(Received for publication, December 7, 1994; and in revised form, January 23, 1995)
From the
RNA editing in Trypanosoma brucei results in the
addition and deletion of uridine residues within several mitochondrial
mRNAs. Editing is thought to be directed by guide RNAs and may proceed
via a chimeric guide RNA/mRNA intermediate. We have previously shown
that chimera-forming activity sediments with 19 S and 35-40 S
mitochondrial ribonucleoprotein particles (RNPs). In this report we
examine the involvement of RNA ligase in the production of chimeric
molecules in vitro. Two adenylylated proteins of 50 and 57 kDa
cosediment on glycerol gradients with RNA ligase activity as components
of the ribonucleoprotein particles. The two adenylylated proteins
differ in sequence and contain AMP linked via a phosphoamide bond. Both
proteins are deadenylylated by the addition of ligatable RNA substrate
with the concomitant release of AMP and by the addition of
pyrophosphate to yield ATP. Incubation with nonligatable RNA substrate
results in an accumulation of the adenylylated RNA intermediate. These
experiments identify the adenylylated proteins as RNA ligases. AMP
release from the mitochondrial RNA ligase is also concomitant with
chimera formation. Inhibition by nonhydrolyzable analogs indicates that
both RNA ligase and chimera-forming activities require -
bond
hydrolysis of ATP. Deadenylylation of the ligase inhibits chimera
formation. These results strongly suggest the involvement of RNA ligase
in in vitro chimera formation and support the
cleavage-ligation mechanism for kinetoplastid RNA editing.
In kinetoplastid protozoa, RNA editing is a post-transcriptional
RNA-processing event that occurs in the mitochondrion and results in
the addition and deletion of uridine residues at specific sites in mRNA
transcripts. Editing produces the sequence information necessary for
functional RNAs by correcting frameshifts, creating start codons, or in
some cases, forming complete reading
frames(1, 2, 3, 4) . The genetic
information necessary to direct the insertion or deletion of uridine
residues in these mRNAs is present in small, 55-70-nucleotide
primary transcripts called guide RNAs (gRNAs). ()The gRNAs
are complementary to the edited mRNAs and contain a nonencoded poly(U)
tail of 5-15
nucleotides(5, 6, 7, 8, 9) .
The uridine tail may donate or accept uridines during the editing
process through the formation of a chimeric gRNA/mRNA molecule
consisting of the gRNA covalently linked via the poly(U) tail to the 3`
segment of the
mRNA(10, 11, 12, 13) . The gRNA also
contains a short 7-10-nucleotide anchor region at its 5`-end
which is complementary to the pre-edited sequences immediately 3` to
the editing site. The anchor region recognizes and base pairs with
unedited mRNA and directs the initiation of the editing process.
Uridine addition and deletion continues until the mRNA is fully
complementary to the gRNA.
Based on the identification of chimeric molecules in vivo(10) and in vitro(11, 12, 13) , two models have been proposed for the mechanism of kinetoplastid mRNA editing. The first, similar to the mechanism of mRNA splicing, involves successive rounds of trans-esterification(10, 14) . In this model the 3`-hydroxyl of the poly(U) tail of the guide makes a nucleophilic attack on the phosphodiester bond at the editing site, producing a free 5` mRNA segment and a chimeric RNA. This chimera is resolved in a second trans-esterification event involving the 3`-hydroxyl of the free 5` mRNA segment to produce an edited mRNA that has one or more uridines inserted or deleted and a gRNA that has the corresponding change in the length of its poly(U) tail.
The second model proposes multiple rounds of cleavage-ligation and draws analogies from the mechanism of tRNA splicing. In this scheme, the gRNA-mRNA chimera is formed by endonuclease and RNA ligase activities(15) . The pre-edited mRNA is cleaved by an endonuclease at the editing site and RNA ligase joins the gRNA to the 3`-fragment of the mRNA producing the chimeric molecule. The chimera is resolved by a second round of cleavage and ligation.
Support for the cleavage-ligation mechanism comes from the identification of mitochondrial RNPs which are thought to be involved in RNA editing(16) . These were shown by glycerol gradient sedimentation of mitochondrial extract to consist of two ribonucleoprotein particles which sedimented as 19 S and 35-40 S complexes containing RNA ligase, chimera formation, and terminal uridylyl-transferase activities. Uridylyl-transferase presumably is required for addition of the nonencoded poly(U) tail to the gRNA. These results led us to examine the role of the RNA ligase in chimera formation.
The mechanism of RNA ligation has been studied for
ligases from wheat germ(17, 18) , yeast(19) ,
and T4-infected Escherichia coli cells (reviewed in (20) ). Even though the enzymes differ in the structures of
their substrates and products, their overall mechanisms are quite
similar. The T4 enzyme, for example, joins RNAs with a 5`-phosphate to
3`-hydroxyl termini in three distinct and reversible steps:1. E + ATP &lrhar2; E-AMP +
PP
2. E-AMP + pN- &lrhar2; E + AppN-
3. -N + AppN- &lrhar2; -NpN-
+ AMP
The first step involves the adenylylation of the enzyme (E), by the transfer of AMP from ATP, via phosphoamide linkage with a lysine residue on the protein. In the second step, AMP from the adenylylated enzyme is transferred to the 5`-phosphate of the RNA molecule forming an activated RNA molecule with a 5`,5`-phosphoanhydride bond. In the third and final step, the 3`-hydroxyl of the same or different RNA molecule attacts the activated RNA forming a phosphodiester bond and releasing AMP.
In this report we provide the first direct evidence for the involvement of mitochondrial RNA ligase in the production of RNA editing intermediates. The RNA ligase activity present in the mitochondria of Trypanosoma brucei has similar substrate requirements and the same overall mechanism as previously characterized RNA ligases. We identify adenylylated RNA ligase intermediates in the 19 S and 35-40 S complexes, along with chimera activity, and demonstrate their involvement in the mechanism of in vitro chimera formation. The involvement of RNA ligase in the in vitro reaction provides strong evidence for a cleavage-ligation type mechanism of RNA editing.
Mitochondria were isolated as described by Braley et al.(25) and Rohrer et al.(26) .
Mitochondrial extract was prepared and sedimented on a 10-30%
glycerol gradient as described by Pollard et al.(16) except that 1 10
cell
equivalents of mitochondria were loaded onto each gradient and
centrifuged for 8 h.
Free nucleotides were also removed by dialysis. The extract, which
was adenylylated with [-
P]ATP and chased
with ATP as described above, was dialyzed against 1 liter (three
changes) of 25 mM HEPES, pH 7.9, 10 mM magnesium
acetate, 50 mM potassium chloride, 5% glycerol, 0.4 mg/ml
bovine serum albumin, and 0.1% Triton X-100 at 4 °C for 16 h.
Incubation of the
isolated adenylylated proteins (as described in Fig. 2, B and C) also resulted in the formation of an adenylylated
RNA intermediate. However, with the use of extract directly off the
glycerol gradient and excess [-
P]ATP, we
were able to increase the yield for further analysis.
Figure 2:
Identification of the adenylylated
proteins as RNA ligase intermediates. Preadenylylated extract (with
[-
P]ATP and ATP) was isolated by gel
filtration and utilized in the RNA ligase assay as described under
``Experimental Procedures.'' A, reactions contained
15,000 cpm of 5`-
P ligatable (Lig.) RNA substrate (lanes 1 and 3) or 5`-
P nonligatable (non-Lig.) RNA substrate (lanes 2 and 4).
Control reactions consisted of the RNAs incubated in the absence of
extract (lanes 3 and 4). Ligation products
corresponding to linear (L) and circular (C)
molecules are indicated. B and C, ligase reactions
were performed as above except the extract was incubated in the absence (lanes 1 and 2) or presence of unlabeled 5`-P
ligatable (lanes 3 and 4) or nonligatable (lanes
5 and 6) RNA substrates. Aliquots (10 µl) were taken
at the start (lanes 1, 3, and 5) and end (lanes 2, 4, and 6) of the 90-min incubation
at 26 °C and stopped with the addition of 5 µl of stop buffer
(EDTA, 8 mM final concentration) followed by heating to 80
°C. 2 µl of each reaction were spotted onto a PEI-cellulose
plate (B) while 13 µl were run on a 10% SDS-PAGE (C). An autoradiograph of each is shown. Regions of the TLC
analysis corresponding to authentic marker compounds AMP, ADP, and ATP,
located by ultraviolet illumination, are indicated. Background release
of AMP is presumably due to the action of the ligase on endogenous
RNAs.
The
adenylylated substrate was also chemically synthesized essentially as
described by Sninsky et al.(22) .
[5`-P]RNA substrate (1.5
10
cpm) was dried down and dissolved in 100 µl of 0.1 M AMP, 0.1 M MgCl
(pH 5.0) and mixed with
1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide
metho-p-toluenesulfonate (40 mg). The reaction was incubated
for 24 h at 26 °C and diluted with 100 µl of H
O,
and the RNA was recovered by ethanol precipitation. The resulting RNA
was then treated with 0.1 unit of bacterial alkaline phosphatase for 1
h at 37 °C. After proteinase K treatment, extraction with
phenol-chloroform, followed by chloroform, the RNA was recovered by
ethanol precipitation.
Figure 1:
Cosedimentation
of adenylylated proteins, RNA ligase and chimera-forming activities. T. brucei mitochondrial extract was partially purified by
glycerol gradient sedimentation and aliquots of each fraction were
assayed for: A, in vitro chimera formation; B, RNA ligase activity; C, protein adenylylation as
described under ``Experimental Procedures.'' Samples from the
chimera and ligase assays were subjected to PAGE and autoradiography,
while samples from protein adenylylation were subjected to SDS-PAGE and
autoradiography. A, the chimeric molecule (155 nt) is
indicated by an arrow. Also visible are aberrant chimeras
consisting of the gRNA attached at cryptic sites in the cytochrome b mRNA substrate and ligation products (
230 nt)
consisting of the gRNA ligated to the 5`- or 3`-end of the cytochrome b mRNA substrate. B, RNA ligase is assayed by the
ability to [5`-
P]pCp label the synthetic
cytochrome b pre-mRNA. C, two proteins which
incorporate [
-
P]ATP are approximately 50
and 57 kDa in size. Fractions corresponding to complex I and II, as
described previously by Pollard et al.(16) , are
indicated.
To confirm the
identification of the adenylylated proteins as RNA ligases, we examined
the release of label upon ligation. Adenylylated proteins from the 19 S
fraction (complex I) were incubated with either
5`-P-labeled ligatable RNA substrate or the same
[5`-
P]RNA substrate that was periodate-treated
to remove the 3`-hydroxyl groups (Fig. 2A). Ligation of
the RNA substrate results in linear and circular molecules (Fig. 2A), identified by nearest neighbor and gel
mobility shift experiments (data not shown). However, no ligation
products are seen with the periodate-treated RNA (Fig. 2A). To determine whether ligation resulted in
deadenylylation of the 50- and 57-kDa proteins, samples were incubated
without RNA, with unlabeled ligatable RNA, or with unlabeled
nonligatable RNA (Fig. 2, B and C). Aliquots
of each reaction were analyzed by thin layer chromatography to
visualize the nucleotides (Fig. 2B) and by SDS-PAGE to
visualize the adenylylated proteins (Fig. 2C). The
release of AMP from both proteins coincided with their incubation in
the presence of ligatable versus nonligatable RNA substrate.
Based on quantitation by phosphorimagery, there was a 5-fold difference
in the AMP release between the ligatable and nonligatable reactions.
Consistent with these results we found that the level of
deadenylylation of both proteins is significantly greater in the
reaction with ligatable RNA. We attribute this difference to an
increase in turnover of the ligase in the presence of the ligatable
substrate. There is no deadenylylation of either the 50- or 57-kDa
protein or release of AMP following their incubation with DNA (data not
shown), indicating that both proteins are RNA ligases.
Figure 3:
Formation of an adenylylated RNA
intermediate. Reaction mixtures (30 µl) containing 20 µl of
glycerol gradient fraction (complex I) and components described under
``Experimental Procedures'' were incubated with
[-
P]ATP and the indicated RNA substrate for
90 min at 26 °C. A, samples were proteinase K-treated and
subjected to PAGE and autoradiography. Lanes: 1 and 8, pM of 5`-P-ligatable RNA substrate; 2 and 8 pM 5`-P nonligatable RNA substrate. B, the
nucleic acids from the gel of Panel A were eluted, digested
with nuclease P1, and examined by thin layer chromatography on PEI
plates and 1.5 M LiCl, lane 3. Identical RNA that was
labeled upon incubation with T4 RNA ligase and
[
-
P]ATP was also digested with P1, lane
1. Sequential digestion by P1 and snake venom phosphodiesterase (SVPDE) of each of these RNAs is shown in lanes 2 and 4. The identity of digestion products are indicated on the left. The AppG dinucleotide was identified by P1 digestion of
the synthetic adenylylated substrate.
Figure 4: Release of AMP from RNA ligase concomitant with chimera formation. A and B, complex I from the glycerol gradient was pre-adenylylated, dialyzed, and utilized in a chimera assay as described under ``Experimental Procedures.'' Reactions containing 30 µl of extract, 0.5 mM ATP, and components described under ``Experimental Procedures'' (40 µl final volume) were incubated for 90 min at 26 °C. A, RNAs were recovered from the reaction and separated by PAGE; an autoradiograph is shown. The arrow denotes the chimeric molecule. B, aliquots (5 µl) were taken at the start (lane 1) and completion (lane 2) of the reaction, were stopped as indicated in Fig. 2, and spotted onto a PEI-cellulose plate that was developed with 1.5 M LiCl. An autoradiograph is shown. The positions of AMP, ADP, and ATP, located by ultraviolet illumination, are indicated.
The predicted involvement of an RNA ligase in the
mechanism of chimera formation requires an adenylylated ligase
intermediate. Formation of this intermediate requires the hydrolysis of
ATP at the -
bond. To investigate the involvement of this
reaction intermediate in chimera formation, we examined the nucleotide
requirements of RNA ligase and chimera activities. We found that both
activities share a requirement for ATP which could not be substituted
with CTP, GTP, UTP, or dideoxy-ATP (data not shown). To determine the
requirement for ATP hydrolysis, either at the
-
or
-
position, each of these activities were assayed in the
presence of the
,
-methylene (AMP-CPP) and
-
-imino
(AMP-PNP) analogs of ATP (Fig. 5). AMP-CPP, which is
nonhydrolyzable at the
-
bond was not utilized in either
assay. AMP-PNP, which is hydrolyzable at the
-
but not the
-
bond, supported both ligase and chimera formation. AMP-CPP
was not utilized but was able to compete with ATP in both the RNA
ligase and chimera assays. Thus, the nonutilization of this analog is
due to the importance of
-
bond hydrolysis and not the
inability of the enzyme to bind the analog. These results indicate that
both RNA ligase and chimera activities require the hydrolysis of ATP at
the
-
bond but not the
-
bond. The differential
ability of the AMP-CPP analog to compete with ATP in the RNA ligase versus chimera assay indicates additional ATP requirements,
other than the adenylylation of the ligase, in the mechanism of chimera
formation.
Figure 5:
Importance of -
bond versus
-
bond hydrolysis of ATP. ATP analogs that are
nonhydrolyzable either at the
-
bond or at the
-
bond were investigated for their utilization in the RNA ligase and
chimera assays. Ligase (A) and chimera (B) assays
were performed as described under ``Experimental Procedures''
in the presence of: lane 1, no ATP; lane 2, AMP-CPP
(0.25 mM); lane 3, ATP (0.25 mM); lanes
4-11, ATP (0.25 mM) and increasing amounts of
AMP-CPP, as indicated; lane 12, AMP-CPP (125 mM); lane 13, no ATP; lane 14, AMP-PNP (0.25 mM); lane 15, ATP (0.25 mM). Reaction products were
separated by PAGE and autoradiographs are shown. Arrow denotes
the chimeric molecule.
The formation of the adenylylated ligase intermediates
resulted in the release of pyrophosphate. The finding that chimera
formation also specifically requires the -
bond hydrolysis of
ATP provides a strong argument for the involvement of a similar
reaction intermediate. Further evidence was provided by the inhibition
of chimera formation with pyrophosphate (Fig. 6) which
deadenylylates the ligase (Table 1). Pyrophosphate inhibits both
RNA ligase and chimera activities in a dose dependent manner (Fig. 6). On the other hand, inorganic phosphate at the highest
concentration tested has no effect on either assay (Fig. 6). The
inhibition by pyrophosphate can be partially reversed with the addition
of pyrophosphatase (Fig. 6). The ability of pyrophosphatase to
reverse the level of inhibition indicates that this inhibition is in
fact due to the pyrophosphate, most likely by deadenylylating the
ligase-AMP. The ability of pyrophosphate to deadenylylate the ligase
and inhibit the formation of the chimeric molecule is indicative of
ligase involvement in chimera formation.
Figure 6:
Dose dependent inhibition of chimera
formation and RNA ligase activity by pyrophosphate. Chimera assays were
performed as described under ``Experimental Procedures'' in
the presence of: lane 1, no sodium pyrophosphate
(PP); lanes 2-5, increasing amounts of
PP
as indicated; lane 6, 2.0 mM PP
and 2 units of yeast pyrophosphatase; lane 7, 3.0 mM sodium phosphate. Ligase assays, lanes 8-14, were
performed as described under ``Experimental Procedures'' with
treatments as described above and as indicated. Reaction products were
separated by PAGE; an autoradiograph is shown. Arrow denotes
the chimeric molecule.
We have previously shown that the gRNA/mRNA chimeric molecules produced in vitro are analogous to those observed in steady-state kinetoplastid mitochondrial RNA(12) . The chimeras formed in vitro consist of the poly(U) tail of the gRNA linked to the mRNA specifically at editing sites and are dependent upon the correct anchor sequence(11, 12, 13) . It is believed that the chimeric molecule represents an intermediate in the editing process. Thus, determination of the mechanism of its formation would provide an indication of the mechanism of kinetoplastid RNA editing.
Previous work by Pollard et al.(16) characterized mitochondrial RNPs that contained potential editing activities including RNA ligase and chimera forming activities. The cosedimentation of RNA ligase and chimera activities suggested that ligase was involved in the production of chimeric molecules. This paper reports on the direct involvement of this RNA ligase activity in the mechanism of chimera formation. We find two distinct proteins which become adenylylated with ATP, cosediment with the ligase activity in glycerol gradients, and have been identified as ligase intermediates by the release of AMP concomitant with ligation. Deadenylylation of these intermediates results in the inhibition of chimera formation. In addition, AMP release from the ligase is concomitant with production of the chimeric molecule. These results argue for a cleavage ligation-type mechanism of chimera formation in vitro and suggests a similar mechanism for RNA editing in kinetoplastids.
The identification of the adenylylated proteins as
RNA ligase intermediates is consistent with the mechanism of other
ligases. Thus, it is not surprising that the RNA ligase activity
present in the chimera-forming complexes specifically requires the
-
bond hydrolysis of ATP. The requirement for
-
bond hydrolysis of ATP in chimera formation could be explained by the
involvement of the RNA ligase intermediate. The finding that
pyrophosphate, which deadenylylates the intermediate, inhibits chimera
formation suggests its direct involvement in the chimera-forming
mechanism. Release of AMP from the ligases upon chimera formation is
also consistent with this argument.
In the initial characterization
of the editing complexes the editing site-specific endonuclease did not
appear to comigrate with either complex(16) . This enzymatic
activity, which cleaves pre-edited mRNA at sites where editing occurs,
would be an essential component of the editing complex for editing to
occur by a cleavage ligation-type mechanism. Preliminary evidence
indicates that our previous inability to detect the endonuclease within
the complexes was due to the masking of endonuclease activity by RNA
ligase. ()Work is currently being done to investigate the
involvement of the endonuclease in chimera formation.
The involvement of RNA ligase in the production of the chimeric molecule is consistent with the type of mechanism proposed in Fig. 7. The first step in chimera formation would involve cleavage of the pre-edited mRNA at the editing site by the editing site-specific endonuclease. The adenylylated RNA ligase would then transfer the AMP to the 5`-phosphate of the 3`-cleavage fragment, forming a 5`,5`-phosphoanhydride bond. This activated phosphate is then attacked by the 3`-hydroxyl of the uridine at the end of the gRNA forming the chimeric molecule. It is conceivable that a second round of cleavage and ligation similar to that outlined above would produce an edited product.
Figure 7: Proposed mechanism of in vitro chimera formation. A model of the possible mechanism involving RNA ligase and endonuclease is presented.
The proposed cleavage-ligation mechanism outlined above is
similar to the two stage mechanism of tRNA splicing in
yeast(33) . In yeast, the mechanism involves polynucleotide
kinase and cyclic phosphodiesterase activities as well as the
endonuclease and ligase(19) . In the first stage the
endonuclease cleaves the RNA generating the 5`-half, terminating with a
2`,3`-cyclic phosphate, the 3`-half, terminating with a 5`-hydroxyl,
and the intervening sequence. In the second stage the 2`,3`-cyclic
phosphate is opened by a cyclic phosphodiesterase and the 5`-hydroxyl
is the substrate for a kinase activity. The resulting species are then
finally substrates for the ligase forming a 2`-phosphomonoester
3`,5`-phosphodiester linkage where the phosphate in the newly formed
bond comes from the position of the nucleotide cofactor.
The
lack of requirement for -
bond hydrolysis of ATP (Fig. 5), the inability to incorporate label from
[
-
P]ATP into the chimera product, as well
as the lack of detectable polynucleotide kinase activity in
mitochondrial extract, (
)describe an overall reaction scheme
similar to tRNA splicing but with several distinct differences. The
ability to digest the ligation products to mononucleotides with P1
nuclease
is consistent with a typical 3`-5`-phosphodiester
linkage produced by the trypanosomal RNA ligase where the 2`-position
is not blocked by a phosphoryl group. These results, along with the
ability to pCp label the 5`-fragment with T4 RNA ligase,
suggest the endonuclease cleaves RNAs leaving 3`-hydroxyls and
5`-phosphates. The 3`-hydroxyl on the gRNA has previously been shown to
be required for chimera formation(12, 13) . This may
reflect the substrate requirement of the ligase for RNAs with
3`-hydroxyls and 5`-phosphates. Thus, the endonuclease may directly
provide the substrates for the ligase in chimera formation as opposed
to the mechanism of yeast tRNA splicing.
The involvement of RNA ligase in the in vitro reaction provides the first direct evidence for a cleavage-ligation mechanism of chimera formation. Further purification of the components of these complexes will allow the determination of other proteins involved in the in vitro reaction. The ability to resolve the chimeric molecule to the edited product would characterize them as true editing intermediates and verify the cleavage-ligation model as the mechanism of kinetoplastid RNA editing.