Is the Trypanosoma brucei REL1 RNA Ligase Specific for U-deletion RNA Editing, and Is the REL2 RNA Ligase Specific for U-insertion Editing?*
Guanghan Gao
and
Larry Simpson
¶
From the
Howard Hughes Medical Institute,
University of California, 6780 MacDonald Research Laboratories, Los Angeles,
California 90095 and
Department of Microbiology,
Immunology and Molecular Genetics, University of California, Los Angeles,
California 90095
Received for publication, March 31, 2003
, and in revised form, May 13, 2003.
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ABSTRACT
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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 RNA interference and found a 4050% 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, R., Aphasizheva, I., Nelson, R. E., Gao, G., Simpson, A. M.,
Kang, X., Falick, A. M., Sbicego, S., and Simpson, L. (2003) EMBO J.
22, 913924), 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.
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INTRODUCTION
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Uridine
(U)1-insertion/deletion
RNA editing occurs in the mitochondrion of kinetoplastid protozoa
(1,
2). The mechanism in all cases
but one involves the annealing of a trans-acting guide RNA to the pre-edited
mRNA just downstream of the initial editing site, specific cleavage of the
mRNA at the unpaired editing site, deletion of unpaired uridines or addition
of uridines to the 3' end of the 5' cleavage fragment, which can
form base pairs with guiding A or G nucleotides in the guide RNA, and ligation
of the 5' and 3' cleavage fragments
(38).
Two mitochondrial RNA ligases have been identified in Trypanosoma
brucei
(911).
Conditional disruption of both alleles of the REL1 ligase in bloodstream
T. brucei was lethal and affected in vivo RNA editing
(12). Conditional expression
of a mitochondrial targeted REL1 transgene with a K86R mutation at
the AMP-binding site in procyclic T. brucei also produced a growth
phenotype and disrupted RNA editing
(13). Significant effects on
ND7 and MURF2 editing were reported, both of which involve
both U-insertions and U-deletions, but no detectable effects on Cyb
and COII editing were reported, both of which involve only
U-insertions (13). Full round
in vitro U-deletion editing using mitochondrial extract from cells
expressing the dominant negative REL1 transgene was inhibited
50%, whereas U-insertion editing was apparently unaffected
(13). It was also reported
that more ATP was required for in vitro U-deletion editing than for
U-insertion editing, and that this correlated with the levels of ATP required
for adenylation of REL1 and REL2, respectively. In addition, we showed that
overexpression of tandem affinity purification-tagged REL1 in Leishmania
tarentolae causes the appearance of a minor L-subcomplex containing REL1,
LC-3, LC6a, and LC-4, which is the homologue of MP63 in T. brucei
(14),2
and others have found another L-subcomplex consisting of REL2, RET2, and
MP81.3
These observations led to the model that REL1 mediates ligation at
U-deletion editing sites and REL2 mediates ligation at U-insertion sites
(13,
15). The fact that
down-regulation of expression of the REL2 RNA ligase by conditional RNAi in
procyclic or bloodstream T. brucei showed no phenotype, either in
terms of cell growth or editing
(16), was interpreted as a
substitution of the REL2 role by REL1, although the reverse does not
occur.
In this paper, we have re-analyzed the effects of RNAi down-regulation of
REL1 and REL2 in terms of this hypothesis and we conclude that the situation
may be more complex than originally envisioned.
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EXPERIMENTAL PROCEDURES
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Trypanosome Culture and RNAiTo construct the pREL1-H2H
vector for inducible RNAi, two 495-bp PCR fragments from the 5' end of
the T. brucei REL1 coding region were inserted in a head-to-head
configuration adjacent to a green fluorescent protein stuffer fragment under
the control of a tetracycline-regulatable procyclic acidic repetitive protein
promoter (17). The vector was
transfected into procyclic T. brucei strain 2913 cells
(18), and drug-resistant cell
clones were selected by plating on agarose
(19). Cells were cultured in
SDM-79 medium, and RNAi was induced with 1 µg/ml tetracycline. Cells were
maintained in log phase growth by daily dilution. The pREL2-H2H vector was
constructed similarly with a 456-nucleotide fragment (nucleotides
69524) of REL2 in a head to head configuration.
Oligonucleotides used for PCR of REL1 and REL2 (added restriction sites are
in boldface) are as follows: for REL1, 5076, 5'-AAG CTT
ATG CAA CTC CAA AGG TTG GG-3' (1st forward primer with
HindIII site); 5077, 5'-TCT AGA ATA CTT GGC ACC AAA CAG
TT-3' (1st reverse primer with XbaI site); 5078,
5'-GGA TCC ATG CAA CTC CAA AGG TTG GG-3' (2nd
forward primer with BamHI site); and 5079, 5'-GGA TCC
ATA CTT GGC ACC AAA CAG TT-3' (2nd reverse primer with
BamHI site); for REL2, 5093, 5'-AAG CTT CAT TTT
TGA GCG CTA CAC AG-3' (1st forward primer with
HindIII site); 5094, 5'-GCT CTA GAA TGT CGA ATG CGT AAA
AGT G-3' (1st reverse primer with XbaI site); 5095,
5'-GGA TCC CAT TTT TGA GCG CTA CAC AGA-3' (2nd
forward primer with BamHI site); and 5096, 5'-GGG ATC
CAT GTC GAA TGC GTA AAA GTG-3' (2nd reverse primer with
BamHI site).
Protein Expression and Western BlottingThe full-length
REL1 and REL2 genes were cloned into the pMAL c2x vector
(New England Biolabs). The plasmids were transformed into Escherichia
coli BL21 DE3 (Stratagene). Expression was induced with 0.3 mM
isopropyl-1-thio-
-D-galactopyranoside at 37 °C for 3 h.
The recombinant REL1 and REL2 proteins fused with the maltose-binding protein
were purified by binding to amylose resin (New England Biolabs) and elution
with 10 mM maltose. The recombinant proteins were further purified
by SDS-PAGE gel electrophoresis. Polyclonal antibodies against REL1 and REL2
were prepared by Covance Research Products Inc. Monoclonal antibodies against
the T. brucei MP81, MP63, and MP42 proteins were kind gifts from Ken
Stuart. Western blotting was performed using the Super-Signal West Pico
chemiluminescent system (Pierce).
RNA AnalysisTotal RNA was purified by the acid guanidium
isothiocyanate method (20).
Primer extension was performed as described previously
(21). Cyb
(oligonucleotide 3812) and Murf2 (oligonucleotide 3807) mRNAs were
analyzed by run-off extensions. COI (oligonucleotide 3808),
COII (oligonucleotide 3809), ND7 (oligonucleotide 4282),
A6 (oligonucleotide 3882), and calmodulin (oligonucleotide 3813)
mRNAs were analyzed by poisoned primer extension using ddGTP to
chain-terminate the extensions at the first C residue after 26 editing sites
for A6, the entire editing domain for COII, and 9 editing
sites for ND7 (22).
For normalization, oligonucleotides 3813 and 3808 for the cytosolic calmodulin
mRNA and the never-edited COI mRNA were extended in the same
reaction. The following oligonucleotides were used: 3807,
5'-CAACCTGACATTAAAAGAC-3'; 3808,
5'-GTAATGAGTACGTTGTAAAACTG-3'; 3809,
5'-ATTTCATTACACCTACCAGG-3'; 3812,
5'-GTTCTAATACATAACAAATCAAAAACACG-3'; 3813,
5'-GTTGATCGGCCATCGTAAATCAAGTGGATG-3'; 3882,
5'-ATAAACTAGAATAAGATATTGAGG-3'; and 4282,
5'-CTTTTCTGTACCACGATGC-3'.
Northern AnalysisTotal RNA (30 µg) was fractionated on a
1.5% agarose-formaldehyde gel in 20 mM MOPS, 5 mM sodium
acetate, and 1 mM EDTA, pH 7.0, and the gel was blotted onto a
Zeta-probe membrane (Bio-Rad). The filter was hybridized with full-length
REL1 or REL2 PCR-amplified DNA labeled with
[
-32P]ATP using the Prime-It II random primer labeling kit
(Stratagene).
Extract Preparation, Glycerol Gradient Sedimentation, and Native Gel
ElectrophoresisPurified mitochondria (25 mg protein/ml) were lysed
with 0.5% Nonidet P-40 in 10 mM Tris-HCl, pH 8.0, 10 mM
MgCl2, and 60 mM KCl. The clarified extract (300 µl)
was centrifuged on a 1030% glycerol gradient in the SW41 rotor
(Beckman) for 20 h at 30,000 rpm, and 150.75-ml fractions were collected from
the top using the Isco density gradient fractionator. Aliquots (10 µl) of
each fraction were mixed with 0.5 µl (10 µCi/µl) of
[
-32P]ATP for 30 min at 27 °C. The reaction was mixed
with SDS loading buffer for denaturing gradient gel analysis, and the gels
were blotted onto nitrocellulose membranes (Protran) for Western analysis. For
both adenylation (23) and
Western blot analysis, an aliquot from fraction 9 of an identical gradient of
mitochondrial extract from untransfected 2913 cells was used as an
internal loading control in each gel. Sedimentation values were calculated
using aldolase (9 S), thyroglobulin (19 S) and E. coli ribosome 30 S
subunits.
RNA Substrates and in Vitro EditingThe following RNA
substrates were chemically synthesized (Oligos Etc. and Xeragon) and
gel-purified: 5' fragment, 5'-GCACUACACGAUAAAUAUAAAAAG-3';
5'-UU fragment, 5'-GCACUACACGAUAAAUAUAAAAAGUU-3'; 3'
fragment, 5'-AACAUUAUGCUUCUUCGddC-3'; AG brRNA,
5'-AAGAAGCAUAAUGUUAGCUUUUUAUAUUUAUCGUGUAGUCddG-3'; and 0 brRNA,
5'-AAGAAGCAUAAUGUUCUUUUUAUAUUUAUCGUGUAGUCddG-3'.
RNAs were 5'-phosphorylated with T4 polynucleotide kinase
(Invitrogen) and [
-32P]ATP. Complementary RNAs were annealed
by heating and slow-cooling. For in vitro editing assays, the
L-complex fractions
(810)
were pooled and concentrated to 300 µl. The concentrated gradient fractions
were stored at 20 °C in 50% glycerol, 1 mg/ml bovine serum albumin,
and 1 mM dithiothreitol. The fractions were fractionated by
electrophoresis on 816% denaturing gradient gels, and the gels were
stained with Coomassie Blue (Sigma) to monitor the protein concentrations. The
in vitro editing reactions were performed at 27 °C for 2 h in 20
µl of 50 mM Tris-HCl, pH 7.5, 10 mM MgCl2,
1 mM dithiothreitol, and 1 mM ATP. 2 mM UTP
was added for the insertion reaction. The products were separated in a
sequencing gel and detected by autoradiography.
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RESULTS
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Down-regulation of REL1 Ligase Expression Inhibits Both U-insertion and
U-deletion Editing in VivoDown-regulation of REL1 by conditional
RNAi produced a growth inhibition by day 6 of induction
(Fig. 1A), which was
preceded by a selective degradation of REL1 mRNA and down-regulation
of REL1 protein expression by day 3 (Fig.
1B). A correlated down-regulation of the MP63 L-complex
protein was also observed (Fig.
1C). The relative abundances of edited and pre-edited
mRNAs were examined for five genes by primer extension analysis
(Fig. 2). Editing of
Cyb and ND7 mRNAs decreased 4070% after 7 days of
RNAi induction, and editing of COII, MURF2, and A6 mRNAs
decreased slightly. There was no change in the abundance of pre-edited RNAs or
the never-edited COI mRNA, suggesting a specific effect on
editing.

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FIG. 1. A, effect of tetracycline induction of RNAi of REL1 and REL2
expression on growth of procyclic T. brucei. The cells were
maintained in log phase by daily dilutions. The cumulative number of cell
divisions is plotted versus the time after the addition of
tetracycline (tet). Open triangles, REL1 RNAi + tet;
filled triangles, REL1 RNAi no tet; open circles, REL2 RNAi
+ tet; filled circles, REL2 RNAi no tet. B, specific
degradation of REL1 mRNA by induction of REL1 RNAi and specific degradation of
REL2 mRNA by induction of REL2 RNAi. Total cell RNA was isolated from cells 0,
3, and 7 days after addition of tetracycline and fractionated by
electrophoresis. The gels were blotted, and the blots were hybridized with a
labeled REL1 probe (left panel) or a labeled REL2 probe (right
panel). Hybridization of -tubulin mRNA was used as a loading
control. C, specific down-regulation of REL1 protein expression by
REL1 RNAi and REL2 protein expression by REL2 RNAi. Mitochondrial extract from
2913 cells and from the transfected cells 0, 3, or 5 days after the
addition of tetracycline was fractionated by SDS acrylamide gel
electrophoresis. Western analysis was performed using antibodies against the
MP81, MP63, REL1, and REL2 proteins. Left, REL1 RNAi. Right,
REL2 RNAi.
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FIG. 2. Effect of REL1 and REL2 RNAi on RNA editing in vivo. The
abundance of pre-edited and edited mRNAs was assayed using primers downstream
of the editing domains. A, primer extensions of never-edited
COI mRNA and cytosolic calmodulin mRNA were used as loading controls.
B, ND7 editing. C, Cyb editing. D, COII editing.
E, A6 editing. F, MURF2 editing. Quantitation of the
autoradiographs are shown below each figure. The ordinate represents the
percent of editing (edited band/the edited plus pre-edited bands x 100).
The fully edited band is indicated by E, and the pre-edited band is
indicated by P.
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As reported previously
(16), down-regulation of REL2
by RNAi produced no growth phenotype (Fig.
1A) or editing phenotype
(Fig. 2), although there was a
selective decrease of REL2 mRNA by day 3
(Fig. 1B) and REL2
protein by day 3 (Fig.
1C).
Down Regulation of REL1 Affects Both U-insertion and U-deletion
Pre-cleaved in Vitro EditingMitochondrial extracts from cells
induced for REL1 RNAi for 0, 3, or 5 days were tested for pre-cleaved in
vitro editing activity (Fig.
3). U-insertion editing and U-deletion editing were inhibited to
similar extents. There was no effect on the addition or deletion of
3'-terminal uridines to or from the 5'-mRNA cleavage fragment. In
the case of REL2 RNAi, there were no detectable changes in the extent of
U-insertion or U-deletion pre-cleaved in vitro editing
(Fig. 3).

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FIG. 3. Effect of REL1 RNAi and REL2 RNAi on pre-cleaved in vitro
editing. Both +2U-guided U-insertion and 2U-guided U-deletion
editing were assayed. Mitochondrial extract was isolated from untransfected
2913 cells and from transfected cells 0, 3, and 5 days after the
addition of tetracycline. The extracts were fractionated in glycerol gradients
and fractions 810 (of 15 total fractions) containing the peak of the
L-complex and were pooled and concentrated. The concentrated fractions were
used for in vitro editing assays. Input lane, no enzyme. The
relative amounts of the fully edited species were determined by PhosphorImager
analysis, and the quantitation is plotted beneath the figures. The ordinate
represents the percent of editing (mature edited band/input band x
100).
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Down Regulation of REL1 Has No Effect on the S Value or Abundance of
the L-ComplexOne possible explanation for the inhibition of both
U-insertion and U-deletion in vitro editing by down-regulation of
REL1 would be that there was a decrease in stability of the
L-complex. In fact, it was previously reported that knocking out of one
REL1 allele in T. brucei procyclics produced a partial
breakdown of the L-complex
(13). To test this
possibility, mitochondrial extract from cells induced for REL1 RNAi for 0, 3,
or 5 days was fractionated in a glycerol gradient and each fraction was
incubated with [
-32P]ATP to label the REL1 and REL2 RNA
ligases, which represent markers for the L-complex. It should be noted that
the REL1 ligase was labeled to a greater extent than the REL2 ligase because
of the latter being pre-charged with AMP
(11). The S value of the
L-complex did not change as the REL1 protein decreased in abundance
(Fig. 4A). Western
analysis of each fraction using antibodies against MP81, MP63, and REL1 was
also performed (Fig.
4A). Despite the loss of detectable REL1 protein, the
L-complex was unaffected in terms of location in the gradient and relative
abundance. Down-regulation of REL2 likewise had no effect on the S value or
relative abundance of the L-complex (Fig.
4B).

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FIG. 4. Effect of REL1 RNAi and REL2 RNAi on S value and relative abundance of
L-complex. The direction of sedimentation is indicated by an arrow.
A, REL1 RNAi. Mitochondrial extracts from transfected cells 0, 3, and 5
days after addition of tetracycline were fractionated in glycerol gradients
and aliquots (10 µl) of each fraction electrophoresed in an SDS acrylamide
gel, which was blotted for Western analysis. 81, 63, and
REL1 represent antibodies against MP81, MP63, and REL1, respectively.
The lower panel in each case is an autoradiograph of an identical gel
of 10-µl aliquots of each fraction labeled with
[ -32P]ATP. The adenylated REL1 and REL2 bands are seen. The
location of the L-complex is indicated by brackets. Lane C, fraction
9 from gradient of mitochondrial extract from 2913 control cells.
B, REL2 RNAi. See A for details.
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DISCUSSION
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The presence of two mitochondrial RNA ligases, both of which are components
of the L-complex, has raised an interesting question. Several lines of
evidence have led to the model that REL1 is involved in U-deletion editing and
REL2 is involved in U-insertion editing
(13,
15,
24). The complete absence of
any phenotype with REL2 down-regulation has been explained by the suggestion
that REL1 can substitute for REL2 in the REL2 down-regulated cells and mediate
both types of editing (13,
15). The lethality of the REL1
knock-out has been explained by assuming that REL2 is not capable of
substituting for REL1 in U-deletion editing.
The results presented in this paper raise some doubts about this model. We
showed that down-regulation of REL1 expression affected the in vivo
editing of different mRNAs to different extents, but the inhibitory effect was
apparently correlated with the percentage of editing of the specific mRNA
rather than with the presence of U-insertions alone or U-insertions and
U-deletions. For example, the editing of Cyb mRNA, which only
involves U-insertions, was inhibited to a similar extent as the editing of
ND7 mRNA, which involves both U-insertions and U-deletions. The
editing of A6, MURF2, and COII mRNAs, the former two
involving both insertions and deletions and the latter involving only
insertions, was inhibited to a lesser extent, both decreasing
20% by day
7. The magnitude of the effect on editing appeared to be inversely correlated
with the normal extent of editing, in that A6, MURF2, and
COII editing is normally very efficient (>90% edited RNA), whereas
Cyb and ND7 editing is less efficient (
70% edited RNA).
The reason for this variation in the extent of inhibition of editing with
different mRNAs is not known. Furthermore, we showed that pre-cleaved
U-insertion and U-deletion in vitro editing were inhibited to similar
extents. These results differ from those reported previously both in terms of
the effect on Cyb mRNA editing in vivo and full-round in
vitro editing (13), the
reasons for which are not clear but may be related to the different modes of
gene knockdown and to the different in vitro assays. We eliminated
the possibility that RNAi down-regulation of REL1 affected the overall
stability of the L-complex and thereby also affected U-insertion editing by
showing that there was no large change in S value or abundance of the
L-complex in the REL1 RNAi-induced cells. We conclude that the simplest
interpretation is that REL1 performs ligations for both U-insertion and
U-deletion editing and that REL2 is less active or even inactive in
vivo, at least under these physiological conditions. We realize that the
simplest interpretation is not always valid and that, for example,
down-regulation of REL1 may affect the functional interaction of REL2 with
other L-complex proteins and thereby also affect U-insertion editing, but this
would have to be established. It is clear from both the apparently functional
suborganization of proteins in the L-complex
(14) and from the striking ATP
titration correlations (15)
that REL1 and REL2 probably have differing biological roles, but the precise
nature of these roles is yet unclear. We speculate that REL2 may be active
under different physiological or developmental conditions. The precise roles
of these two RNA ligases await reconstitution of activities with recombinant
proteins.
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FOOTNOTES
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* The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section 1734
solely to indicate this fact. 
¶
To whom correspondence should be addressed: UCLA, 6780 MacDonald Research
Laboratories, 675 Charles Young Dr., S., Los Angeles, CA 90095. E-mail:
simpson{at}kdna.ucla.edu.
1 The abbreviations used are: U, uridine; RNAi, RNA interference; MOPS,
4-morpholinepropanesulfonic acid; ND, NADH dehydrogenase; MURF, maxicirels
unidentified reading frame. 
2 R. Aphasizhev and L. Simpson, unpublished results. 
3 K. Stuart, A. Panigrahi, and A. Schnaufer, personal communication. 
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ACKNOWLEDGMENTS
|
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We thank Agda Simpson for performing the initial transfections and Ken
Stuart for the gift of the monoclonal antibodies. We thank all members of the
Simpson laboratory for advice and discussion, especially Ruslan Aphasizhev for
assistance with the in vitro editing assay.
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