THE MITOCHONDRIAL GENOME OF TRYPANOSOMES
Research in the Simpson Laboratory
The trypanosomes or kinetoplastid protozoa comprise a large group of
parasitic flagellated cells that are the causal agents for a variety of human
and animal diseases. Research in Dr. Simpson's laboratory has focused on the
molecular biology of the mitochondrial genome in these organisms from several
points of view. These cells represent one of the earliest eukaryotic lineages containing mitochondria and, as such, possess many unusual physical
and biochemical features, among which is a mitochondrial genome known as
'kinetoplast DNA' that consists of a network of thousands of catenated mini-
and maxicircles, transcripts of which are modified by a novel process termed
'RNA editing'. Click here to see several
micrographs of kDNA networks.
The cells can be plated on agar. Click
here to see an avi movie of T.
brucei procyclic colonies on agar.
Click on "kinetoplast" or on "kDNA"
to see electron micrographs of the DNA.
RNA Editing of Mitochondrial Transcripts in the Mitochondrion of
RNA editing in trypanosomatid protozoa
involves the insertion and deletion of uridine residues (U's) at specific sites
within coding regions of mRNA transcripts of the
maxicircle genome. The sequence information
for editing is contained in a class of small RNAs termed guide RNAs(gRNAs), which were previously discovered in this
laboratory. gRNAs are small RNAs which, at
the 5' end have a region of complementarity with mRNA sequence just downstream
of the sites to be edited, and at the 3' end have a non-encoded oligo[U] tail.
Click here to see several examples of
edited sequence aligned with the cognate gRNAs.
The majority of the gRNAs are encoded in the thousands of minicircle
molecules which are catenated together into a single giant network of DNA. This
laboratory showed previously that the 3' to 5'
polarity of editing is due to the creation of upstream gRNA anchor
sequences by downstream editing.
Click here to see
the sequential editing of the A6 mRNA of L. tarentolae by 6 overlapping
Two basic models have been proposed by our laboratory for the mechanism
of editing, one of which involves a cleavage, 3'-terminal U addition, and
The other, which was proposed
independently by Cech, involves two successive transesterifications such as
occur in RNA splicing, with a transfer of U's from the 3' end of the gRNA or
directly from UTP to the editing site. gRNA/mRNA chimeric molecules, which were
discovered by the Simpson laboratory, were initially proposed to be
intermediates in the transesterification model, but recent evidence suggests
that these molecules represent non-productive byproducts of the
The Evolution of RNA Editing
A rooted phylogenetic tree of the kinetoplastid protozoa was
constructed from nuclear rRNA sequences, which, together with a comparative
analysis of editing of three maxicircle genes in several trypanosomatid
species, led to the surprising conclusion that extensive or pan-editing,
mediated by multiple overlapping guide RNAs, is phylogenetically more ancient
than the limited editing which occurs at the 5' end of editing domains
(5'-editing), and that RNA editing preceded the evolution of parasitism in this
The maxicircle genomic organization
in all analyzed trypanosomatid species is identical, but the extent of editing
varies considerably (Click here for NAR cover
showing a comparison of maxicircle genomes). This can be visualized in a
comparison of the extent of editing of the A6 or MURF4
mRNA in three species. The editing appears to become limited to the 5' end of
the editing domain. This can be also seen in a comparison of the editing of the
ND7 mRNA in T. brucei and
L. tarentolae. In T. brucei there is
pan-editing of two domains, whereas in L. tarentolae, editing is limited to the
5' end of each domain.
The phylogenetic analysis also showed that the bodonid/cryptobiids
represent an early diverged sister group to the trypanosomatids, as was
proposed previously by classical analyses. Click
here to see a diagram of the taxonomy of kinetoplastid protozoa. Analysis
of one cryptobiid species, Trypanoplasma borreli, which is discussed below,
showed the presence of the U addition/deletion type of RNA editing of several
mitochondrial transcripts, in spite of a complete dissimilarity of the
mitochondrial gene order.
The evidence indicates that ancestral kinetoplastid cryptogenes were
probably pan-edited and the 5'-edited homologues were possibly generated by
several independent retroposition events from partially edited RNAs. A
of the extent of editing in an old laboratory strain of Leishmania tarentolae
and a recently isolated strain has provided additional evidence for this
This research was done by Dimitri Maslov and Otavio Thiemann.
Disruption of RNA Editing by Prolonged Culture of the
RNA editing in kinetoplastids appears to be a labile genetic trait that
is affected by prolonged cell culture. The transcripts of the G1-G5 cryptogenes
are pan-edited in the recently isolated LEM125 strain of Leishmania tarentolae,
but not in the UC strain which has been in culture for 55 years. At least 32
minicircle-encoded guide RNAs (gRNAs) for
the editing of G1-G5 transcripts are present in LEM125 and absent in UC. The
hypothesis was presented that specific minicircle sequence classes encoding
gRNAs for the editing of these transcripts were lost during the culture history
of the old lab strain, probably due to the absence of a selective pressure for
the protein products, which include subunits of complex I of the respiratory
Click here to see a diagram of the
construction of the gRNA library which was used to detect the additional gRNAs
in the LEM125 strain..
The absence of gRNAs for the editing of G5 in the UC strain led to the
existence of extensively misedited RNAs. Some
of these misedited RNAs showed correct editing of block I, and misediting
upstream. The editing of Block I was mediated by a maxicircle-encoded gRNA,
which was present in both strains. Several non-cognate gRNAs were identified in
the UC strain which could account for specific misedited upstream sequences.
This research was done by Otavio Thiemann.
Detection and Identification of Human Pathogenic
Leishmania and Trypanosoma Species by Hybridization of PCR-amplified Mini-Exon
A single pair of PCR primers within a conserved region of the mini-exon
repeat was used to amplify the repeats from 10 species of pathogenic Leishmania
belonging to 4 major clinical groups and also from 3 species of Trypanosoma.
Oligonucleotide hybridization probes for the detection and identification of
the PCR-amplified repeats were constructed from alignments of mini-exon intron
and intergenic sequences. The probes generated from mini-exon intergenic
regions of the L. (V.) braziliensis, L. (L.) donovani and L. (L.) mexicana
species hybridized specifically to their cognate groups without discriminating
between the species within the groups. The probes for L. (L.) major and L. (L.)
aethiopica were species-specific, while the L. (L.) tropica probe also
hybridized with the L. (L.) aethiopica mini-exon repeat. The mini-exon
intron-derived probes for T. cruzi, T. rangeli and T. brucei were
species-specific. This method involving the detection of specific PCR-amplified
products produced using a single primer set represents a novel sensitive and
specific assay for multiple trypanosomatid species and groups. It essentially
represents a multiplex assay and should complement the minicircle-based
diagnostic assay previously developed by this laboratory..
This research was done by Anthea Ramos, Dmitri A. Maslov, Octavio
Fernandes, David A. Campbell.
RNA Editing in Trypanosoma cruzi
Trypanosoma cruzi is the causal agent of Chagas Disease, an important
disease of the American tropics, for which there is no adequate chemotherapy or
vaccine. Dr. Simpson's laboratory previously developed a sensitive
diagnostic test for the presence of these parasites in
chronically ill patients, by PCR amplification of parasite-specific but highly
polymorphic DNA fragments from kinetoplast minicircle molecules.
A knowledge of the RNA editing system and the genetic function of these
molecules could aid in development of a rational chemotherapy for this disease.
The previously observed extensive sequence heterogeneity of the kinetoplast
minicircle DNA in Trypanosoma cruzi, both intra- and inter-strain, has raised
the question as to how the minicircle DNA in this species can have any
gRNA-coding capacity at all, since there does not appear to be any variable
region sequences conserved between different strains. To address this question,
the complete edited sequence of maxicircle unidentified reading frame 4 mRNA
gRNAs were obtained from gRNA libraries constructed from two clonal strains
of T. cruzi - Sylvio X10/CL1 and CAN III/CL1. Libraries of PCR-amplified
minicircle variable regions were also constructed for both strains. A single
gene for each gRNA was identified in the same polarity within specific
variable regions from both strains, 60-100 nucleotides downstream from the
conserved 12mer sequence. GTP-capped total gRNA from
one strain failed to cross-hybridize with minicircle DNA from the other strain.
The explanation for this proved to be the number of polymorphisms, mainly
transitions, within the homologous gRNAs in the two strains. In most cases
these transitions did not destroy the edited mRNA/gRNA base-pairing, as a
result of the allowed G-U wobble base-pairing. The sequences of the variable
regions containing homologous gRNAs in the two strains probably derived from an
ancestral sequence, and each has accumulated sufficient polymorphisms so as not
to allow hybridization. Within a strain, multiple redundant gRNAs were
identified which encode identical editing information but have different
This research was done by Herbert Avila.
RNA Editing in Crithidia fasciculata
Although the mitochondrial uridine insertion/deletion, guide
RNA-mediated type of RNA editing has been described in Crithidia fasciculata,
no evidence for the encoding of guide RNAs in the kinetoplast minicircle DNA
has been presented. There has also been a question as to the capacity of the
minicircle DNA in this species to encode the required variety of gRNAs, since
the kinetoplast DNA from the C1 strain has been reported as essentially
containing a single minicircle sequence class. To address this problem, the
genomic and mature edited sequences of the MURF4 and RPS12 cryptogenes were
determined, and a gRNA library was constructed from mitochondrial RNA. Five
specific gRNAs were identified, two of which edit blocks within the MURF4 mRNA,
and three of which edit blocks within the RPS12 mRNA. The
genes for these gRNAs are all localized with
identical polarity within one of the two variable regions of specific
minicircle molecules, approximately 60 bp from the 'bend' region. These
minicircles were found to represent minor sequence classes representing
approximately 2% of the minicircle DNA population in the network. The major
minicircle sequence class also encodes a gRNA at the same relative genomic
location, but the editing role of this gRNA was not determined. These results
confirm that kinetoplast minicircle DNA molecules in this species encode gRNAs,
as is the case in other trypanosomatids, and suggest that the copy number of
specific minicircle sequence classes can vary dramatically without an overall
effect on the RNA editing system.
This research was done by Shinji Yasuhira.
Guide RNAs and guide RNA genes in the cryptobiid
kinetoplastid protozoan, Trypanoplasma borreli
Trypanoplasma borreli belongs to the bodonid/cryptobiid group of
kinetoplastid protozoa, which represents a sister group to the trypanosomatids.
RNA transcripts from several mitochondrial genes in this organism undergo the
trypanosomatid type of uridine addition/deletion RNA editing. A gRNA cDNA
library was constructed and five gRNAs
were identified, one for editing the ribosomal protein S12 mRNA, three for
editing the cytochrome oxidase subunit I mRNA and one for editing the
cytochrome b mRNA. All of the gRNAs contained non-encoded oligo[U] sequences at
their 5' end, as well as at the 3' end as is common with gRNAs in
trypanosomatids. The mechanism for addition of the 5' non-encoded oligo[U]
sequence and the function of this sequence are unknown. The T. borreli gRNAs
were shorter (25-35 nt, excluding the 5' oligo[U]) than gRNAs in
trypanosomatids (45-50 nucleotides), indicating a smaller size of editing
blocks in this organism. Genomic sequences for two gRNAs were cloned and
sequenced. These two gRNA-encoding sequences were shown to originate from the
170-200 kb Component I molecules which represent a possible homologue of
minicircle DNA in trypanosomatids, and not from the 80 kb Component II
molecules which contain the structural genes and cryptogenes.
This research was done by Shinji Yasuhira
Phylogenetic affinity of mitochondria of Euglena
gracilis and kinetoplastids using cytochrome oxidase I and hsp60.
The mitochondrial DNA-encoded cytochrome oxidase subunit I (COI) gene
and the nuclear DNA-encoded hsp60 gene from the euglenoid protozoan Euglena
gracilis were cloned and sequenced. The COI sequence represents the first
example of a mitochondrial genome-encoded gene from this organism. This gene
contains seven TGG tryptophan codons and no TGA tryptophan codons, suggesting
the use of the universal genetic code. This differs from the situation in the
mitochondrion of the related kinetoplastid protozoa, in which TGA codes for
tryptophan. In addition, a complete absence of CGN triplets may imply the lack
of the corresponding tRNA species. COI cDNAs from E. gracilis possess short 5'
and 3' untranslated transcribed sequences and lack a 3' poly[A] tail. The COI
gene does not require uridine insertion/ deletion RNA editing, as occurs in
kinetoplastid mitochondria, to be functional, and no short guide RNA-like
molecules could be visualized by labeling total mitochondrial RNA with
[alpha-32P]GTP and guanylyl transferase. In spite of the differences in codon
usage and the 3' end structures of mRNAs, phylogenetic analysis using the
COI and hsp60
protein sequences suggests a monophyletic relationship between the
mitochondrial genomes of E. gracilis and of the kinetoplastids, which is
consistent with the phylogenetic relationship of these groups previously
obtained using nuclear ribosomal RNA sequences.
This research was done by Shinji Yasuhira.
Involvement of mitochondrial ribonucleoprotein complexes
in RNA Editing
By analysis of mitochondrial extracts on glycerol gradients, two types
of ribonucleoprotein (RNP) complexes were identified containing gRNAs. The
T-class sediments at approximately 10S and consists of approximately six
complexes, the endogenous RNA of which can be self-labeled with
[32P]UTP. The most abundant T-complex, T-IV, was visualized by
electron microscopy as 980-140 A particles. This complex exhibits terminal
uridylyl transferase (TUTase) activity and contains gRNAs. The other
T-complexes also contain mRNA fragments. The function of T-IV may be to add U's
to the 3' end of the gRNA. The second class of RNP complexes consists of
170-300 A particles which show little TUTase activity, and exhibit an in vitro
RNA editing-like activity.
Two mitochondrial proteins of 18 kD and 51 kD which appeared to show
interaction with exogenous gRNAs were isolated and the genes cloned. These
proteins were components of complex T-I and T-VI, respectively. The proteins
possess 17 and 9 amino acid N terminal cleavable mitochondrial targeting
sequences. The p18 protein localized throughout the entire tubular
mitochondrion. and has been identified by the Benne lab as a subunit of the
mtochondrial ATPase. The p51 protein was identified as a mitochondrial aldehyde
dehydrogenase. In addition, 71 kD and 62 kD proteins which comigrated in native
gels with other T-complexes were identified as hsp70 and hsp60 homologs.
The majority of the protein-RNA interactions in the T-complexes were
shown to be low affinity. However, digestion of the extract with micrococcal
nuclease and saturation with rRNA was found to uncover a high affinity RNP
complex, which involves at least two proteins interacting with the endogenous
The second class of complexes sediments at 20S. A gRNA-independent in
vitro editing-like activity which comigrates with the 20S complexes was assayed
by following the incorporation of radioactively labeled U's into the pre-edited
region of a synthetic mRNA substrate by digestion with RNase H and specific
oligonucleotides. The incorporation is limited precisely to the pre-edited
region and is dependent on some type of endogenous RNA, as shown by micrococcal
nuclease digestion experiments. Labeled C residues were incorporated into the
same sites as U residues, but at a 20 fold lower level.
This research was done by Marian Peris, Agda Simpson, Frederic
Bringaud, Elaine Byrne and Georges Frech.
Native gel analysis of ribonucleoprotein complexes from
a Leishmania tarentolae mitochondrial extract
Two polypeptides of 50 and 45 kDa were adenylated by incubation of a
mitochondrial extract from Leishmania tarentolae with [a-32P]ATP. These
proteins were components of the 20S complex, which migrated as a single band of
approximately 1800 kDa in a native gel. The facts that RNA ligase activity
cosedimented at 20S and that the ATP-labeled p45 and p50 polypeptides were
deadenylated upon incubation with a ligatable RNA substrate suggests that these
proteins may represent charged intermediates of a mitochondrial RNA ligase.
Hybridization of native gel blots with guide RNA (gRNA) probes showed the
presence of gRNA in the previously identified T-IV complexes that sedimented in
glycerol at 10S and contained TUTase activity, and also in a previously
unidentified class of heterodisperse complexes that sedimented throughout the
gradient. gRNAs were not detected in the p45+p50-containing complex. The
heterodisperse gRNA-containing complexes were sensitive to incubation at 27oC
and appear to represent complexes of T-IV subunits with mRNA. Polyclonal
antiserum to a 70 kDa protein that purified with terminal uridylyl transferase
activity was generated, and the antiserum was used to show that this p70
polypeptide was a component of both the T-IV and the heterodisperse
gRNA-containing complexes. We propose that the p45+p50-containing 1800 kDa
complex and the p70+gRNA -containing heterodisperse complexes interact in the
This research was performed by Marian Peris, Agda M. Simpson, Jeremy
Grunstein, Joanna E. Liliental and Georges C. Frech
Guide RNA-independent RNA editing in vitro
A mitochondrial extract from Leishmania tarentolae directs the
incorporation of uridylate (U) residues within the pre-edited domain of
synthetic cytochrome b (CYb) and NADH dehydrogenase subunit 7 mRNA. This has
several characteristics of an in vitro RNA editing activity, but no direct
evidence for involvement of guide RNAs was obtained. In fact, evidence obtained
by Greg Connell indicates that this activity is independent of both exogenous
and endogenous gRNAs. The incorporation is limited to the preedited region but
the number and localization of U's inserted is not identical to that found in
mature correctly edited RNA. The activity is selectively inhibited by digestion
of the lysate with micrococcal nuclease, possibly suggesting a requirement for
some type of endogenous RNA. A low level of incorporation of [alpha-32P]CTP
occurs at the same sites as UTP.
Stereochemical evidence for the enzyme cascade model
for RNA editing
Chiral phosphorothioates were used to investigate the
stereoconfiguration requirements and the stereochemical course of an RNA
editing-like internal uridine (U)-incorporation activity and a 3' terminal
U-addition activity using a mitochondrial extract from L. tarentolae. The
extract utilizes SP-[a-S]UTP for both 3' and internal U- incorporation into
substrate RNA. The internal as well as the 3' incorporation of SP-[a-S]UTP
proceeds via inversion of the stereoconfiguration.
The mitochondrial RNA ligase produces an
inversion of the stereoconfiguration. Iternal U-incorporation does not occur at
sites containing thiophosphodiesters of the RP configuration. The
results are compatible with an enzyme cascade model
for this in vitro U-insertion activity involving sequential endonuclease,
uridylyl transferase directly from UTP and RNA ligase steps, and incompatible
with models involving the transfer of U residues from the 3' end of gRNAs.
This research was done by Georges Frech.
The role of mRNA structure in guide RNA-independent RNA
editing in vitro
A primer extension assay was used for
the detection of uridine insertions occurring in vitro in synthetic pre-edited
cytochrome b mRNA during incubation with a Leishmania tarentolae mitochondrial
extract. Two different activities were detected that inserted uridines within
the first two editing sites: one that is
dependent on the secondary structure of the mRNA but is independent of both
exogenous and endogenous guide RNA, and a second that does not put the same
structural constraints on the mRNA, but is dependent on the presence of a
cognate guide RNA. The possibility that contaminating gRNA-mRNA chimeric
molecules were the source of the extension ladders was eliminated by performing
the extension assay on RNAs transcribed from 3'-tagged RT-PCR-amplified cDNAs.
Leaving aside the question of the biological relevance of the gRNA-independent
reaction, it is possible that the structure of the RNAs supporting this in
vitro reaction could be mimicking the RNA structures occurring during
gRNA-mediated editing. For example, the duplex formed by foldback of the Cyb mRNA 5' and 3' extensions,
which is required for the gRNA-independent reaction, may serve the same
function as the anchor duplex created by the gRNA binding to the mRNA.
Annealing of the cognate gRNA containing a complementary anchor sequence with
the 5'-extended mRNA construct would be predicted to disrupt the mRNA secondary
structure, thereby accounting for the observed inhibitory effect of the
addition of exogenous gRNA on the gRNA-independent U-insertion activity. The
creation of a recognition element for the assembly of the U-insertion machinery
may represent an important role of the gRNA in the gRNA-mediated reaction. The
recognition element could be the double stranded RNA formed by the gRNA-mRNA
interaction that may be mimicked by the intramolecular helix necessary for the
This work was performed by Greg Connell and Elaine Byrne.
Guide RNA-directed uridine-insertion RNA editing in
Guide RNAs (gRNAs) have been proposed to mediate uridine (U)
addition/deletion editing of mitochondrial messenger RNAs in kinetoplastid
protozoa. The U's are proposed to be derived either from UTP by two successive
cleavage-ligations or transesterifications, or from the 3' end of the gRNA by
the same mechanisms. By use of a sensitive and specific primer extension
assay, we have demonstrated guide
RNA-dependent U-insertions into a specific
editing site of a preedited mRNA which was incubated in a mitochondrial extract
from Leishmania tarentolae. The predominant number of U-insertions was
determined by the number of guiding nucleotides in the added gRNA, and the
formation of a gRNA-mRNA anchor duplex was necessary for activity. UTP and a-b
bond hydrolysis of ATP were required, and the activity was inhibited above
50-100 mM KCl. A guide RNA-independent insertion of up to approximately 13 U's
occurred in the absence of the added cognate gRNA; the extent of this activity
was affected by sequences upstream and downstream of the edited region. Heparin
inhibited the guide RNA-independent U-insertion activity and had no effect on
the guide RNA-dependent activity. Blocking the 3'OH of the gRNA had little
effect on the gRNA-dependent U-insertion activity. The data are consistent with
a modified enzyme cascade model in which
the U's are derived directly from UTP.
This work was performed by Elaine Byrne and Greg Connell.
Sequence-dependent importation of tRNAs into the
mitochondrion of Leishmania tarentolae
Another topic of research in the Simpson lab is the importation of
tRNAs into the kinetoplast- mitochondrion of L. tarentolae. Previous research
had shown that no mitochondrial tRNAs were encoded in the maxicircle (or
minicircle) genome. Therefore the mitochondrial tRNAs had to be derived from
the cytosol and be encoded in nuclear genes. To investigate this, an in vivo
transfection method was employed. tRNAIle(UAU) was shown previously by Shi et
al., 1994 and Lye et al., 1993 to exclusively localized within the
mitochondrion and tRNAGln(CUG) exclusively in the cytosol. L. tarentolae cells
were transfected with plasmids encoding either tRNAIle or tRNAGln that were
tagged with altered sequences in the D loop, permitting discrimination from the
endogenous tRNAs. Primer extension analysis was used to show that the
plasmid-encoded genes were expressed and that the tagged tRNAs showed a similar
intracellular localization as the endogenous tRNAs. Exchange or deletion of the
5'-flanking genomic sequences had no effect on the expression or mitochondrial
localization of the tagged tRNAIle or on the expression or cytosolic
localization of the tagged tRNAGln, suggesting that the signals for importation
are localized within the tRNA itself. Swapping the D loop+stem from the
exclusively cytosolic tRNAGln with that from the tRNAIle produced a partial
mitochondrial localization of the plasmid-expressed mutated tRNAGln. However, D
loop exchange did not eliminate the mitochondrial localization of the
plasmid-expressed mutated tRNAIle, suggesting that tertiary structure or
additional sequence elements may be involved in the importation signal.
This research was done by Beatriz Lima.
The mitochondrial glutamate dehydrogenase from
Leishmania tarentolae is a guide RNA-binding protein
To identify specific proteins interacting with guide RNAs in
mitochondrial ribonucleoprotein complexes from Leishmania tarentolae,
fractionated and unfractionated mitochondrial extracts were subjected to
UV cross-linking with added labeled gRNA
and also with [a-32P]UTP-labeled endogenous RNA. An
abundant 110-kD protein (p110) localized in the T-V complex, which sediments in
glycerol gradients at the leading edge of
the 10S terminal uridylyl transferase peak, was found to interact with both
types of labeled RNAs. The p110 protein was gel-isolated and subjected to
microsequence analysis, and the gene was cloned. The
sequence revealed significant similarity
with mitochondrial glutamate dehydrogenases. A polyclonal antiserum was raised
against a recombinant fragment of the p110 gene and was used to demonstrate a
stable and specific gRNA-binding activity by co-immunoprecipitation and competitive gel-shift
analyses. Complex formation was strongly
inhibited by competition with poly[U] or by deletion or
substitution of the gRNA 3'-oligo[U]
tail. Also, addition of a 3' oligo[U]
tail to an unrelated transcript was sufficient for p110 binding. Both the
gRNA-binding activity of the p110 protein and in vitro gRNA-independent and
gRNA-dependent uridine insertion activities in the mitochondrial extract were
inhibited by high concentrations of dinucleotides.
This work was performed by Frédéric Bringaud, Renata
Stripecke, Georges C. Frech, Stephen Freedland, Christoph W. Turck and Elaine
Knockout of the glutamate dehydrogenase gene in
bloodstream Trypanosoma brucei in culture has no effect on editing of
Glutamate dehydrogenase (GDH) was shown
to bind the 3' oligo[U] tail of the mitochondrial guide RNAs (gRNAs) of
Leishmania tarentolae, apparently in the dinucleotide pocket.
Bloodstream Trypanosoma brucei cells in culture represent a good system
to investigate the genetic effects of knocking out kinetoplastid nuclear genes
to test a role in RNA editing, since editing of several mitochondrial genes
occurs but is dispensable for viability. Both GDH alleles of bloodstream T.
brucei in culture were replaced by drug resistant markers without any
effect on viability. The ratios of edited to unedited mRNAs for several
cryptogenes were assayed by primer extension analysis. The steady state
abundances of these edited RNAs were unaffected by the double knockout. This
evidence suggests that GDH may not play a role in the editing reaction in
bloodstream trypanosomes in culture, but this conclusion is tentative since
there could be redundant genes for any biological function. We employed a
double allelic replacement technique to generate a tetracycline inducible
conditional expression of an ectopic copy of the deleted gene in bloodstream
trypanosomes in culture. We used this strategy for genes encoding mitochondrial
proteins which are not required during this stage of the life cycle, but as a
general strategy it should be appropriate for generation of conditional null
mutants for essential genes as well.
This work was performed by A. Estevez, F. Kierzenbaum, E. Wirtz, F.
Bringaud and J. Grunstein.
The mitochondrion in dividing Leishmania tarentolae
cells is symmetric and circular and becomes a single asymmetric tubule in
non-dividing cells due to division of the kinetoplast portion
Kinetoplastid protozoa have a single
mitochondrion that extends throughout the cell. The disk-shaped portion of
the mitochondrion adjacent to the basal body of the flagellum contains the
kinetoplast DNA nucleoid body which consists of thousands of catenated
minicircles and a smaller number of catenated maxicircles. The maxicircles
contain structural genes and cryptogenes, rRNA genes, and a few guide RNA genes
The minicircles contain the majority of the guide RNA genes. The long slender
non-dividing stationary phase Leishmania tarentolae cells in culture have an
asymmetric mitochondrion that consists
of a single tubule extending from one edge of the kinetoplast portion. This
presents a problem for cell division, in that one daughter cell will receive
significantly less mitochondrial membranes than the other cell. We show in this
paper that the solution to this problem is that dividing cells, which are normally shorter and rounder than
stationary phase cells, possess a symmetric circular mitochondrion that has mitochondrial tubules extending
from both edges of the kinetoplast which are joined in the posterior region of
the cell. This implies that growth of the mitochondrion occurs after cell
division, either from elongation of the longitudinal tubule towards the
anterior of the cell, or from elongation of the kinetoplast portion of the
mitochondrion towards the posterior region and fusion of the tubules.
This work was performed by Larry Simpson and Frank Kretzer.
Lack of evidence for presence of 5' extentions on
tRNAs imported into the mitochondrion of Leishmania
All mitochondrial tRNAs in kinetoplastid protozoa are encoded in
nuclear DNA and transported into the mitochondrion (Simpson et al., 1989, Nucl.
Acids Res. 17: 5427-5445). It has been proposed by Hancock et al. (J. Biol.
Chem., 1990, 265: 19208-19215) that tRNAs in these cells are imported into the
mitochondrion as 5'-extended precursors which are processed by a mitochondrial
RNase P-like activity. We have examined this hypothesis by cloning and
sequencing primer extension products of mitochondrial tRNAs from L. tarentolae
and T. brucei, and have found that these are derived from circularized mature
tRNA molecules. We suggest that these molecules are produced by the endogenous
RNA ligase activity either in vivo or during mitochondrial isolation. We did
not obtain any evidence for the existence of high molecular weight precursors
of mitochondrial tRNAs. This negative result is consistent with previous in
vivo transfection studies with both L. tarentolae (Lima and Simpson, 1996, RNA
2: 429-440) and T. brucei (Hauser and Schneider, 1995, EMBO J. 14: 4212-4220),
in which mitochondrial targeting of plasmid-expressed tRNAs was independent of
the presence of 5'-flanking sequences. We conclude that the hypothesis for
5'-extended tRNA precursors in kinetoplastid mitochondrial importation remains
to be verified.
This work was performed by Ruslan Aphasizhev.
Cloning and characterization of the Leishmania
tarentolae adenine phosphoribosyltransferase
Adenine phosphoribosyltransferase (APRT) is an important enzyme
involved in the recycling of purine nucleotides in all cells. Parasitic
protozoa of the order Kinetoplastida are unable to synthesize purines de novo
and utilize the salvage pathway for synthesis of ribonucleotides. The aprt gene
was cloned from a Leishmania tarentolae genomic library and the sequence
determined. The L. tarentolae aprt gene contains a 708 nucleotide open
reading frame that encodes a 25 kDa protein. The predicted amino acid
sequence has 85% identity to the APRT of L.
donovani (Allen,T., Hwang, H., Wilson, K., Hanson, S., Jardim, A. and
Ullman, B. (1995) Mol. Biochem. Parasitol. 74, 99-103). A recombinant protein
was expressed in Escherichia coli, purified to
homogeneity and found to retain enzymatic activity. The L. tarentolae
APRT is active as a homodimer in solution. The availability of the APRT
enzyme from another kinetoplastid protozoan and the possibility of expressing
the recombinant protein in large quantities should provide the basis for a
functional and structural analysis of this enzyme which has been suggested as a
potential target for rational drug design.
This work was performed by Otavio Thiemann.
Purification and Characterization of MAR1: a
Mitochondrial Associated Ribonuclease from Leishmania tarentolae
A relatively thermostable 22 kDa endoribonuclease (MAR1) was purified
more than 10,000 fold from a mitochondrial extract of Leishmania
tarentolae and the gene cloned. The purified nuclease has a Km of 100-145
± 33 nM and a Vmax of 1.8-2.9 ± 2 nmoles/min, depending on the
RNA substrate, and yields a 3' OH and a 5' phosphate. Cleavage was limited to
several specific sites in the substrate RNAs tested, but cleavage of pre-edited
RNAs was generally independent of the addition of cognate guide RNA. The MAR1
gene was expressed in Escherichia coli or in Leishmania
tarentolae cells and the recombinant protein was affinity-purified. The
cleavage specificity of the recombinant enzyme from Leishmania
tarentolae was identical to that of the native enzyme. The single copy MAR1
gene maps to an 820 kb chromosome and contains an open reading frame of 579 nt.
The 18 amino acid N-terminal sequence shows characteristics of an uncleaved
mitochondrial targeting sequence. Database searching revealed two homologues of
MAR1 corresponding to unidentified open reading frames in Caenorhabditis
elegans (Z69637) and Archaeoglobus fulgidus (AE000943). The function
of MAR1 in mitochondrial RNA metabolism in L. tarentolae remains to be
determined. Click here and
here to see the figures from this paper.
This work was performed by Juan Alfonzo and Otavio Thiemann.
Phylogenetic Affinities of Diplonema within the
Euglenozoa as Inferred from the SSU rRNA Gene and Partial COI Protein
In order to shed light on the phylogenetic position of diplonemids
within the phylum Euglenozoa, we have sequenced small subunit rRNA (SSU rRNA)
genes from Diplonema (syn. Isonema) papillatum and
Diplonema sp. We have also analyzed a partial sequence of the
mitochondrial gene for cytochrome c oxidase subunit I from D.
papillatum. With both markers, the maximum likelihood method favored a
closer grouping of diplonemids with kinetoplastids, while the parsimony and
distance suggested a closer relationship of diplonemids with euglenoids. In
each case, the differences between the best tree and the alternative trees were
small. The frequency of codon usage in the partial D. papillatum COI was
different from both related groups; however, as is the case in kinetoplastids
but not in Euglena, both the non-canonical UGA codon and the canonical
UGG codon were used to encode tryptophan in Diplonema.
This research was performed by Dmitri Maslov and Shingi
In vitro uridine insertion RNA editing mediated
by cis-acting guide RN
Guide RNAs involved in mediating RNA editing in vivo are provided in
trans except for the CO2 mRNA, which has the gRNA in cis at the 3'
end of the RNA. We have found that a cognate gRNA provided in cis at the
3' end of a pre-edited NADH dehydrogenase 7 (ND7) mRNA substrate can direct U
insertions at editing site 1 when incubated with a mitochondrial lysate from
Leishmania tarentolae. The efficiency of gRNA-dependent U insertion
mediated by a cis-acting is greater on a molar basis than that for a
trans-acting gRNA, as expected for a unimolecular gRNA:mRNA interaction.
Blocking the 3' end of a cis-acting gRNA lacking a 3' oligo[U] tail has no
effect on gRNA-dependent U insertions, nor does providing the gRNA in
cis upstream of the mRNA, confirming the previous observation that the
terminal 2'- and 3'-hydroxyls of the gRNA are not involved in U insertion
activity. These results also establish that the oligo[U] tail is not required
for U insertion in vitro. Increasing the extent of base pairing between
the 3' end of the gRNA and the 5' end of the mRNA significantly increases in
vitro gRNA-dependent U insertion at site 1, presumably by maintaining the
mRNA 5' cleavage fragment within the editing complex. We speculate that, in
vivo, protein:RNA and/or protein:protein interactions may be responsible
for maintaining the mRNA 5' cleavage fragment in close proximity to the mRNA 3'
cleavage fragment, and that such interactions may be rate limiting in
vitro. Click here to see the Figures from
This work was done by Steve Kapuschoc.
The Mitochondrial RNA ligase from Leishmania
tarentolae can join RNA molecules bridged by a complementary RNA
A biochemical characterization was performed with a partially purified
RNA ligase from isolated mitochondria of Leishmania tarentolae. This
ligase has a Km of 25 ± 0.75 nM and a Vmax of 1.0 x10-4 ± 2.4 x
10-4 nmoles/min when ligating a nicked double stranded RNA substrate. Ligation
was negatively affected by a gap between the donor and acceptor nucleotides.
The catalytic efficiency of the circularization of a single stranded substrate
was five-fold less than that of the ligation of a nicked substrate. These
properties of the mitochondrial RNA ligase are consistent with an expected in
vivo role in the process of uridine insertion/deletion RNA editing, in which
the mRNA cleavage fragments are bridged by a cognate guide RNA.
Click here to see the figures from
This work was done by Valerie Blanc, Juan D. Alfonzo, and Ruslan
T7 RNA polymerase-driven transcription in mitochondria
of Leishmania tarentolae and Trypanosoma brucei.
The study of RNA editing and other molecular processes in the
trypanosome mitochondrion would benefit greatly from the ability to insert and
express exogenous DNA in the organelle. However, even with a method to
introduce DNA, the current lack of knowledge about mitochondrial transcription
would hinder efforts to obtain expression. To circumvent this problem, we have
transfected Leishmania tarentolae promastigotes and Trypanosoma brucei
procyclic cells with bacteriophage T7 RNA polymerase targeted to the
mitochondrion. Mitochondria isolated from the transfectants contained active T7
RNA polymerase, as shown by a comigration in density gradients of mitochondrial
marker enzymes and T7 polymerase activity. A DNA cassette under T7 control was
introduced into isolated mitochondria from the transfectants by electroporation
and the DNA was shown to be transcribed. This system should allow the
transcription of foreign genes of choice within the mitochondrial matrix either
in a transient assay using electroporation of DNA into isolated mitochondria,
or in a stable assay using cells transfected with DNA by the biolistic gun
Click here to see the
figures from this work.
This work was done by Antonio M. Estévez, Otavio H. Thiemann and
Juan D. Alfonzo.
C to U editing of Anticodon of Imported Mitochondrial
tRNATrp allows Decoding of UGA Stop Codon in Leishmania
All mitochondrial tRNAs in kinetoplastid protists are encoded in the
nucleus and imported into the organelle. The tRNATrp(CCA) can decode the
standard UGG tryptophan codon but cannot decode the mitochondrial UGA
tryptophan codon. We show that the mitochondrial tRNATrp undergoes a specific C
to U nucleotide modification in the first position of the anticodon which
allows decoding of mitochondrial UGA codons as tryptophan. Functional evidence
for the absence of a UGA suppressor tRNA in the cytosol, using a reporter gene,
was also obtained, which is consistent with a mitochondrial localization of
this editing event. Leishmania cells have dealt with the problem of a
lack of expression within the organelle of this non-universal tRNA by
compartmentalizing an editing activity which modifies the anticodon of the
This work was performed by Juan D. Alfonzo, Valerie Blanc, Antonio M.
Estévez and Mary Anne T. Rubio.
Evolution of RNA Editing in Trypanosome
Two different RNA editing systems have been described in the
kinetoplast-mitochondrion of trypanosomatid protists. The first involves the
precise insertion and deletion of U residues mostly within the coding regions
of maxicircle-encoded mRNAs to produce open reading frames. This editing is
mediated by short overlapping complementary guide RNAs (gRNAs) encoded in both
the maxicircle and the minicircle molecules, and involves a series of enzymatic
cleavage-ligation steps. The second editing system is a C34 to U34 modification
in the anticodon of the imported tRNATrp, thereby permitting the decoding of
the UGA stop codon as tryptophan. U-insertion editing probably originated in an
ancestor of the kinetoplastid lineage and has evolved in some cases by the
replacement of the original pan-edited cryptogene with a partially edited cDNA.
The driving force for this retroposition was postulated to be the stochastic
loss of entire minicircle sequence classes and their encoded gRNAs upon
segregation of the single kinetoplast DNA network into daughter cells at cell
division. A large plasticity in frequencies of minicircle sequence classes has
been observed during cell culture in the laboratory. Computer simulations
provide theoretical evidence for this plasticity if a random distribution and
segregation model of minicircles is assumed. The specific C to U tRNA editing
probably evolved after the loss of the original endogenous tRNA genes from the
mitochondrial genome and the development of a mechanism for importation of
nuclear-encoded tRNAs into the organelle. The relationship of the two editing
systems is discussed.
This work was performed by Otavio Thiemann, Nickloas Savill, Juan
Alfonzo, and Dmitri Maslov. It was presented at the Colloquium on Variation and
Evolution in Plants and Microorganisms, Jan. 27-29, 2000, Beckman Center of the
National Academies of Sciences and Engineering, Irvine, CA.
Selective Importation of RNA into Isolated Mitochondria
from Leishmania tarentolae
All mitochondrial tRNAs in kinetoplastid protozoa are encoded in the
nucleus and imported from the cytosol. Incubation of two in vitro transcribed
tRNAs, tRNAIle(UAU) and tRNAGln(CUG), with isolated
mitochondria from Leishmania tarentolae, in the absence of any added
cytosolic fraction, resulted in a protease-sensitive, ATP-dependent
importation, as measured by nuclease protection. Evidence that nuclease
protection represents importation was obtained by the finding that Bacillus
subtilis pre-tRNAAsp was protected from nuclease digestion and
was also cleaved by an intra-mitochondrial RNase P-like activity to produce the
mature tRNA. The presence of a membrane potential is not required for in vitro
importation. A variety of small synthetic RNAs were also found to be
efficiently imported in vitro. The data suggest that there is a structural
requirement for importation of RNAs greater than approximately 17 nt, and that
smaller RNAs are apparently non-specifically imported. The signals for
importation of folded RNAs have not been determined, but the specificity of the
process was illustrated by the higher saturation level of importation of the
mainly mitochondria-localized tRNAIle as compared to the level of
importation of the mainly cytosol-localized tRNAGln. Furthermore,
exchanging the D-arm between the tRNAIle and the tRNAGln
resulted in a reversal of the in vitro importation behavior and this could also
be interpreted in terms of tertiary structure specificity.
This work was performed by Mary Anne T. Rubio, Xuan Liu, Harumi Yuzawa
and Juan D. Alfonzo.
End processing precedes mitochondrial importation and
editing of tRNAs in Leishmania tarentolae
All mitochondrial tRNAs in Leishmania tarentolae are encoded in the
nuclear genome and imported into the mitochondrion from the cytosol. One
imported tRNA (tRNATrp) is edited by a C to U modification at the first
position of the anticodon. In order to determine the in vivo substrates for
mitochondrial tRNA importation as well as tRNA editing, we examined the
subcellular localization and extent of 5'- and 3'-end maturation of
tRNATrp(CCA), tRNAIle(UAU), tRNAGln(CUG), tRNALys(UUU), and tRNAVal(CAC).
Nuclear, cytosolic and mitochondrial fractions were obtained with little cross
contamination, as determined by northern analysis of specific marker RNAs. The
tRNAGln was mainly cytosolic in localization, the tRNAIle and tRNALys were
mainly mitochondrial, and the tRNATrp and tRNAVal were shared between the two
compartments. 5'- and 3'-extended precursors of all five tRNAs were present
only in the nuclear fraction, suggesting that the mature tRNAs represent the in
vivo substrates for importation into the mitochondrion. Consistent with this
model, T7-transcribed mature tRNAIle undergoes importation in vitro into
isolated mitochondria more efficiently than the 5'-extended precursor tRNAIle.
The 5'-extended precursor tRNATrp was found to be unedited, which is consistent
with a mitochondrial localization of this editing reaction. T7-transcribed
unedited tRNATrp was imported in vitro more efficiently than edited tRNATrp,
suggesting the presence of importation determinants in the anticodon.
This research was performed by Steve Kapushoc, Juan Alfonzo and Mary
Uridine insertion/deletion RNA editing in L.
tarentolae mitochondria shows cell cycle dependence
L. tarentolae cells were synchronized using hydroxyurea and the
relative abundance of edited and pre-edited transcripts for 4 maxicircle genes
was analyzed by primer extension. The primers in each case hybridized to
unedited sequence downstream of the editing domain, so that both unedited and
edited extension products can be observed on the same gel. The FE/UE ratio was
found to vary from 1.3 to 2.0 fold for all four genes during the cell cycle.
The ratio peaked in S+G1 and then again in the same phase of the second
synchronized cycle. This variation is most likely due to variation in the
extent of editing. The level of this regulation is not known. This phenomenon
may have some relationship to the synchronicity of nuclear and mitochondrial
replication phases, which appears to involve a differential expression of
nuclear-encoded replication proteins due to differential turnover of mRNAs (see
papers from Dan Ray lab).
This work was performed by R. Carrillo, O. Thiemann and J. Alfonzo.
Isolation and Characterization of a U-specific 3'-5'
Exonuclease from Mitochondria of Leishmania tarentolae
We have purified a 3'-5' exoribonuclease from mitochondrial extract of
Leishmania tarentolae over 4000-fold through six column fractionations. This
enzyme digested RNA in a distributive manner, showed a high level of
specificity for 3' terminal U's and was blocked by a terminal dU or pCp; there
was a slight exonucleolytic activity on a 3' terminal A or C, but no activity
on a 3' terminal G residue. The enzyme preferred single-stranded oligo[U] 3'
and did not digest duplex RNA. Two other 3'-5' exoribonuclease activities were
also detected in the mitochondrial extract, one of which was stimulated by a
3'-phosphate and the other of which degraded RNAs with a 3'OH to
mononucleotides in a processive manner. The properties of the distributive
U-specific 3'-5' exoribonuclease suggest an involvement in the U-deletion RNA
editing reaction that occurs in the mitochondrion of these cells.
A and B show the U-specificity of the enzyme. C shows a
comparison of the purified exonuclease and the recombinant TUTase activities.
This work was performed by Ruslan Aphasizhev.
Guide RNAs of the recently isolated LEM 125 strain of
Leishmania tarentolae: an unexpected complexity
Guide RNAs (gRNAs) are encoded both in the maxicircle and minicircle
components of the mitochondrial DNA of trypanosomatid protozoa. These RNAs
mediate the precise insertion and deletion of U residues in transcripts of the
maxicircle DNA. We showed previously that the old UC laboratory strain of
Leishmania tarentolae apparently lost more than 40 minicircle-encoded gRNAs
which are present in the recently isolated LEM125 strain (Thiemann et al.,
1994). We have further analyzed the population of minicircle-encoded gRNAs in
the LEM125 strain. Sau3AI and MspI minicircle libraries were constructed and
screened for novel gRNAs by negative colony hybridization. This search yielded
20 minicircles encoding new gRNAs that covered most of the remaining gaps in
the editing cascades of the ND8, ND9, G3 and G4 genes, and in addition, more
than 30 minicircles containing either unassigned or undetectable gRNA genes. We
also completely sequenced 34 of the 45 minicircle sequence classes encoding
previously identified gRNAs. A total of 19 pairs of redundant gRNAs, which are
gRNAs of different sequences covering the same editing blocks, were identified.
The redundant gRNA pairs showed a differential steady-state abundance and
mismatches may represent one of the factors determining this abundance
differences. Alignments of the minicircles encoding redundant gRNAs yielded 59
to 93% matching nucleotides, suggesting an origin from duplication of ancestral
minicircles and subsequent genetic drift. We propose a functional explanation
for the existence of redundant gRNAs in this strain.
Differential localization of nuclear-encoded tRNAs
between the cytosol and mitochondrion in Leishmania tarentolae
All mitochondrial tRNAs of the kinetoplastid protozoan Leishmania
tarentolae are encoded in the nucleus and are imported from the cytosol into
the mitochondrion. We previously reported the partitioning of five tRNAs and
found that all were shared between the two compartments to different extents.
In order to increase our knowledge of the tRNAs of this organism, and to
attempt to understand the signals involved in their subcellular localization, a
method to RT-PCR amplify new tRNAs was developed. Various tRNAs were 3'
polyadenylated and reverse transcribed with a sequence tagged primer. The cDNA
was tagged by ligation to an anchor oligonucleotide, and the resulting
double-tagged cDNA was amplified by PCR. Four new tRNAs were obtained, bringing
to 20 the total number of L. tarentolae tRNAs identified to date. The
subcellular localization of 17 tRNAs was quantitatively analyzed by
two-dimensional gel electrophoresis and Northern hybridization. In general, the
previously suggested operational classification of tRNAs into three groups
(mainly cytosolic, mainly mitochondrial, and shared between the two
compartments) is still valid, but the relative abundance of each tRNA in the
cytosol or mitochondrion varies greatly as does the level of expression.
This work was performed by Steve Kapushoc and Juan Alfonzo.
Trypanosome Mitochondrial 3' Terminal Uridylyl
Transferase (TUTase): A Key Enzyme in U-insertion/deletion RNA Editing
A 3' terminal RNA uridylyltransferase was purified from mitochondria of
Leishmania tarentolae and the gene cloned and expressed from this species and
from Trypanosoma brucei. The enzyme is specific for 3' U-addition in the
presence of Mg++. TUTase is present in vivo in at least two stable
configurations: One contains a ~500 kDa TUTase oligomer and the other a ~700
kDa TUTase complex. Anti-TUTase antiserum specifically co-precipitates a small
portion of the p45 and p50 RNA ligases and approximately 40% of the guide RNAs.
Inhibition of TUTase expression in procyclic T. brucei by RNAi down-regulates
RNA editing and appears to affect parasite viability.
This work was performed by Ruslan Aphasizhev et al.
Modification of the universally unmodified uridine-33 in
a mitochondria-imported edited tRNA and the role of the anticodon arm structure
on editing efficiency
Editing of tRNA has a wide phylogenetic distribution among eukaryotes
and in some cases serves to expand the decoding capacity of the target tRNA. We
previously described C to U editing of the wobble position of the imported
tRNATrp in Leishmania mitochondria which is essential for decoding UGA codons
as tryptophan. Here we show the complete set of nucleotide modifications in the
anticodon arm of the mitochondrial and cytosolic tRNATrp as determined by
electrospray ionization mass spectrometry. This analysis revealed extensive
mitochondria-specific post-transcriptional modifications, including the first
example of thiolation of U33, the "universally unmodified" uridine. In light of
the known rigidity imparted on sugar conformation by thiolation, our discovery
of a thiolated U33 suggests that conformational flexibility is not a universal
feature of the anticodon structural signature. In addition, the in vivo
analysis of tRNATrp variants presented shows a single base pair reversal in the
anticodon stem of tRNATrp is sufficient to abrogate editing in vivo, indicating
that subtle changes in anticodon structure can have drastic effects on editing
This work was performed by Crain, P.F., Alfonzo, J.D., Rozenski, J.,
Kapushoc, S.T., McCloskey, J.A. and Simpson, L.
Wobble modification differences and subcellular
localization of tRNAs in Leishmania tarentolae: implication for tRNA sorting
In Leishmania tarentolae, all the tRNAs required for mitochondrial
translation are encoded in the nuclear genome and imported from the cytosol. It
is known that tRNAGlu(UUC) and tRNAGln(UUG) are localized in both cytosol and
mitochondria. We investigated structural differences between affinity-isolated
cytosolic (cy) and mitochondrial (mt) tRNAs for Glu and Gln by direct enzymatic
sequencing and mass spectrometric analysis. A unique modification difference in
both tRNAs was identified at the anticodon wobble position: cy tRNAs have
5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U), whereas mt tRNAs have 5-
methoxycarbonylmethyl-2'-O-methyluridine (mcm5Um). In addition, a trace portion
(4%) of cy tRNAs was found to have 5-methoxycarbonylmethyluridine (mcm5U) at
its wobble position, which could represent a common modification intermediate
for both modified uridines in cy and mt tRNAs. We also isolated a trace amount
of mitochondria-specific tRNALys(UUU) from the cytosol and found mcm5U at its
wobble position, while its mitochondrial counterpart has mcm5Um. We found that
mt tRNALys and in vitro-transcribed tRNAGlu were imported much more efficiently
into isolated mitochondria than the native cy tRNAGlu in an in vitro
importation assay, indicating that cytosol specific 2-thiolation could play an
inhibitory role in tRNA import into mitochondria.
This work is the result of a collaboration and was performed by
Tomonori Kaneko, Takeo Suzuki, Stephan T. Kapushoc, Mary Anne Rubio, Jafar
Ghazvini, Kimitsuna Watanabe and Tsutomu Suzuki
A 100 kDa complex of two RNA-binding proteins from
mitochondria of Leishmania tarentolae catalyzes RNA annealing and interacts
with several RNA editing components
A stable 100 kD complex from mitochondria of Leishmania tarentolae
containing two RNA-binding proteins, Ltp26 and Ltp28, was identified by
cross-linking to unpaired 4-thiouridine nucleotides in a partially duplex RNA
substrate. The genes were cloned and expressed and the complex was
reconstituted from recombinant proteins in the absence of RNA or additional
factors. The Ltp26 and Ltp28 proteins are homologues of gBP27 and gBP29 from
Crithidia fasciculata, and gBP25 and gBP21 from Trypanosoma brucei,
respectively. The purified Ltp26/Ltp28 complex, the individual recombinant
proteins and the reconstituted complex are each capable of catalyzing the
annealing of complementary RNAs, as was previously shown for gBP21 from T.
brucei. A high molecular weight RNP complex consisting of the Ltp26/Ltp28
complex and several 55-60 kDa proteins together with guide RNA could be
purified from mitochondrial extract of L. tarentolae transfected with
Ltp28-TAP. This complex also interacted in a less stable manner with the RNA
ligase-containing L- complex and with the 3' TUTase. The Ltp26/Ltp28 RNP
complex is a candidate for catalyzing the annealing of guide RNA and pre-edited
mRNA in the initial step of RNA editing.
Update 6-16-2003: These proteins are now labeled MRP1 and MRP2 and the
complex is labeled MRP1/2.
This work was performed by R. Aphasizhev, I. Aphasizhev and R.
Is the Trypanosoma brucei REL1 RNA ligase specific for
U-deletion RNA editing and the REL2 RNA ligase specific for U-insertion
It was shown previously that the REL1 mitochondrial RNA ligase in
Trypanosoma brucei was a vital gene and disruption affected RNA editing in vivo
whereas the REL2 RNA ligase gene could be down regulated with no effect on cell
growth or on RNA editing. We performed down regulation of REL1 in procyclic T.
brucei (midgut insect forms) by RNAi and found a 40-50% inhibition of Cyb
editing, which has only U-insertions, as well as a similar inhibition of ND7
editing, which has both U-insertions and U-deletions. In addition, both
U-insertion and U-deletion in vitro pre-cleaved editing were inhibited to
similar extents. We also found little if any effect of REL1 down regulation on
the sedimentation coefficient or abundance of the RNA ligase-containing
L-complex (Aphasizhev et al. (2003) EMBO J. 22:913-924), suggesting that the
inhibition of both insertion and deletion editing was not due to a disruption
of the L-complex. Together with the evidence that down regulation of REL2 has
no effect on cell growth or on RNA editing in vivo or in vitro, these data
suggest that the REL1 RNA ligase may be active in vivo in both U-insertion and
U-deletion editing. The in vivo biological role of REL2 remains obscure.
This work was performed by G. Gao.
Isolation of an RNA Editing Complex Active in Both
U-insertion and U-deletion in vitro Editing
A multiprotein, high molecular weight complex active in both
U-insertion and U-deletion as judged by a pre-cleaved RNA editing assay was
isolated from mitochondrial extracts of Leishmania tarentolae by the tandem
affnity purification (TAP) procedure, using three different TAP-tagged proteins
of the complex. This editing- or E-complex consists of at least three
protein-containing components interacting via RNA: the RNA ligase-containing
L-complex, a 3' TUTase (terminal uridylyltransferase) and two RNA-binding
proteins, Ltp26 and Ltp28. Thirteen approximately stoichiometric components
were identified by mass spectrometric analysis of the core L-complex: two RNA
ligases; homologs of the four Trypanosoma brucei editing proteins; and seven
novel polypeptides, among which were two with RNase III, one with an AP
endo/exonuclease and one with nucleotidyltransferase motifs. Three proteins
have no similarities beyond kinetoplastids.
shows the isolated L-complex sediments as a single band in a glycerol gradient
and contains around 15 proteins.
This work was performed by Ruslan Aphasizhev, Inna Aphasizheva, Robert
E.Nelson, Guanghan Gao, Agda M.Simpson, Xuedong Kang, Arnold M.Falick, and
RBP38, an RNA-binding protein from trypanosomatid
mitochondria, modulates RNA stability
A novel RNA-binding protein, RBP38, was isolated from Leishmania
tarentolae mitochondria. This protein does not contain any known RNA binding
motifs and is highly conserved among the trypanosomatids but no homologues were
found in other organisms. Recombinant LtRBP38 binds single and double stranded
RNA substrates with dissociation constants in the 100 nanomolar range, as
determined by fluorescence polarization analysis. Down regulation of expression
of the homologous gene, TbRBP38, in procyclic T. brucei using conditional RNAi
resulted in 80% reduction of steady-state levels of RNAs transcribed from both
maxicircle and minicircle DNA. In organello pulse-chase labeling experiments
were used to determine the stability of RNAs in mitochondria that were depleted
of TbRBP38. The half-life of metabolically labeled RNA decreased from ~160 min
to ~60 min after depletion. In contrast, there was no change in transcriptional
activity. These observations suggest a role of RBP38 in stabilizing
RNAi down regulation of RBP38 is shown on the left. The effect of down
regulation on RNA stability in organello is shown on the right.
This work was performed by Sandro Sbicego, Juan D. Alfonzo, Antonio M.
Estévez, Mary Anne T. Rubio, Xuedong Kang, Christoph W. Turck and Marian
Genomic Organization of Trypanosoma brucei Kinetoplast
The sequences of seven new Trypanosoma brucei kinetoplast DNA
minicircles were obtained. A detailed comparative analysis of these sequences
and those of the 18 complete kDNA minicircle sequences from T. brucei available
in the database was performed. These 25 different minicircles contain 86
putative gRNA genes. The number of gRNA genes per minicircle varies from 2 to
5. In most cases, the genes are located between short imperfect inverted
repeats, but in several minicircles there are inverted repeat cassettes that
did not contain identifiable gRNA genes. Five minicircles contain single gRNA
genes not surrounded by identifiable repeats. Two pairs of closely related
minicircles may have recently evolved from common ancestors: KTMH1 and KTMH3
contained the same gRNA genes in the same order, whereas KTCSGRA and KTCSGRB
contained two gRNA genes in the same order and one gRNA gene specific to each.
All minicircles could be classified into two classes on the basis of a short
substitution within the highly conserved region, but the minicircles in these
two classes did not appear to differ in terms of gRNA content or gene
organization. A number of redundant gRNAs containing identical editing
information but different sequences were present. The alignments of the
predicted gRNAs with the edited mRNA sequences varied from a perfect alignment
without gaps to alignments with multiple mismatches. Multiple gRNAs overlapped
with upstream gRNAs, but in no case was a complete set of overlapping gRNAs
covering an entire editing domain obtained. We estimate that a minimum set of
approximately 65 additional gRNAs would be required for complete overlapping
sets. This analysis should provide a basis for detailed studies of the
evolution and role in RNA editing of kDNA minicircles in this species.
This work was done by Min Hong.
The RET1 TUTase adds U's to the gRNA 3' end and the RET2
TUTase adds U's at editing sites of mRNAs
We have described two TUTases, RET1 (RNA Editing TUTase 1) and RET2
(RNA Editing TUTase 2) as components of different editing complexes. TAP-tagged
Trypanosoma brucei RET2 was expressed and localized to the cytosol in
Leishmania tarentolae cells by removing the mitochondrial signal
sequence. Double affinity isolation yielded tagged TbRET2 together with a few
additional proteins. This material exhibits a U-specific transferase activity
in which a single uridine is added to the 3' end of a single-stranded RNA,
thereby confirming that RET2 is a 3' TUTase.
We also found that RNA interference of RET2 expression in T.
brucei inhibits in vitro U-insertion editing and has no effect on the
length of the 3'-oligo[U] tails of the gRNAs, whereas down regulation of RET1
has a minor effect on in vitro U-insertion editing but produces a decrease in
the average length of the oligo[U] tails.
This suggests that RET2 is responsible for U-insertions at editing
sites and RET1 is involved in gRNA 3'-end maturation, which is essential for
creating functional gRNAs. From these results we have functionally relabelled
the previously described TUT-II complex containing RET1 as the GP-complex
(Guide RNA Processing).
This work was done by Ruslan Aphasizhev and Inna Aphasizheva.
here to return to Simpson lab main web site.
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