely, that a separate 50mt-LSU rRNA piece exists.
Whereas the mitoribosome-enriched fraction analysed
here contains a highly abundant 350-nt molecule (not
shown), this RNA species lacks 2?structure motifs
typical for mt-LSU rRNA, but instead displays remote
similarity to phylogenetically conserved mt-SSU rRNA
signatures [helices h18, h44 and h45; numbering as in
(28)]. Whether this molecule represents indeed mt-SSU
RNA is currently uncertain, because its 50tier consists
virtually exclusively of Gs and Ts impeding meaningful
secondary structure modeling, and its length is much
shorter than ever reported for this rRNA. These issues
could be re-examined rigorously once a protocol is avail-
able for isolating pure mitoribosomes from Diplonema
and by sequencing a mitoribosomal library prepared spe-
cifically for small RNAs.
The 30half is the most conserved portion of all mt-LSU
rRNAs. The corresponding 2?structure of Diplonema mt-
LSU rRNA was modeled based on comparison with the
mitochondrial consensus structure—the homologs from
kinetoplastids and E. gracilis are too divergent for a mean-
ingful comparison of covariant residues. Overall, the fold
of domains V and IV is less deviant in Diplonema than in
kinetoplastids, where the PTC-abutting helices H89 and
H91 are considerably truncated. The absent masses of
these two helices appear to be the cause of the positionally
shifted a-sarcin/ricin loop (H95) toward the PTC (19),
seen in the cryo-electron microscopy map of the
mitoribosome from Leishmania tarentolae. We posit that
the extremely short single-stranded segment between
helices H73 and H95 in Diplonema mt-LSU rRNA
induces an even more pronounced overall shift of H95
together with H89 and H91 and stronger domain V/IV
compaction in the mitoribosome.
models is shownin
Role of extensive U-‘insertion’ editing in mt-LSU rRNA
mitochondria is the only example of a massively edited
rRNA and represents the most extensive editing ever
observed at a single site. Other cases of rRNA editing
that mostly restore secondary structure elements and
conserved sequence motifs (43,44). In kinetoplastids, mt-
rRNAs are virtually never edited. Eukaryotic cytosolic
rRNAs are chemically modified [guided by small nucleolar
RNAs; ref. (45)], but sensu stricto RNA editing has not
been described for these molecules.
The region occupied by the U-tract in our model of
Diplonema mt-LSU rRNA corresponds to the 30half of
H61 in the E. coli structure, a helix that plays an import-
ant role in the ribosome. The part of this helix abutting
our knowledge,LSU rRNAof Diplonema
H64 ensures correct positioning of the SSU/LSU-connect-
ing domain IV (14,16,28), whereas the part adjacent to
H72 is deeply embedded in the ribosome (as are H72
According to a most recent 2?structure model of LSU
rRNA (46), the segment corresponding to the six 30ter-
minal nucleotides in the U-tract together with the three
first nucleotides in module 2 constitute the 30half of the
newly proposed helix H26a. The corresponding 50half of
this helix is a stretch traditionally modeled as single-strand
connecting H26 and H47 in domain II. Helix 26a
is thought to be a pivotal structural element of the
proposed core domain 0, to which the traditional
domains I-VI would be rooted. With the U-tract not
only substituting the 30half of H61 but also being part
of H26a, RNA editing of Diplonema mt-LSU rRNA
would be function-critical.
GenBank KF633465, KF633466, KF633467, KF633468.
Supplementary Data are available at NAR Online.
The authors acknowledge M. Aoulad-Aissa for excellent
technical assistance and thank M.W. Gray (Dalhousie
University, Halifax, Canada) for help in modeling the
rRNA secondary structure and B.F. Lang (Universite ´ de
Montreal) for reading the article. G.B. designed and
supervised the study. G.N.K. conducted RT-PCR. Both
G.N.K. and M.V prepared RNA samples for RNA-Seq.
Isolation of mitochondria and mitoribosomes, secondary
structure analyses and cytosolic rRNA depletion was per-
formed by M.V. Preliminary RNA-Seq data analysis was
conducted by G.B. and M.V. S.M. performed the detailed
data analyses including read mapping and statistics. All
authors contributed to the final manuscript version.
Canadian Institute for Health Research [CIHR, grant
MOP-79309; to G.B.]; Ph. D. scholarship from the
Programme Canadien de Bourses de la Francophonie
(PCBF; to G.N.K.). Funding for open access charge:
Canadian Institute for Health Research.
Conflict of interest statement. None declared.
1. Gray,M.W., Lang,B.F. and Burger,G. (2004) Mitochondria of
protists. Annu. Rev. Genet., 38, 477–524.
2. Feagin,J.E., Harrell,M.I., Lee,J.C., Coe,K.J., Sands,B.H.,
Cannone,J.J., Tami,G., Schnare,M.N. and Gutell,R.R. (2012) The
fragmented mitochondrial ribosomal RNAs of Plasmodium
falciparum. PLoS One, 7, e38320.
2670Nucleic Acids Research, 2014,Vol.42, No. 4
3. Jackson,C.J., Norman,J.E., Schnare,M.N., Gray,M.W.,
Keeling,P.J. and Waller,R.F. (2007) Broad genomic and
transcriptional analysis reveals a highly derived genome in
dinoflagellate mitochondria. BMC Biol., 5, 41.
4. Jackson,C.J., Gornik,S.G. and Waller,R.F. (2012) The
mitochondrial genome and transcriptome of the basal
dinoflagellate Hematodinium sp.: character evolution within the
highly derived mitochondrial genomes of dinoflagellates. Genome
Biol. Evol., 4, 59–72.
5. Gillespie,D.E., Salazar,N.A., Rehkopf,D.H. and Feagin,J.E.
(1999) The fragmented mitochondrial ribosomal RNAs of
Plasmodium falciparum have short A tails. Nucleic Acids Res., 27,
6. Adler,B.K., Harris,M.E., Bertrand,K.I. and Hajduk,S.L. (1991)
Modification of Trypanosoma brucei mitochondrial rRNA by
posttranscriptional 3’ polyuridine tail formation. Mol. Cell. Biol.,
7. Vlcek,C., Marande,W., Teijeiro,S., Lukesˇ,J. and Burger,G. (2011)
Systematically fragmented genes in a multipartite mitochondrial
genome. Nucleic Acids Res., 39, 979–988.
8. Kiethega,G.N., Yan,Y., Turcotte,M. and Burger,G. (2013) RNA-
level unscrambling of fragmented genes in Diplonema
mitochondria. RNA Biol., 10, 301–313.
9. Lang,B.F. and Burger,G. (2007) Purification of mitochondrial and
plastid DNA. Nat. Protoc., 2, 652–660.
10. Shen,Y.-Q., O’Brien,E.A., Koski,L., Lang,B.F. and Burger,G.
(2009) EST databases and web tools for EST projects.
In: Parkinson,J. (ed.), Methods in Molecular Biology: Expressed
Sequence Tags (ESTs), Vol. 533. Humana Press, Totowa, NJ.
11. Milne,I., Stephen,G., Bayer,M., Cock,P.J., Pritchard,L., Cardle,L.,
Shaw,P.D. and Marshall,D. (2013) Using tablet for visual
exploration of second-generation sequencing data. Brief
Bioinform., 14, 193–202.
12. Cannone,J.J., Subramanian,S., Schnare,M.N., Collett,J.R.,
D’Souza,L.M., Du,Y., Feng,B., Lin,N., Madabusi,L.V.,
Muller,K.M. et al. (2002) The comparative RNA web (CRW)
site: an online database of comparative sequence and structure
information for ribosomal, intron, and other RNAs. BMC
Bioinform., 3, 2.
13. Lorenz,R., Bernhart,S.H., Honer Zu Siederdissen,C., Tafer,H.,
Flamm,C., Stadler,P.F. and Hofacker,I.L. (2011) ViennaRNA
Package 2.0. Algorithms Mol. Biol., 6, 26.
14. Ban,N., Nissen,P., Hansen,J., Moore,P.B. and Steitz,T.A. (2000)
The complete atomic structure of the large ribosomal subunit at
2.4 A resolution. Science, 289, 905–920.
15. Eperon,I.C., Janssen,J.W., Hoeijmakers,J.H. and Borst,P. (1983)
The major transcripts of the kinetoplast DNA of Trypanosoma
brucei are very small ribosomal RNAs. Nucleic Acids Res., 11,
16. Mears,J.A., Cannone,J.J., Stagg,S.M., Gutell,R.R., Agrawal,R.K.
and Harvey,S.C. (2002) Modeling a minimal ribosome based on
comparative sequence analysis. J. Mol. Biol., 321, 215–234.
17. de la Cruz,V.F., Simpson,A.M., Lake,J.A. and Simpson,L. (1985)
Primary sequence and partial secondary structure of the 12S
kinetoplast (mitochondrial) ribosomal RNA from Leishmania
tarentolae: conservation of peptidyl-transferase structural elements.
Nucleic Acids Res., 13, 2337–2356.
18. Sloof,P., Van den Burg,J., Voogd,A., Benne,R., Agostinelli,M.,
Borst,P., Gutell,R. and Noller,H. (1985) Further characterization
of the extremely small mitochondrial ribosomal RNAs from
trypanosomes: a detailed comparison of the 9S and 12S RNAs
from Crithidia fasciculata and Trypanosoma brucei with rRNAs
from other organisms. Nucleic Acids Res., 13, 4171–4190.
19. Sharma,M.R., Booth,T.M., Simpson,L., Maslov,D.A. and
Agrawal,R.K. (2009) Structure of a mitochondrial ribosome with
minimal RNA. Proc. Natl Acad. Sci. USA, 106, 9637–9642.
20. Vaidya,A.B., Akella,R. and Suplick,K. (1989) Sequences similar
to genes for two mitochondrial proteins and portions of
ribosomal RNA in tandemly arrayed 6-kilobase-pair DNA of a
malarial parasite. Mol. Biochem. Parasitol., 35, 97–107.
21. Feagin,J.E., Werner,E., Gardner,M.J., Williamson,D.H. and
Wilson,R.J. (1992) Homologies between the contiguous and
fragmented rRNAs of the two Plasmodium falciparum
extrachromosomal DNAs are limited to core sequences. Nucleic
Acids Res., 20, 879–887.
22. Boer,P.H. and Gray,M.W. (1988) Scrambled ribosomal RNA
gene pieces in Chlamydomonas reinhardtii mitochondrial DNA.
Cell, 55, 399–411.
23. Denovan-Wright,E.M. and Lee,R.W. (1994) Comparative
structure and genomic organization of the discontinuous
mitochondrial ribosomal RNA genes of Chlamydomonas
eugametos and Chlamydomonas reinhardtii. J. Mol. Biol., 241,
24. Nedelcu,A.M., Lee,R.W., Lemieux,C., Gray,M.W. and Burger,G.
(2000) The complete mitochondrial DNA sequence of
Scenedesmus obliquus reflects an intermediate stage in the
evolution of the green algal mitochondrial genome. Genome Res.,
25. Fan,J. and Lee,R.W. (2002) Mitochondrial genome of the
colorless green alga Polytomella parva: two linear DNA molecules
with homologous inverted repeat termini. Mol. Biol. Evol., 19,
26. Spencer,D.F., Collings,J.C., Schnare,M.N. and Gray,M.W. (1987)
Multiple spacer sequences in the nuclear large subunit ribosomal
RNA gene of Crithidia fasciculata. EMBO J., 6, 1063–1071.
27. Schnare,M.N. and Gray,M.W. (2011) Complete modification
maps for the cytosolic small and large subunit rRNAs of Euglena
gracilis: functional and evolutionary implications of contrasting
patterns between the two rRNA components. J. Mol. Biol., 413,
28. Yusupov,M.M., Yusupova,G.Z., Baucom,A., Lieberman,K.,
Earnest,T.N., Cate,J.H. and Noller,H.F. (2001) Crystal structure
of the ribosome at 5.5 A resolution. Science, 292, 883–896.
29. Niemann,M., Kaibel,H., Schluter,E., Weitzel,K., Brecht,M. and
Goringer,H.U. (2009) Kinetoplastid RNA editing involves a 3’
nucleotidyl phosphatase activity. Nucleic Acids Res., 37,
30. Hashimi,H., Zimmer,S.L., Ammerman,M.L., Read,L.K. and
Lukes,J. (2013) Dual core processing: MRB1 is an emerging
kinetoplast RNA editing complex. Trends Parasitol., 29, 91–99.
31. Wassenegger,M. and Krczal,G. (2006) Nomenclature and
functions of RNA-directed RNA polymerases. Trends Plant Sci,
32. Cogoni,C. and Macino,G. (1999) Gene silencing in Neurospora
crassa requires a protein homologous to RNA-dependent RNA
polymerase. Nature, 399, 166–169.
33. Ding,B. (2010) Viroids: self-replicating, mobile, and fast-evolving
noncoding regulatory RNAs. Wiley Interdiscip. Rev. RNA, 1,
34. Polashock,J.J. and Hillman,B.I. (1994) A small mitochondrial
double-stranded (ds) RNA element associated with a hypovirulent
strain of the chestnut blight fungus and ancestrally related to
yeast cytoplasmic T and W dsRNAs. Proc. Natl Acad. Sci. USA,
35. Finnegan,P.M. and Brown,G.G. (1986) Autonomously replicating
RNA in mitochondria of maize plants with S-type cytoplasm.
Proc. Natl Acad. Sci. USA, 83, 5175–5179.
36. Bracht,J.R., Fang,W., Goldman,A.D., Dolzhenko,E., Stein,E.M.
and Landweber,L.F. (2013) Genomes on the edge: programmed
genome instability in ciliates. Cell, 152, 406–416.
37. Rechavi,O., Minevich,G. and Hobert,O. (2011) Transgenerational
inheritance of an acquired small RNA-based antiviral response in
C. elegans. Cell, 147, 1248–1256.
38. Klimov,P.B. and Knowles,L.L. (2011) Repeated parallel evolution
of minimal rRNAs revealed from detailed comparative analysis.
J. Hered., 102, 283–293.
39. Waeschenbach,A., Telford,M.J., Porter,J.S. and Littlewood,D.T.
(2006) The complete mitochondrial genome of Flustrellidra hispida
and the phylogenetic position of Bryozoa among the Metazoa.
Mol. Phylogenet. Evol., 40, 195–207.
40. He,Y., Jones,J., Armstrong,M., Lamberti,F. and Moens,M. (2005)
The mitochondrial genome of Xiphinema americanum sensu stricto
(Nematoda: Enoplea): considerable economization in the length
and structural features of encoded genes. J. Mol. Evol., 61,
41. Min,G.S. and Park,J.K. (2009) Eurotatorian paraphyly: revisiting
phylogenetic relationships based on the complete mitochondrial
Nucleic Acids Research,2014, Vol.42, No. 42671
genome sequence of Rotaria rotatoria (Bdelloidea: Rotifera:
Syndermata). BMC Genomics, 10, 533.
42. Spencer,D.F. and Gray,M.W. (2011) Ribosomal RNA genes in
Euglena gracilis mitochondrial DNA: fragmented genes in a
seemingly fragmented genome. Mol Genet Genomics, 285, 19–31.
43. Mahendran,R., Spottswood,M.S., Ghate,A., Ling,M.L., Jeng,K.
and Miller,D.L. (1994) Editing of the mitochondrial small subunit
rRNA in Physarum polycephalum. EMBO J., 13, 232–240.
44. Barth,C., Greferath,U., Kotsifas,M. and Fisher,P.R. (1999)
Polycistronic transcription and editing of the mitochondrial small
subunit (SSU) ribosomal RNA in Dictyostelium discoideum. Curr.
Genet., 36, 55–61.
45. Decatur,W.A. and Fournier,M.J. (2003) RNA-guided nucleotide
modification of ribosomal and other RNAs. J. Biol. Chem., 278,
46. Petrov,A.S., Bernier,C.R., Hershkovits,E., Xue,Y.,
Waterbury,C.C., Hsiao,C., Stepanov,V.G., Gaucher,E.A.,
Grover,M.A., Harvey,S.C. et al. (2013) Secondary structure and
domain architecture of the 23S and 5S rRNAs. Nucleic Acids
Res., 41, 7522–7535.
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