(Received for publication, May 28, 1996, and in revised form, October 10, 1996)
From the 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 mitochondrial genome of Leishmania tarentolae, like
that of all other trypanosomatids, consists of a single network of catenated DNA maxicircles and minicircles (1-3). The maxicircle contains genes encoding proteins required for respiration and mitochondrial translation. The majority of the mRNAs transcribed from the maxicircle, however, must be edited by the addition and/or deletion of uridines (U nucleotides) to create an open reading frame
(4-7).
The genetic information determining the number and location of
U-insertions and deletions has been proposed to be located within the
guide RNAs (gRNAs),1 a class of small RNA
molecules transcribed from both maxicircles and minicircles (8-11).
gRNAs contain an anchor sequence that is complementary to mRNA
immediately 3 Since L. tarentolae is not readily accessible to genetic
analysis, the development of an in vitro system is crucial
to understanding the mechanism of RNA editing in this organism. A
mitochondrial lysate prepared from the related trypanosomatid,
Trypanosoma brucei, has been shown to delete U nucleotides
at site 1 of the mRNA encoding ATPase subunit 6 (= maxicircle
unidentified reading frame 4 or MURF4) when the appropriate gRNA is
added (12). The deletions can be manipulated in a predictable manner by
altering the guiding nucleotides of the gRNA. Guide RNA-dependent
U-insertion, which is by far the most common form of modification in
kinetoplastid RNA editing, has also been recently demonstrated in
vitro (36, 40).
An in vitro internal U-insertion activity has been
demonstrated for two synthetic pre-edited mRNAs. The mRNAs
incorporated [ We describe in this paper a primer extension assay that permits the
detection of U-insertions at individual editing sites. We used this
assay to characterize two different reactions. The internal U-insertion
activity initially detected with the RNase H-based assay is shown to be
highly dependent on the secondary structure of the mRNA and also
not to be mediated by gRNA. A second U-insertion reaction is also
described, which is dependent on the addition of the correct gRNA to
the mitochondrial lysate.
The oligonucleotides used in these
studies are listed below together with a brief description of their
function: FP = forward or 5 S194: TAATACGACTCACTATAGGG S1743:
CACCATATTTCGCTTA S1820: S1916:
TAATACGACTCACTATAGGG Kinetoplast DNA
was purified from stationary cells by sedimentation through a cesium
chloride step gradient (15). The maxicircle sequence encoding the
gCyb-I gRNA was amplified from 0.5 ng of kinetoplast DNA with 30 cycles
of PCR using primers S1820 and S1821. The same two oligonucleotides
were used to sequence the DNA fragment.
Kinetoplast RNA was extracted from purified mitochondria (16). A
poly(C) tail was added to 5 µg of the kinetoplast RNA with poly(A)
polymerase (Pharmacia Biotech Inc.). After phenol-chloroform extraction
and ethanol precipitation, the RNA was annealed to S1873 and
reverse-transcribed with avian myeloblastosis virus reverse
transcriptase (Promega). The cDNA product was PCR-amplified using
primers S1872 and S1873 and was cloned into pAMP1 (Life Technologies,
Inc.) and transformed into DH5 The Cyb mRNA constructs
were transcribed from PCR products that had been amplified from plasmid
pNB2 (17) using the following primers: S194 and S2085 for the 21-nt 5 The gRNAs were transcribed directly from synthesized oligonucleotide
templates, as described previously (18), and the 3 PCR was performed in 100-µl reactions containing 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 2.5 mM MgCl2, 200 µM of each dNTP,
0.5 µM of each primer, and 2.5 units of Taq
polymerase (Perkin Elmer). The PCR profiles were 5 min at 95 °C,
followed by a variable number of cycles of 95 °C for 30 s,
55 °C for 30 s or 60 s, and 72 °C for 60 s.
The Cyb mRNA
construct (3 pmol) was denatured at 65 °C for 5 min in 1 mM EDTA, 50 mM HEPES (pH 7.5). For native
conditions, the solution was adjusted to a final concentration of 20 mM KCl, 10 mM MgCl2, 50 mM HEPES (pH 7.5), and 2 mM spermidine in a
200-µl volume. Following a 10-min 27 °C preincubation, 0.5 µl of
DMS (Aldrich) was added, and the solution was incubated for another 8 min. For denaturing conditions, the RNA in 200 µl of 1 mM
EDTA, 50 mM Hepes (pH 7.5) was heated with 0.5 µl of DMS
at 95 °C for 45 s. Reactions were terminated with the addition
of 100 µl of 1 M Approximately 0.2 pmol of the modified RNA was extended with primers
S1685, S1786, S2073, and S2085 as described by Inoue and Cech (19). The
extension products were resolved on a 12% polyacrylamide, 7 M urea gel and scanned on a PhosphorImager (Molecular Dynamics). A ratio of the intensity of the RT termination signal quantitated under native conditions to that measured under denaturing conditions was determined for the modified bases (A and C nucleotides). This ratio in each reaction was normalized to the base of maximal activity (highest native/denatured value). A mean normalized ratio for
each site was obtained from at least two independent modification reactions. Positions complicated by modification-independent RT terminations were not quantitated.
L. tarentolae (UC
strain) cells were grown to a late log phase density of 108
cells/ml as described previously (20). After washing twice in 0.25 M sorbitol, 20 mM HEPES, 2 mM EDTA
(pH 7.5) (SHE), the cells were resuspended at a density of
109 cells/ml and disrupted in a Stansted Power Fluid
apparatus (Energy Service Co., Washington, DC) at 1200 p.s.i. The
crude mitochondrial fraction was pelleted, resuspended in 0.25 M sorbitol, 20 mM Hepes (pH 7.5), 2 mM MgCl2, digested with DNase I to remove
contaminating nuclear DNA, and washed by centrifugation in SHE. The
crude fraction was taken up in 76% Renografin and a purified
mitochondrial fraction isolated by flotation in Renografin density
gradients as described previously for hypotonically broken cells (15),
and resuspended in 20 mM HEPES (pH 7.5), 10% glycerol, and
100 mM KCl for storage at In a 20-µl volume containing 12 mM HEPES (pH 7.5) and 0.1 mM EDTA, 3 pmol of
the mRNA construct were added to 30 pmol of the appropriate gRNA
and denatured at 65 °C for 3 min. The RNA solution was adjusted in a
50-µl volume to a final concentration of 5 mM HEPES (pH
7.5), 0.04 mM EDTA, 20 mM KCl, 3 mM
potassium phosphate (pH 7.5), 10 mM MgCl2, 20 mM dithiothreitol, 2 mM spermidine, 10 µg/ml
leupeptin, 1 mg/ml Pefabloc SC, 1 mM ATP, 1 mM
GTP, and 1 mM UTP. Reactions were initiated by the addition
of approximately 109 cell equivalents of the mitochondrial
lysate containing about 30 µg of protein. After incubation at
27 °C for 50 min (Cyb), reactions were terminated by
phenol-chloroform extraction followed by ethanol precipitation.
The lysate-treated Cyb mRNA
constructs were purified in a 6% polyacrylamide, 7 M urea
gel. A gel slice containing the full-length RNA plus molecules up to
40% longer was excised to ensure that those RNAs with U-additions
would also be included. RNA was eluted from the gel at 37 °C for
1 h in 1 mM EDTA, 0.1% SDS, 0.5 M
NH4 acetate, 10 mM MgCl2, and 0.3 M sodium acetate and then ethanol-precipitated using 10 µg of glycogen (Boehringer Mannheim) as a carrier. Approximately 0.7 pmol of each treated RNA sample was assayed for U-insertions by RT
using a primer annealed immediately 3 Two different oligonucleotides, which annealed immediately 3 Guide RNA-independent U-insertions in Cyb
mRNA constructs. A, upper panel, diagram of
primer extension assay of fully edited mRNA. U nucleotides added by
editing are in boldface. Lower panel, primer
extension assay of Cyb mRNA constructs after incubation of RNAs in
mitochondrial lysate. No exogenous gRNA was added. The substrate RNAs
are indicated above each lane: edited Cyb, a Cyb transcript
edited at sites 1 and 2; endogenous, no RNA added to lysate;
5
RT-PCR of the 3 The assay
described in Fig. 1A was used to detect
U-insertions that occur within the first two editing sites of the Cyb
pre-edited transcript. The mature in vivo edited mRNA
has a single U at each of these sites. A DNA oligonucleotide annealed
to the RNA immediately downstream of the first editing site was
extended with RT in the presence of [ As shown in Fig. 1A, the major cDNA extension product of
a synthetic Cyb transcript edited at sites 1 and 2 was of the expected size: the length of the primer + 4 bases (P+4, lane 1).
Assay of the Cyb mRNA endogenous to the lysate resulted in a
product of the same size (lane 2). This is expected, since
approximately 95% of the Cyb mRNA from late log phase L. tarentolae cells is fully edited (21). The bands migrating between
the P+4 band and the major normalization N band, which are present in
all lanes (see "Experimental Procedures"), represent artifacts of
this normalization extension and are probably the result of the priming
of the assay primer at an ectopic site on the normalization primer
(Fig. 1A).
A major advantage of the primer extension assay is that insertions
occurring in less than 1 in 20,000 molecules can be detected. At this
level of sensitivity, however, artifacts resulting from misincorporation by the RT become significant. An example of such an
artifact is the primer + 2 (P+2) band that appeared in the assays of
all of the pre-edited Cyb mRNA constructs tested, independent of
exposure to the mitochondrial lysate (lanes 3-12). The
artifact band was not seen with the edited Cyb transcript in
lanes 1 and 2 since extensions containing the
misincorporation would still be extended up to the dideoxy termination
site (primer + 4). The appearance of this band is consistent with the
misincorporation of a labeled adenosine (A) opposite the cytidine (C)
immediately 5 The internal
U-additions initially detected by an RNase H-based assay (13) were
within a Cyb mRNA construct (pNB2 RNA) that contained at the 5 Removal of the upstream sequence from the 5 Substitution of both the 5 The U-insertions occurred in the absence of added gRNA. It was
previously proposed that endogenous gRNAs could be mediating the
U-insertions into the Cyb pre-edited region as assayed by the RNase H
method (13). However, a change in the anchor binding site of the Cyb
mRNA construct that should completely inhibit the binding of
endogenous gRNA ( A potential
complication of the direct primer extension assay is the difficulty in
differentiating between U nucleotides inserted within an editing site
of an mRNA and U nucleotides present at the assayed sites as part
of a gRNA-mRNA chimeric molecule. Chimeras result from the covalent
linkage of the 3 Even though gRNA was not added to the reactions of Fig. 1, it is
possible that chimeric molecules could be formed from the small pool of
gRNAs that are endogenous to the mitochondrial lysate. The majority of
the chimeras formed in vitro copurify with the Cyb mRNA
construct containing the natural 5 The method diagrammed in Fig. 2A was used to
completely eliminate any possible contribution of chimeric molecules
and endogenous edited Cyb mRNA to the U-insertion signal. The
mRNA construct used in this assay was modified at the 3
When the pre-edited Cyb transcript containing the 5 The information required to correctly edit Cyb
mRNA is potentially contained within two overlapping gRNAs. A
single nucleotide error in the L. tarentolae maxicircle
sequence (22) resulted in the original published sequence of the
maxicircle-encoded gCyb-I gRNA being truncated by 20% (8). The
corrected sequence of this gRNA potentially is able to template the
U-insertions for the first seven editing sites and part of site 8, instead of the first five editing sites as previously suggested (Fig.
3A). This would create an anchor for the
overlapping gCyb-II gRNA that could then be used to edit the remaining
sites, in agreement with the scheme proposed for Crithidia
fasciculata (27).
Cloning and sequencing of the endogenous gCyb-I gRNAs from the
mitochondrial fraction revealed a limited heterogeneity of the 3 The gCyb-I gRNA used in the assay for in vitro U-insertions
was transcribed from a DNA template that did not encode an oligo(U) tail, since in vitro transcription of gRNA by T7 RNA
polymerase directly from a template encoding an oligo(U) 3 Addition of
the cognate gCyb-I gRNA to the reaction with the Cyb mRNA construct
containing the 5 Addition of the cognate Cyb gRNA to the Cyb mRNA containing the
natural 5 As described in the previous section, gRNA was synthesized without a 3 The presence of the primer + 2 artifact band partially obscures the
region of interest in these experiments and makes quantitation difficult, but it is clear that gRNA-dependent U-insertion
does occur, and this is particularly evident with the mRNA
substrate containing the natural 5 Sensitive and specific primer extension assays were used to detect
U-insertions occurring in vitro at sites 1 and 2 of
pre-edited Cyb mRNA. The insertions were dependent on incubation of
the RNAs in a detergent lysate of a purified mitochondrial fraction
from L. tarentolae. With a 5 These in vitro U-insertions are unlikely to have been
mediated by misguiding by the endogenous gRNA pool (26, 28), as is
known to occur at a very low level during in vivo editing of the Cyb mRNA (29), for several reasons. First, U-insertions were
detected in the Cyb mRNA constructs having the natural 5 Second, most, if not all, of the gRNAs in the UC laboratory strain of
L. tarentolae used in this study have been identified (30,
31), and none of these is capable of mediating the insertions detected
in the Cyb RNAs with a mutated anchor binding site (Fig. 1B,
lanes 11 and 12). For example, a search of the UC
strain gRNA collection for those complementary to the mutated anchor
binding sites did not find any interactions with a
In this regard, it has been proposed that additional factors must be
required to stabilize the gRNA-mRNA interaction, since some of the
reported gRNA anchor sequences are too short to form a stable duplex on
their own (32). The 8-base anchor binding site previously proposed for
gCyb-II gRNA of L. tarentolae was one of the shortest in the
literature (8). However, editing of the first seven sites by the
corrected gCyb-I gRNA sequence potentially extends this anchor to 14 bases, which is closer to the mean anchor length of the other L. tarentolae gRNAs (33). Likewise, a 4-base anchor has been proposed
for the same gRNA in C. fasciculata (32), but this would
also be increased by the editing mediated by gCyb-I gRNA (27). There
are probably other examples of putative gRNAs in the literature that
are either a result of sequencing errors or fortuitous sequence
similarity. Short anchor sequences might be considered an indicator of
these complications, and this will eventually be tested with the
in vitro system.
There is no evidence that the 5 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 with the Cyb mRNA 5
Addition of exogenous synthetic gCyb-I gRNA was found to stimulate the
insertion of U nucleotides into Cyb mRNA constructs with the
natural 5 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 gRNA-independent
reaction. Alternatively, the recognition element could be an indirect
consequence of the formation of the double-stranded RNA. However, DMS
modification of the editing sites within the Cyb mRNA was not
significantly altered by the addition of the 5 We thank J. Alfonzo and O. Thiemann for
helpful discussions. We also thank G. Frech for supplying the S1
construct.
Howard Hughes Medical Institute, the
§ Department of Molecular,
Department of Medical Microbiology and
Immunology, University of California, Los Angeles,
California 90095-1662
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES
of the editing sites, and they also have a sequence that
can function as a template for the insertions and deletions (provided
G:U base pairs are allowed) (8). The 3
end of the gRNA has a
posttranscriptionally added oligo(U) tail that has been
postulated to be important for editing (8).
-32P]UTP when incubated with a
mitochondrial lysate from L. tarentolae (13). Digestion with
RNase H was used to show that U nucleotides were incorporated
internally into a synthetic pre-edited cytochrome b (Cyb)
transcript, as well as at the 3
terminus. The 3
end-labeling is due
to a terminal uridylyl transferase activity that has been identified in
mitochondrial extracts (14). Approximately 95% of the internal
insertions were localized within a region containing the 15 editing
sites of this RNA (13). Although the RNase H-based assay was well
suited to detect internally labeled RNA, the assay does not permit the
detection of U-insertions at individual editing sites. Furthermore,
since the U-insertions occurred independent of added gRNA, the
relationship of the in vitro reaction to biological editing
was unclear.
Oligodeoxynucleotides
PCR primer, RP = reverse or 3
PCR primer. The locations of the underlined sequences in the L. tarentolae maxicircle sequence (GenBank entry LEIKPMAX) are
indicated.
(21 nt 5
extended Cyb mRNA FP, nt 5350-5365 in LEIKPMAX). S656:
TAATACGACTCACTATAGGG
(gCyb-I gRNA
FP, nt 16780-16800 in LEIKPMAX). S1206: TAATACGACTCACTATA (T7 RNA polymerase promoter). S1395:
T
T
(Cyb mRNA edited at sites 1+2 FP, nt 5398-5450 in LEIKPMAX).
S1520: TAATACGACTCACTATAGGGCC
(Cyb mRNA with 5-bp G-C stabilized helix FP, nt 5371-5410 in
LEIKPMAX). S1521: GGGCC
(Cyb
mRNA with 5-bp G-C stabilized helix RP, nt 5456-5480 in LEIKPMAX).
S1685: AAATT
(Cyb mRNA with
disrupted helix RP, nt 5460-5480 in LEIKPMAX).
(Cyb reverse transcriptase (RT) normalization primer, nt 5372-5416 in
LEIKPMAX). S1786:
(RT of
DMS-treated RNA, nt 5400-5426 in LEIKPMAX). S1790:
GGGCC
TAATTGATTTATG
(Cyb mRNA with mutated anchor binding site RP, nt 5440-5480
and nt 5410-5426 in LEIKPMAX). S1795:
AAAAAAAAAAAAAAA
CCTATAGTGAGTCGTATTA (transcription of gCyb-I gRNA plus U-tail, nt 16758-16803 in
LEIKPMAX). S1796:
CCTATAGTGAGTCGTATTAC (transcription of gCyb-I gRNA without U-tail, nt 16758-16803 in LEIKPMAX).
(maxicircle fragment encoding gCyb-I gRNA RP, nt 16801-16845 in
LEIKPMAX). S1821:
(maxicircle
fragment encoding gCyb-I gRNA FP, nt 16705-16744 in LEIKPMAX). S1872:
(CUACUACUACU)ATGACTTGAAGTTAAAAGA (3
RACE of gCyb-I gRNA FP, nt
16780-16798 in LEIKPMAX). S1873: CAUCAUCAUCAUGGGGGGGGGGGGGGG
(3
RACE of poly(C)-tailed gCyb-I gRNA RP). S1894:
CCTATAGTGAGTCGTATTAC (transcription of gCyb-I gRNA with 3
extension, nt 16754-16803 in LEIKPMAX). S1899:
TATTATTTAAAAATTTATATTACTTTAACTTCAAGTCATATGTGCCTATAGTGAGTCGTATTAC (transcription of gCyb-I(0,0) gRNA).
(Cyb mRNA with natural 5
end FP, nt 5371-5408 in LEIKPMAX). S2003: A
CC (RNase H digestion of gCyb-I gRNA, nt
16758-16803 in LEIKPMAX). S2073:
(Cyb RT assay
primer, nt 5425-5459 in LEIKPMAX). S2074:
(Cyb RT normalization primer, nt
5514-5573 in LEIKPMAX). S2077: CACCATATACTCGTAATAAATAATTGATTTATGAA (RT
of anchor-mutated Cyb mRNA). S2085:
(Cyb mRNAs
RP, nt 5502-5545 in LEIKPMAX). S2093:
TTCGACATGAGACACGGATCGATCCCC
(3
tagged
Cyb RNAs RP, nt 5532-5545 in LEIKPMAX). S2094:
TAATACGACTCACTATAGGGCGAATTGGGTACCGGGCC (5
tagged Cyb mRNA FP).
S2095: TTCGACATGAGACACGGATC (RT of 3
tagged RNAs).
cells for sequencing.
extension, S1916 and S2085 for the natural 5
end, S1520 and S1521 for
the helix stabilized with 5 G-C base pairs, S1520 and S1685 for the
disrupted G-C helix, S1520 and S1790 for the mutated anchor binding
site, S1916 and S2093 for the natural 5
end 3
tag, S2094 and S2093
for the 5
extension 3
tag. The Cyb construct edited at both sites 1 and 2 was generated by PCR amplification with S1395 and S2085, followed by amplification of the purified product with S1916 and S2085; RT
sequencing of the transcribed RNA confirmed the sequence.
ends were sequenced
by the same procedure described for the endogenous L. tarentolae gRNAs. All RNAs were gel-purified prior to use in the
assay.
-mercaptoethanol, 0.5 M
HEPES (pH 7.5) and then ethanol-precipitated.
80 °C. After thawing on ice,
the protease inhibitors leupeptin and Pefabloc SC (Boehringer Mannheim)
were added, for the Cyb assay, to final concentrations of 10 µg/ml
and 1 mg/ml, respectively, and the mitochondria were lysed by addition
of Triton X-100 to a final concentration of 0.3%.
of the editing sites. The RNA
was heated at 65 °C for 3 min in a 7.5-µl volume containing 10 pmol of primer S2073, 10 pmol of a normalization primer, 60 mM NaCl, 10 mM dithiothreitol, and 50 mM Tris-HCl (pH 8.3). The solution was made 5 mM in MgCl2 and annealed 10 min at 42 °C. The extensions were performed in a 5-µl volume containing 2 µl of
the primer/RNA solution and 200 µM ddCTP, 375 µM dGTP, 1.3 µM [
-32P]dATP
(800 µCi/mmol), 5 mM MgCl2, 60 mM
NaCl, 10 mM dithiothreitol, 50 mM Tris-HCl (pH
8.3), and 1 unit of avian myeloblastosis virus reverse transcriptase
(Promega). Reactions were allowed to proceed at 42 °C for 30 min
prior to electrophoresis in a 12% polyacrylamide, 7 M urea
gel. All reactions were quantitated using a PhosphorImager (Molecular
Dynamics).
of an
encoded U, were used to normalize the extension signal; S1743 anneals
5
of the editing sites and S2074 at the 3
end of the RNA. The
extension of S1743 generates some artifact bands that are not present
with S2074, but it was used for Fig. 1A because the
annealing site of S2074 is not present on some of the assayed constructs. S2074 was used for all other reactions. Since the lysate-mediated U-insertions occur infrequently, the signal intensity from the normalization primer is much greater than that from the assay
primer, and it can interfere with quantitation. For this reason, the
intensity of the normalization extensions was decreased with the
addition of ddT to the 3
end of the primers by treatment with terminal
transferase (Boehringer Mannheim). This prevented 80% of primer S2074
and 99% of primer S1743 from being extended. The extension signal from
all of the primers was linear with the RNA concentration provided that
at least a 7-fold molar excess of the oligos were used in the
reactions.
Fig. 1.
extension, Cyb mRNA construct with the 5
extension shown in B; natural 5
end, Cyb mRNA
construct with the mature 5
end; plus GGGCC/CCCGG, Cyb
mRNA construct with substitution of 5
upstream sequence and
terminal 3
65 nt with an artificial 5-bp GC-helix; plus
GGGCC/UUUAA = Cyb mRNA construct lacking a stable helix
as shown in B; mutated anchor, Cyb mRNA
construct with mutated gRNA anchor binding site and the GGGCC/CCCGG
terminal helix as shown in B, lower panel.
Presence or absence of lysate is indicated by + or
. The extension
product used for normalization of RNA quantity is indicated by
N (see "Experimental Procedures"). The primer + 2 (P+2)
and primer + 4 (P+4) extension products are indicated on the
left. The additional bands shown in lanes 1 and 2 migrating between the P+4 band and the N band represent
artifacts of the normalization extension. These artifact bands are also present in experimental lanes 3-12. B,
upper panel, a predicted secondary structure for the Cyb
mRNA construct containing the 21-nt 5
-extended sequence plus 3 G
nucleotides added by T7 transcription. The natural 5
end is indicated
by an arrow, and editing sites are indicated by a bracket; the
first two sites are numbered. The anchor-binding sequence is
in lowercase. DMS modification of this RNA was performed
under both native and denaturing conditions (see "Experimental
Procedures"). A ratio of the intensity of the RT termination signal
measured under native conditions with that under denaturing conditions
was determined for each modified position. A base was defined as
protected from modification (
) if this ratio is less than 50% of
maximum, partially protected (
) if the ratio is between 50% and
75%, and fully reactive (
) if greater than 75%. The structure is 3 kcal/mol removed from the most stable fold, but it is in better
agreement with the DMS modification results; the helix containing the
5
-extended sequence is also present in the optimal fold. Lower
panel, the sequences and stabilities of additional constructs used
in this study.
[View Larger Version of this Image (34K GIF file)]
tagged Cyb
mRNA constructs eliminated complications from endogenous edited
mRNA and also chimeras and other potential ligation products. After
gel purification, the lysate-treated 3
tagged constructs were
reverse-transcribed using primer S2095. The cDNA derived from the
Cyb mRNA construct containing the mature 5
end was treated with
RNase A and amplified with four cycles of PCR using S1916 and S2095.
The 3
tagged construct containing the cytochrome oxidase III extension
also has a 67-base 5
tag taken from the PNB2 vector (17).
Amplification of the cDNA synthesized from this construct was with
primers S2094 and S2095. RNA was produced for use in the RT assay by T7
transcription of the PCR products.
Direct Primer Extension Assay for Cyb mRNA
-32P]dATP and the
appropriate deoxy- and dideoxynucleoside triphosphates. Incorporation
of label into the cDNA extension products is indicative of
U-insertions in the mRNA. The extension of a second primer outside
of the editing sites (indicated by N in Fig. 1A)
was used to normalize the U-insertion signal.
of the first editing site. In any case, the bands of
interest are only those that are dependent on incubation of pre-edited mRNAs with the mitochondrial lysate.
end
73 nt of vector sequence in addition to 24 nt of upstream sequence that
included part of the adjacent cytochrome oxidase III gene (22). As
shown in Fig. 1A (lanes 3 and 4), the
RT assay of an RNA containing 21 nt of this 5
upstream sequence
resulted in a ladder of extension products that is dependent on prior
treatment with the mitochondrial lysate. The ladder is consistent with
the RNA having been modified in the lysate with a heterogeneous number
of U-insertions. Each successive band, progressing up the ladder,
resulted from the primer extension of a subpopulation of RNAs
containing an increasing number of inserted U nucleotides. For
quantitation, these bands must be normalized for the increase in the
amount of incorporated label. Approximately 1 in every 100 molecules
contained at least one U-insertion. The insertions appear to be
restricted to sites within the RNA that are normally edited in
vivo; the extension of several DNA primers through non-edited
regions of the lysate-treated RNA failed to detect inserted U
nucleotides (data not shown).
end of the Cyb mRNA
construct resulted in the loss of the extension ladder (Fig. 1A, lanes 5 and 6); the remaining
primer + 4 signal results from the detection of the Cyb mRNA
endogenous to the lysate (lane 2). The secondary structure
predicted by MFOLD (39), suggested that the role of the upstream
sequence could be to stabilize a helix. As shown in Fig. 1B,
part of this sequence is protected from DMS modification, which is
consistent with the presence of a duplex.
upstream sequence and the terminal 3
65 nucleotides of the construct with an artificial 5-bp G-C helix (Fig.
1B) preserved the U-insertion activity (Fig. 1A, lanes 7 and 8). Disruption of the artificial G-C
helix by substitution of one strand with an AU sequence resulted in
loss of the insertion activity, thus confirming the importance of the
stabilized helix (Fig. 1A, lanes 9 and
10).
Go > 0) only resulted in a
40% decrease in the level of U-insertion into sites 1 and 2, as
assayed by primer extension (Fig. 1A, lanes 11 and 12). In addition, a second independent Cyb mRNA
construct also having a completely disrupted anchor binding site (S1
construct in Ref. 13) showed the same level of U-insertions as the wild type RNA (data not shown). This evidence suggests that the in vitro U-insertion activity occurring in sites 1 and 2 is
independent of endogenous gRNA.
oligo(U) tail of the gRNA to the mRNA at an
editing site and are known to exist in steady state kinetoplast RNA and
to be created in vitro by incubation of synthetic gRNA with
mitochondrial extract (23-26). It is unclear whether chimeras
represent true intermediates of the editing process or are a side
product of the reaction.
end; the distance between the 5
end of this RNA and the first editing site is approximately the same
size as the gRNA. The maximal possible contribution of these chimeric
molecules to the primer extension signal, therefore, would not exceed
that in lane 5 of Fig. 1A.
end with a
tag sequence. After incubation with the lysate, a cDNA copy of this
RNA was synthesized using a primer complementary to the tag sequence, thereby eliminating any reverse transcription of endogenous Cyb mRNA. The cDNA was then PCR-amplified using the tagged 3
primer together with a 5
primer specific to the 5
end of the Cyb
mRNA; since chimeras and other potentially complicating ligation
products do not contain the 5
end of the mRNA, they would not be
PCR-amplified. The RT primer extension assay using
[
-32P]dATP was performed on RNA produced by T7
transcription of the RT-PCR DNA product.
Fig. 2.
Indirect primer extension assay using RNA
transcribed from RT-PCR amplified cDNA. A, a cDNA
copy of lysate-treated RNA was synthesized using a primer complementary
to a tag sequence on the 3 end of the substrate mRNA, thereby
preventing reverse transcription of endogenous RNAs. The cDNA was
amplified using a 5
PCR primer sense to the 5
end of the Cyb
mRNA; molecules such as chimeras that do not contain both ends of
the Cyb mRNA construct would not be amplified. RNA for the RT
primer extension assay was transcribed from a T7 promoter incorporated
into the 5
PCR primer. B, use of the indirect assay to
detect U-insertions. The substrate RNA is indicated above each lane.
Lanes 10-13 represent a separate experiment using the Cyb
mRNA construct with the natural 5
end in which the effect of
addition of 1 mM UTP and 10 mM Mg2+
were analyzed. The primer+4 (P+4) bands are indicated.
[View Larger Version of this Image (43K GIF file)]
upstream sequence
and the 3
tag was incubated with mitochondrial lysate in the absence
of added gRNA, the indirect assay produced a ladder of primer extension
products similar to that obtained from the direct assay of the
non-tagged Cyb mRNA (compare lane 4 in Fig. 2A and lane 3 in Fig. 1A). This
confirms that chimeras did not significantly contribute to the
extension signal obtained with the direct primer extension assay in
Fig. 1A. The absence of a band in lane 2 of Fig.
2B also indicates that this indirect assay succeeded in
eliminating the primer + 4 signal arising from the edited Cyb mRNA
that is endogenous to the lysate.
Fig. 3.
gCyb-I gRNA can potentially template
U-insertions for the first seven editing sites of the Cyb mRNA.
A, editing sites 5-8 of fully edited Cyb mRNA are
shown, with U nucleotides added by editing indicated by u.
The sequence at the 3 end of gCyb-I gRNA was obtained by directly
sequencing maxicircle (Mc) DNA and several cDNA clones;
the guiding nucleotides are in lowercase, and the T residue
not present in the originally reported gRNA sequence is indicated by an
asterisk. The cDNA clones showed a limited heterogeneity
at the 3
end of the genomically encoded sequence and a heterogeneity
in the number of posttranscriptionally added U nucleotides
(bold). B, a 32P-labeled gCyb-I gRNA
lacking an oligo(U) tail was incubated in mitochondrial lysate under
the conditions used to detect the internal U-insertions, and the RNA
was isolated and analyzed by acrylamide gel electrophoresis. Sequencing
of the 3
end of the RNA by 3
RACE as described under "Experimental
Procedures" confirmed that the elongation is caused by
U-addition.
[View Larger Version of this Image (31K GIF file)]
uridylylation site, as well as heterogeneity in the number of
posttranscriptionally added 3
terminal U nucleotides (Fig. 3A) as is common with other gRNAs.
tail
resulted in extensive misincorporation (data not shown). Cloning and
sequencing of the 3
ends of individual gRNA molecules lacking the
oligo(U) tail indicated that 90% of these RNAs were correctly
transcribed and terminated. Fig. 3B shows that incubation of
these transcripts with the mitochondrial extract under the conditions
employed to detect U-insertions resulted in the 3
addition of 15 ± 5 U nucleotides to approximately 20% of the transcript population
by the endogenous mitochondrial terminal uridylyl transferase.
-extended sequence dramatically decreased the overall
level of U-insertions, leaving a primer + 4 band and a background
ladder (Fig. 2B, lane 6). Little insertion was
seen on incubation with the gCyb-I(0,0) gRNA lacking guiding nucleotides for editing sites 1 and 2 (Fig. 2B, lane
7). Incubation with a non-cognate gRNA for editing of NADH
dehydrogenase subunit 7 mRNA had a small inhibitory effect on the
gRNA-independent U-insertion ladder.
end brought about the appearance of a primer + 4 U-insertion
band and a faint background ladder (Fig. 2B, lane 12). This gRNA-dependent U-insertion activity requires
both Mg2+ and added UTP (Fig. 2B, lanes
10-12). Although the insertions are dependent on the presence of
guiding nucleotides for sites 1 and 2, increasing the number of guiding
nucleotides failed to result in a corresponding increase in the number
of U nucleotides inserted into these sites (data not shown).
oligo(U) tail, but it was added during the incubation by the endogenous
terminal uridylyl transferase activity (Fig. 3). In a control
experiment, the addition of an oligo(U) tail (15 ± 5 U
nucleotides) with poly(A) polymerase to the 3
end of the synthetic
gCyb-I gRNA prior to incubation with lysate did not increase the level
of insertion (data not shown).
end in lane 12 of Fig.
2B.
-extended Cyb mRNA
transcript, a ladder of extension products representing insertions of
up to 15 U nucleotides was observed in the absence of added gRNA. The
extension ladder was not observed with a transcript having the 5
end
characteristic of fully edited Cyb mRNA ("natural 5
end"). 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. Evidence was also presented that the U-insertions with the
5
-extended mRNA substrate can occur in the absence of a cognate
anchor sequence, which indicates that the activity is independent of
endogenous as well as exogenous gRNA.
end only
after exogenous gRNA was added to the lysate; the endogenous gRNA pool
was not sufficient to mediate the reaction. When exogenous gRNA was
added to the lysate, the presence of the 5
extension on the Cyb
mRNA construct did not significantly increase the level of
U-insertion, suggesting that it does not strengthen the interaction with gRNA. Thus, if endogenous gRNAs were mediating insertions into the
Cyb mRNAs with the 5
upstream sequence, they would also have been
expected to mediate insertions into the RNAs with the natural 5
end.
G27 better than
6.7 kcal/mol. The
G27 for the binding of gCyb-I gRNA to its
anchor binding site is
19.1 kcal/mol. As a result, the
Kd for the fortuitous binding of gRNAs to the
mutated anchor binding sites would be approximately 109
times greater than that of the cognate gRNA interacting with its
mRNA. Since the most abundant gRNA in the UC strain is only 500 times more abundant than gCyb-I gRNA,2 the
disfavorable dissociation constant for potential fortuitous interactions would not be compensated by a greater gRNA abundance. In
the absence of other factors, insertions mediated by endogenous gRNAs
interacting with the mutated anchor binding sites should be relatively
rare events. Although it is possible that other components could be
stabilizing the gRNA-mRNA interaction, the binding energy
contributed by the anchor sequence and the evolutionary pressure
required to maintain it cannot be ignored.
-extended pre-edited mRNAs required
for the gRNA-independent insertions in vitro exist in vivo. Primer runoff analysis of L. tarentolae
endogenous edited Cyb RNA localized the 5
end to the position shown in
Fig. 1B (data not shown), which is in agreement with the
previous mapping of the major Cyb transcript (34). Mapping of the 5
end of pre-edited Cyb RNA from L. tarentolae gave ambiguous
results, as the primer used to extend the pre-edited transcript
cross-hybridized with additional RNAs (data not shown). The 5
ends of
both the major edited and pre-edited transcripts from T. brucei, however, were reported to be located at approximately the
same position as that of the L. tarentolae edited transcript
(35). Although it is possible that the Cyb transcript in L. tarentolae is made as part of a rapidly processed polycistronic
RNA, there is not yet any evidence to support this hypothesis. It is
interesting, however, that similar in vitro results have
been obtained with ND7 pre-edited mRNA, in which a 5
-extended
construct showed a higher level of gRNA-independent U-insertions than
the non-extended construct (36).
extension, 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, as
diagrammed in Fig. 4. 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.
Fig. 4.
Possible relationship of secondary structure
of the 5-extended Cyb mRNA to the secondary structure of the
postulated gRNA-mRNA hybrid. Upper panel, secondary
structure of the Cyb mRNA construct. Editing sites 1 and 2 are
indicated, and the entire pre-edited region is underlined.
The anchor-binding sequence is in lowercase. The helix
required for gRNA-independent U-insertion into this RNA is indicated.
Lower panel, secondary structure of the postulated gCyb-I
gRNA/Cyb mRNA hybrid. Editing sites 1 and 2 are indicated, and the
entire pre-edited region is underlined. The anchor helix is
indicated, which we speculate fulfills a similar role in
gRNA-dependent U-insertion activity as the indicated helix
in the mRNA sequence shown above does in the gRNA-independent
activity. The guiding A and G nucleotides in the single-stranded loop
are in lowercase.
[View Larger Version of this Image (30K GIF file)]
end, and this stimulation was dependent on the presence of
guiding nucleotides in the cognate gRNA (data not shown). The major
primer + 4 band observed by the indirect assay in Fig. 2B,
lane 12, could be interpreted as representing a single U
insertion into each of the two editing sites as in vivo, or
alternatively, as two insertions occurring within a single site. A
clear demonstration of in vitro U-insertions into pre-edited mRNA being determined by base pairing with guiding nucleotides has
been obtained recently using a pre-edited mRNA for NADH
dehydrogenase subunit 7 and the cognate gRNA (36).
extension, suggesting
that the stabilized duplex supporting the gRNA-independent reaction
does not result in major global changes to the structure (data not
shown). Other parts of the mRNA in addition to the stabilized
duplex, however, may still be involved in editing site recognition. For
example, it has previously been proposed that the presence of the
editing sites within a single-stranded loop is important for an
endonucleolytic cleavage hypothesized by the enzyme cascade model to be
part of the editing reaction (17, 37), but only limited evidence has been provided in support of this hypothesis (38).
*
This work was supported in part by Grant AI09102 from the
National Institutes of Health (to L. S.). The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
Current address: Dept. of Pharmacology, University of
Minnesota, MN 55455.
**
To whom correspondence should be addressed: Howard Hughes Medical
Institute-UCLA, 6780 MacDonald Research Laboratories, 6775 Circle Dr.
S., Los Angeles, CA 90024.
1
The abbreviations used are: gRNA, guide RNA;
PCR, polymerase chain reaction; RT, reverse transcription; nt,
nucleotide(s); Cyb, cytochrome b; bp, base pair(s); DMS,
dimthyl sulfate; FP, forward or 5 PCR primer; RP, reverse or 3
PCR
primer.
2
O. Thiemann and L. Simpson, unpublished
results.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.