(Received for publication, August 17, 1995; and in revised form, October 18, 1995)
From the
RNA editing corrects a C
A
mismatch to a conventional
T-A
Watson-Crick base pair in the acceptor stem of the
mitochondrially encoded tRNA
in plants. In vitro processing of edited and unedited Oenothera tRNA
precursor RNAs with pea mitochondrial protein extracts shows a
significant effect of this RNA-editing event on the efficiency of 5`
and 3` processing. While mature tRNA molecules are rapidly generated by in vitro processing from edited precursors, the formation of
mature tRNAs from unedited pre-tRNAs is considerably reduced. Primer
extension analyses of in vitro processing products show that
processing at both 5` and 3` termini is governed by the RNA-editing
event. Investigation of edited and unedited precursor RNAs by lead
cleavage experiments reveals differences in the higher order structures
of the pre-tRNAs. The differing conformations are most likely
responsible for the altered processing efficiencies of edited and
unedited precursor molecules. RNA editing of the tRNA
precursors is thus a prerequisite for efficient excision of the
mature tRNA
in vitro. Hence RNA editing might be
involved in regulating the amount of mature tRNA
in the
steady state RNA pool of mitochondria in higher plants.
Plant mitochondrial RNAs have to undergo various posttranscriptional processing events to achieve their mature forms. These include splicing, 5` and 3` processing of mRNAs and rRNAs, excision of tRNAs from precursor molecules, and RNA editing (Gray et al., 1992). RNA editing has been described in different genetic systems and unlike all other processing steps alters the primary sequence of the affected RNA (for review see Bonnard et al., 1992; Wissinger et al., 1992; Gray et al., 1992). In plant mitochondria the editing process results in a conversion of genomically encoded cytidines (C) to uridines (U), however, in some rare cases reverse reactions have also been observed (Bonnard et al., 1992; Hiesel et al., 1994). Although specificity and mechanism of the RNA-editing reaction remain elusive, recent studies indicate that either deamination or transglycosylation of the ribosyl residue is involved in the conversion and that the sugar-phosphate backbone of the RNA remains intact during the editing process (Rajasekhar and Mulligan, 1993).
Most of the editing sites
described in higher plant mitochondria are found in mRNA molecules and
usually change the encoded amino acid identities. This frequently
results in an evolutionary better conservation of the protein than
predicted by the genomic sequence. The alteration of rRNA primary
sequence by RNA editing remains unclear, since so far only a single
edited cDNA has been found in Oenothera (Schuster et
al., 1991). However, tRNA editing has unambiguously been
identified in several instances and different plant species. One
editing site located in the anticodon stem of tRNA is
only found in Oenothera, while a C to U conversion has been
observed in the acceptor stem of tRNA
in several plants, i.e. potato, bean, and Oenothera. This nucleotide
transition corrects a
C
A
mismatch to a
regular
T-A
Watson and Crick base pair
(Maréchal-Drouard et al., 1993; Binder et al., 1994). It has been speculated that this editing event
might interfere with other maturation processes such as 5` and 3` end
processing, in which endonucleases are implicated in both dicot and
monocot plant species (Marchfelder et al., 1990, Hanic-Joyce
and Gray, 1990). Such a potential connection between RNA editing and
the generation of mature 5` and 3` ends was indicated by the
observation that all investigated mature tRNA
molecules
are edited (Maréchal-Drouard et al.,
1993), while in only 53.3% of the precursors is this nucleotide
conversion detected (Binder et al., 1994).
In vitro processing assays with edited and unedited tRNA precursor molecules and plant mitochondrial protein lysates now
clearly show that the efficiency of 5` and 3` processing depends on RNA
editing. Lead cleavage studies of the pre-tRNA molecules indicate that
the single nucleotide transition caused by RNA editing leads to
significant changes in higher order structures that are responsible for
the differing processing efficiencies.
Plasmids
containing the tRNA gene were derived by polymerase chain
reaction from cDNA reverse transcribed from edited and unedited
precursor RNAs. cDNA synthesis used Superscript
reverse
transcriptase (Life Technologies, Inc.) primed with oligonucleotide P7
5`-ATAAGCTTGAATTTCCAAATCCGG-3` complementary to the 3`-terminal part of
the precursor. Using primer P6 5`-CAAAAGACGGAAGACAAGAG-3`
(complementary to the 5`-terminal sequences of the precursor) and
primer P7, single strand cDNA was amplified on a Biomed thermocycler
under the following conditions: 1 min at 94 °C, 1 min at 50 °C,
and 2 min at 72 °C. Polymerase chain reaction was performed using
2.5 units of Taq DNA polymerase in a buffer supplied by the
manufacturer (Boehringer) with 50 nM dNTP and 500 ng of each
primer. After 30 cycles and a final incubation at 72 °C for 5 min,
DNA fragments were digested with HindIII and cloned into
pBluescript II KS+ cut with SmaI/HindIII. Edited
(ptrnfbs300+) and unedited (ptrnfbs300-) tRNA
cDNA clones were confirmed by DNA sequencing. The tRNA precursor
molecules transcribed with T7 RNA polymerase from a HindIII-linearized template consist of 55-nt (
)vector and 180-nt mitochondrial sequences, which
correspond to sequences between nucleotide positions 313 and 492 in
EMBL data bank entry X74449 (Binder and Brennicke, 1993).
Figure 1:
Schematic
structure of the tRNA precursor. Both edited and unedited
pre-tRNA molecules are 235 nt long and consist of a 117-nt-long 5`
leader, the 73-nt-long tRNA
(indicated by the classic
clover leaf structure) and a 45-nt 3` trailer sequence. The indicated
C
(cytidine) to U
(uridine) editing site is
located at position 121 of the precursor molecule, corresponding to
nucleotide 4 in the tRNA sequence.
Distinct RNA molecules are observed after
incubation of the edited precursor for 15 and 30 min, respectively (Fig. 2, right side). Signal 3 corresponds to RNA
molecules migrating in the size range of the mature tRNA (73 nt). Signal 1 most likely represents a reaction intermediate
processed at the 5` end but unprocessed at the 3` end of the tRNA (128
nt), while signal 2 corresponds to the 5` leader (117 nt). RNAs
detected in the range of about 45 nt (signal 4) probably represent the
3` trailer of the precursor.
Figure 2:
In vitro processing of tRNA precursor RNAs. Reaction products from processing assays
performed with edited (right part) and unedited (left
part) pre-tRNAs were separated on a 6% polyacrylamide gel.
Incubation times are given at the top. Control reactions were
carried out under identical conditions for 30 min without protein. Numbers on the right indicate specific processing
products derived from the edited precursor. Sizes of coelectrophoresed
DNA length markers (left) are given in
nucleotides.
Processing assays with unedited
pre-tRNAs reveal a complex pattern of different reaction products (Fig. 2, left side). The very weak signal in the size
range of the mature tRNA indicates a very diminished
correct and complete processing reaction. All stronger signals observed
after 15 min of incubation represent RNAs also arising in control
reactions and thus most likely derive from nonspecific cleavage. After
30 min the mitochondrial lysate has degraded the vast majority of the
unedited precursor RNAs to nonspecific reaction products.
Investigation of the 5` terminus of total mtRNA reveals a clear major signal at the precise 5` end of the tRNA predicted by the secondary structure model. The major signal obtained in the primer extension from the same primer P5 on in vitro processed edited RNAs coincides with the in vivo 5` end, thus confirming correct in vitro excision (Fig. 3, lanes 1 and 2). Only a minor signal is observed at the mature 5` end of the tRNA molecule in the analysis of processing products from the unedited precursor (Fig. 3, lane 3). These relations confirm the reduced 5`-processing efficiency of the unedited precursor RNA seen in the in vitro product analyses (Fig. 2). Accuracy of 5` processing, however, appears not to be affected by the editing event, since the weak signal at the mature 5` end is precise without scattering of the cleavage site to neighboring nucleotides. Mature 5` termini are not detected in the control reactions, confirming that the signals corresponding to the mature 5` tRNA ends result from genuine in vitro processing reactions of the mitochondrial protein lysate (Fig. 3, lanes 4-6).
Figure 3:
Analysis of 5` end processing of
tRNA. Primer extension analysis of in vitro processing products from edited (lanes 2 and 5)
and unedited (lanes 3 and 6) precursor RNAs and total in vivo mitochondrial RNA (lane 1). All in vitro processing assays and control reactions (lanes 2, 3, 5, and 6) were incubated for 30 min.
Control reactions were performed with nucleic acids extracted from the
mitochondrial protein isolated (lane 4) and with the
degradation products of in vitro synthesized precursors after
incubation without mitochondrial protein lysate (lanes 5 and 6). Extension products were coelectrophoresed with sequencing
reactions identically primed (primer P5) on an edited cDNA clone (cDNA). For easier interpretation sequencing reactions (CTAG) are labeled inverted to show the sequence of the sense
strand. The 5` end of the mature tRNA
and the editing
site are indicated on the left.
The 3` ends of mitochondrial tRNAs from plants and
other species are generated by endonucleolytic cleavage, allowing
indirect investigation of this processing event by primer extension
analysis of the 5` termini of 3` trailer fragments (Manam and van
Tuyle, 1987; Chen and Martin, 1988; Hanic-Joyce and Gray, 1990; Binder
and Brennicke, 1993). Analysis of steady state Oenothera mtRNA
identifies a guanosine as the 5`-terminal nucleotide of the trailer
molecule, which coincides with the first nucleotide downstream of the
mature 3` end of tRNA (without the posttranscriptionally
added CCA; Fig. 4, lane 1). This 3` terminus is also
detected with in vitro processed products derived from edited
precursor molecules, suggesting correct cleavage at the 3` end in
vitro (Fig. 4, lanes 1 and 2). One of the
additional primer extension products also indicates incorrect
processing, generating a tRNA molecule shortened by one nucleotide at
the 3` end.
Figure 4:
Accuracy of 3` end processing. The
position of the endonucleolytic processing event generating the 3` end
of tRNA is indirectly investigated by primer extension
analysis of the 5` end of the 3` trailer from primer P1. The extension
products were coelectrophoresed with sequencing reactions performed
with the same primer on a cDNA clone (cDNA, CTAG).
For easier interpretation labeling of the sequence is inverted to represent the sense strand. Primer extension reactions were
performed with isolated total in vivo mtRNA (lane 1)
and with processing products from edited and unedited precursors (lanes 2 and 3). Control reactions were carried out
both with precursors incubated without lysate (lanes 5 and 6) and with the lysate fraction without added pre-tRNA
substrate (lane 4). The 3` end of the mature tRNA
is indicated on the left. The 5`-terminal nucleotide of
the 3` trailer is marked by an arrow.
Only a very faint signal of correct 3` cleavage is observed with in vitro processing products derived from unedited precursor RNAs, indicating an almost completely reduced 3` tRNA processing activity (Fig. 4, lane 3). Slightly smaller extension products detected with both edited and unedited RNAs are also observed in the control reactions in the absence of protein lysate, identifying these signals as nonspecific breakage in the in vitro transcribed RNAs. However, no mature tRNA 3` termini were detected in these control reactions, confirming the genuine enzymatic origin of these 3` ends in the in vitro processing reactions (Fig. 4, lanes 4-6).
These primer extension
analyses indicate that the single nucleotide transition by RNA editing
is indispensable for efficient in vitro processing at both 5`
and 3` ends of tRNA.
Figure 5:
Lead cleavage analysis of
tRNA precursor molecules. A, lead cleavage
products derived from edited and unedited pre-tRNAs were analyzed by
primer extension using primer P1. The two different precursor molecules
were incubated either in the presence (0.4 mM Pb
) or absence of lead acetate (control). To
identify the locations of the lead cleavage sites extension products
were coelectrophoresed with the respective sequencing reactions. 5` and
3` ends of tRNA
are indicated on the left. Major
variations in the lead cleavage patterns are marked by arrows. B, schematic structure of mitochondrial tRNA
.
While the main lead cleavage site in the unedited precursor is located
between G
and U
(indicated by the arrow labeled 2 and ed-), the edited pre-tRNA
molecule is predominantly cleaved between nucleotides U
and G
(1a, ed+) and C
and U
(1b, ed+),
respectively. Designation of the cleavage sites corresponds to Fig. 5A, aside from the major variation 1, which is
here resolved into cleavage sites 1a and 1b.
Numbering of the nucleotides (given in parentheses) follows
the standards given in Steinberg et
al.(1993).
Accuracy of the 3` reaction on the other hand differs between the
two templates. The majority of edited precursor is correctly processed,
with a minor adjacent primer extension product indicating cleavage
products shortened by one nucleotide at the 3` tRNA terminus. In
contrast, almost no correct 3` cleavage is observed upon processing of
the unedited pre-tRNA. A weak primer extension signal indicates an
incorrect cleavage at a cytidine residue, which generates a 3` terminus
2 nucleotides shorter than the correct 3` end determined with isolated in vivo mtRNA. This suggests that the altered conformation of
the acceptor stem caused by the CA mismatch influences the 3`
cleavage specificity. It is also possible that the higher order
structure of the unedited precursor molecule makes this RNA susceptible
to cleavage by other nucleases not normally involved in 3` processing
of tRNAs.
The major breakpoint between nucleotides
U and G
detected in the analysis of the
edited precursor (Fig. 5B, breakpoint 1a)
coincides with cleavage sites observed in unmodified and modified
mature yeast tRNA
(Behlen et al., 1990). This
specific sensitivity substantiates the significance of the cleavage
sites and further supports the extrapolation that only edited pre-tRNAs
are capable of forming correct secondary and tertiary structures.
The cleavage sites differing between edited and unedited precursor molecules are considered significant for the individual conformations, since these differences are consistently observed in the comparison of the results of different lead cleavage experiments of the same RNA well above the normal experimental variations (Fig. 5, data not shown). Primer extension signals occurring independently of lead cleavage are due to pauses of the reverse transcriptase on highly structured RNAs and have also been observed in the lead cleavage analysis of RNase P RNAs from different organisms (Zito and Pace, 1992).
Conformation of the precursor seems to be the crucial distinguishing feature for the binding and/or substrate specificities of the different enzymes including the RNA editing specificity, which may thus in part be guided by higher order RNA structure.
Figure 6:
Northern blot analysis of
tRNA transcripts. A DNA fragment containing tRNA
as well as 360-nt upstream and 230-nt downstream sequences,
respectively, was hybridized to total mtRNA from Oenothera. A
signal corresponding to an RNA of about 75 nt indicates mature
tRNA
molecules. In addition a series of highly abundant
larger RNAs with sizes up to 2,600 nt were detected. Different exposure
times of the same Northern blot are indicated at the top.
Sizes of coelectrophoresed DNA length standards are given in
nucleotides on the left.
Posttranscriptional regulation during maturation has also
been suggested to control steady state abundance of tRNA in mitochondria of HeLa cells. Although a short primary
transcript containing this tRNA is transcribed in a 25-fold higher rate
than most other tRNAs, the steady state level of the mature
tRNA
is only 2-4 times higher than the average
level of other less frequently transcribed tRNAs (King and Attardi,
1993). In Oenothera mitochondria a promoter has been
identified just upstream of the tRNA
gene, which could
lead to a similarly selectively elevated transcription of this gene.
The downstream-located tRNA
, however, is cotranscribed
and would thus be present in similar amounts and therefore likewise
require extensive specific posttranscriptional regulation. Specific
promoters located immediately upstream of other tRNA genes could
analogously imply additional posttranscriptional regulation.