(Received for publication, April 21, 1997)
From the Department of Microbiology, Ohio State University, Columbus, Ohio 43210
We have investigated the requirements for mature tRNA structure in the in vivo splicing of the Haloferax volcanii, intron-containing tRNATrp RNA. A partial tRNATrp gene, which contained only the anticodon stem-loop region of the mature tRNA, was fused to a carrier yeast tRNA gene for expression in H. volcanii. Transcripts from this hybrid gene were found to be processed by endonuclease and ligase at the tRNATrp exon-intron boundaries. These results verify that the substrate recognition properties of the halobacterial endonuclease observed in vitro reflect the properties of this enzyme in vivo, namely that mature tRNA structure is not essential for recognition by the endonuclease. The independence of these reactions on mature tRNA provides further support for a relationship between archaeal tRNA and rRNA intron-processing systems and highlight a difference in the substrate recognition properties between the archaeal and eucaryal processing systems. The significance of these differences is discussed in light of the observation that the tRNA endonucleases of these organisms are related.
While intron-containing tRNAs are present in the Archaea (formerly
the Archaebacteria), Eucarya, and Bacteria, these sequences do not
represent a single homogeneous class of introns. Bacterial and
chloroplast tRNA introns are either group I or group II introns, whereas the eucaryal nuclear and archaeal tRNA introns lack any identifiable sequence or structural relationship to the group I, group
II, group III, or mRNA introns (1). In the absence of defining
sequence or structural characteristics in the archaeal and eucaryal
tRNA introns, speculation on the relatedness of these introns has been
based primarily on comparisons of their splicing systems. Until
recently, it was thought that the archaeal and eucaryal splicing
enzymes were distinct systems that were related in function only, and
consequently that eucaryal and archaeal introns potentially represented
two separate classes of introns. This argument was based on the
observations that the archaeal and eucaryal tRNA intron endonucleases
differed in subunit composition and substrate recognition mechanisms.
The eucaryal endonuclease was observed to be a heterotrimer (2), which
has recently been shown to be a tetramer (3), whereas the archaeal
enzyme is a homodimer (4). Recognition of the exon-intron boundaries by
the eucaryal enzyme involves a complex mechanism that is dependent on
the presence of mature tRNA structure. All eucaryal tRNA introns are
located in the anticodon loop between positions 37 and 38 of the
mature tRNA, extending the anticodon helix, while maintaining the
overall mature tRNA structure (1). The eucaryal enzyme senses the
distance from the top of the anticodon stem to the 5 and 3
cleavage
sites (5, 6) and requires the formation of a three-nucleotide bulge
loop at the intron-exon 2 cleavage site, the A-I interaction (7). This
mechanism is well suited for the identification of eucaryal tRNA
introns where all introns are located in the same relative position. In
contrast, in vitro studies with the Haloferax
volcanii intron endonuclease showed that this enzyme does not
require complete mature tRNA structure in its substrate (8, 9). This
enzyme requires a defined structural element at the exon-intron
boundaries, the bulge-helix-bulge motif (9). In this structure each
cleavage site is located in a three-nucleotide bulge loop, and the two
loops are separated by 4 base pairs. The enzyme senses the distance
between the bulge loops, rather than the length of the anticodon stem
(9). This mechanism is well suited for the archaeal tRNA intron since
these introns are not restricted to a single location in the mature domain of the tRNA. In the Archaea, tRNA introns have been observed in
the anticodon loop, the anticodon stem, and the extra arm (10-16). Despite their variability in location, all archaeal intron-containing tRNAs can assume the bulge-helix-bulge structure at their intron-exon boundaries.
With such fundamental differences in subunit composition and
recognition mechanisms, the proposal that the archaeal and eucaryal tRNA processing systems were different appeared justified. However, the
recent characterization of the genes encoding the H. volcanii and Saccharomyces cerevisiae tRNA intron
endonucleases has unexpectedly revealed that these two enzymes are
related (3, 4). A comparison of the amino acid sequences of the
halobacterial endonuclease monomer and the yeast endonuclease complex
revealed that the halobacterial protein shared sequence similarity with
two subunits of the yeast tetramer. This similarity extended over an
approximately 115-amino acid region, and in each case, this sequence
was located in the carboxyl terminus of the protein (4). Knowing that
the archaeal and eucaryal endonucleases are related underscored the
need to verify that the substrate recognition properties of the
archaeal endonuclease defined in vitro are the same as those
used in vivo. In this report we describe experiments to test
the proposal that the halobacterial tRNA intron endonuclease can
process a tRNA intron from a RNA molecule that lacks full tRNA
structure. As an in vivo test for this model, we have
constructed an H. volcanii expression module that is capable
of producing a hybrid RNA that encodes a partial H. volcanii
tRNATrp RNA fused to the 5 leader region of the non-processing yeast
tRNAProM RNA (17). The tRNATrp
13115
-tRNAProM hybrid
RNA encodes the tRNATrp anticodon stem-loop region and intact intron
fused to the carrier RNA. This represents the minimum exon sequences
required for in vitro cleavage. Analysis of RNA from cells
carrying this hybrid gene demonstrate that this partial tRNATrp RNA is
processed by both endonuclease and ligase enzymes in the absence of a
complete mature tRNA structure. We discuss the implications of this
observation in defining the relationships between archaeal and eucaryal
tRNA processing systems and the roles of these archaeal enzymes in
cellular RNA processing.
H. volcanii
strain WFD11 (18) was grown aerobically at 37 °C in complex medium
(19), and when necessary to ensure maintenance of pWL-based expression
plasmids, this medium was supplemented with 20 µM
mevinolin (a gift from Merck). Escherichia coli strains DH5-F
and JM110 were cultured in Luria Broth (LB) medium or LB
medium supplemented with 100 µg/ml ampicillin when cells carried pUC-
or pWL-based plasmids.
T4 polynucleotide kinase, T4 DNA ligase, Klenow DNA polymerase,
SuperScriptTMII, Moloney murine leukemia virus reverse
transcriptase, and all restriction enzymes were purchased from Life
Technologies, Inc.; SequenaseTM, 5-bromo-4-chloro-3-indolyl
-D-galactopyranoside and
isopropyl-1-thio-
-D-galactopyranoside were obtained from
U. S. Biochemical Corp.; AmpliTaqTM DNA polymerase and
GeneAmpTM core reagents were purchased from Perkin-Elmer,
and Zeta-Probe nylon membrane was obtained from Bio-Rad Laboratories.
Oligonucleotides used in this study were synthesized by The Ohio State
University Biochemical Instrument Center or Ransome Hill Biosciences,
Inc.
The intron-containing H. volcanii tRNATrp13115
gene, which contains a complete intron
and only the anticodon stem and loop regions of the mature tRNATrp RNA,
was isolated from plasmid pVT22-
13115
(20) as a 150-base pair
EcoRI-HindIII restriction fragment. Protruding 5
and 3
ends of the tRNATrp
13115
fragment were filled in using
Klenow DNA polymerase, and this fragment was cloned into the
HincII restriction site of the vector pUC1318 (21). The
tRNATrp
13115
gene was recovered from the pUC1318 vector as a
XbaI-XbaI restriction fragment, which was then
subcloned into the XbaI site of the H. volcanii
expression vector pWL302A1 (22) to yield the plasmid
pWL302A1-
13115
. The A3 to T3 mutation of the tRNATrp
13115
gene
(Fig. 1B) was prepared using the polymerase chain reaction
(PCR).1 The PCR reaction
contained 30 mM Tricine, pH 8.4, 2 mM
MgCl2, 5 mM
-mercaptoethanol, 0.01%
gelatin, 0.1% Thesit, 200 µM each dNTP, 1 µM of the mutagenic primers T7O168A3-T
(5
-AGCTCTAGATAATACGACTCACTATAGGCATGGCGACTGACTCCAGTGGCT-3
), 1 µM of the primer O168short
(5
-AGGGATCTAGACCCGATCGACTG-3
), and 2.5 units of
AmpliTaqTM DNA polymerase. The reaction mixture was
incubated at 95 °C for 5 min, and polymerization was carried out for
30 cycles. Each cycle consisted of incubation at 94 °C for 1 min,
55 °C for 1 min, and 72 °C for 2 min. The resulting fragment was
cloned into pUC1318 and subcloned into pWL302A1 as described for the
wild-type tRNATrp
13115
gene, yielding the plasmid
pWL302A1-
13115
T3. These plasmids were introduced into E. coli strain JM110, and the plasmids isolated from these strains
were then used to transform H. volcanii strain WFD11 (22).
Passage through strain E. coli JM110
(dam
) reduces restriction during
transformation of H. volcanii (23).
In Vitro Endonuclease Assay
Radiolabeled substrate RNAs
were generated by T7 RNA polymerase transcription of PCR-amplified DNAs
and gel-purified as described previously (9). The tRNATrp13115
and
tRNATrp
13115
T3 genes were amplified using the primers T7O168
(5
-AGCTCTAGATAATACGACTCACTATAGGCATGGCGACTGACTCCA-3
) and
T7O168A3-T (5
-AGCTCTAGATAATACGACTCACTATAGGCATGGCGACTGACTCCAGTGGCT-3
) as forward primers, respectively, and O1683
(5
-AACCCCGATCGATCG-3
) as the reverse primer. Reactions were carried
out as described above. A typical endonuclease reaction contained 2 µl of an H. volcanii S100 extract (8) and
32P-labeled substrate RNA (10,000 cpm) in a buffer
containing 40 mM Tris-HCl, pH 7.5, 20 mM
MgCl2, and 2% glycerol. Reactions were incubated at
37 °C for 30 min. The products were separated by denaturing (8 M urea) polyacrylamide gel electrophoresis and visualized by autoradiography.
For analysis of
tRNATrp13115
-tRNAProM and tRNATrp
13115
T3-tRNAProM RNAs,
H. volcanii cells were grown to an
A560 of 1.0 at 37 °C in complex medium (22)
containing 20 µM mevinolin. Cells from a 25-ml culture
volume were harvested by centrifugation at 5000 × g
and lysed by adding 1.25 ml of lysis buffer (10 mM Tris-HCl, pH 8.0, 10 mM NaCl, 1 mM trisodium
citrate, and 1.5% SDS) to the cell pellet. The lysate was incubated
for 10 min at 37 °C, followed by an additional 10-min incubation on
ice. DEPC (25 µl) and NaCl-saturated double distilled H2O
(625 µl) were added to the mixture, and the incubation was continued
for an additional 15 min. DEPC was omitted from preparations to be used in reverse transcription reactions. The mixture was then transferred to
a RNase-free Corex tube, and the contents were centrifuged at
12,000 × g for 10 min at 4 °C. The aqueous phase
was recovered, and the RNA was ethanol-precipitated. The resulting RNA
pellet was washed once with 2 ml of 95% ethanol and resuspended in
RNase-free double distilled H2O or RNA loading buffer (7 M urea, 10% glycerol, 0.05% bromphenol blue, and 0.05%
xylene cyanol). A typical yield was 120 µg of total RNA per 25 ml of
original culture volume.
For Northern analysis, total RNA isolated from H. volcanii
cells, approximately 50 µg in 50 µl of RNA loading buffer, was separated by electrophoresis in a 6% denaturing (7 M urea)
polyacrylamide gel. After separation, the RNA was electrophoretically
transferred to a Zeta-Probe membrane as described by the manufacturer
(Idea Scientific Co., Minneapolis, MN). Northern blots were
prehybridized at 50 °C for 10 min in a solution containing 5 × SSC (1 × SSC contains 150 mM NaCl and 15 mM trisodium citrate), 20 mM
NaH2PO4, pH 7.0, 7% SDS, 10 × Denhardt's solution, and 100 µg/ml single-stranded salmon sperm DNA.
The prehybridization mixture was discarded and replaced with the same
buffer at 150 µl/cm2 Zeta-Probe membrane. Transcripts
were detected by probing with one of either two 5 end-labeled
oligonucleotides: ProExI (5
-CCCAAAGCGAGAATCATACCAC-3
) specific for
the 3
reporter gene tRNAProM or IntDel (5
-GGACTCTAGAATTCGAG-3
) specific for the splice junction formed by exons 1 and 2. Oligonucleotides were labeled with [
-32P]ATP and T4
polynucleotide kinase. Hybridizations were conducted overnight at
50 °C for ProExI and 45 °C for IntDel. Following hybridization
the membranes were washed three times for 15 min in 400 ml of wash
buffer (2 × SSC, 0.5% SDS) at 22 °C. Hybrids were detected by
autoradiography. For sequential hybridizations, blots were stripped
between hybridizations by washing the membrane twice in a solution
containing 0.1 × SSC, 0.5% SDS at 95 °C.
A reverse
transcription-PCR approach was used to verify that the 180-nucleotide
RNA species resulted from accurate exon-intron cleavage and exon
ligation at the tRNATrp13115
processing site of the hybrid RNA.
ProExI (20 pmol) was annealed with 25 µg of total RNA in a 20-µl
reaction volume of 1 × Moloney murine leukemia virus reverse
transcriptase buffer (50.0 mM Tris-HCl, pH 8.3, 40 mM KCl, 6.0 mM MgCl2, 1.0 mM dithiothreitol) at 80 °C for 4 min. This mixture was
then allowed to cool to room temperature over a course of 5 min. First
strand cDNA synthesis was initiated with the addition of 30 µl of
cDNA extension mixture (50.0 mM Tris-HCl, pH 8.3, 40 mM KCl, 6.0 mM MgCl2, 1.0 mM dithiothreitol, 0.1 mg/ml bovine serum albumin, and 1.25 mM of each dNTP) and 200 units of Moloney murine leukemia
virus reverse transcriptase buffer; reactions were incubated for 1 h at 50 °C. Reverse transcriptase was then inactivated by raising
the reaction temperature to 95 °C for 2 min. RNA was degraded by
adding 1 µl of RNase A (10 mg/ml) and incubation of the mixture for
10 min at 42 °C. Proteins were removed from the reaction by
extracting the mixture once with an equal volume of phenol/chloroform
and once with chloroform. cDNAs were ethanol- precipitated, and the
precipitate was washed once with 400 µl of 75% ethanol, dried, and
redissolved in 20 µl of double distilled H2O.
cDNAs were amplified by PCR as described above using the primers
Prol5 (5
-GCAAGGGGACTCTAGAGT-3
) and ProExI. Reverse transcriptase DNA
products from this reaction were cloned into the SmaI site of pUC19 and sequenced using the SequenaseTM system. Five
individual pUC19 clones were examined, and each was determined to have
the identical insert.
Based on in vitro processing studies, the
tRNATrp13115
variant of the H. volcanii tRNATrp gene was
chosen as a model RNA to investigate the requirements for mature tRNA
structure in in vivo tRNA splicing. This gene encodes an RNA
having the complete tRNATrp intron and only the anticodon stem-loop of
the mature tRNA. This RNA is accurately and efficiently cleaved by a
partially purified H. volcanii tRNA intron endonuclease (8).
To test whether these sequences and structures were sufficient for
cleavage in vivo we needed a carrier RNA to express this
potentially unstable form of the tRNATrp RNA. Previous in
vivo expression studies showed that a modified version of the
yeast tRNAPro(UGG) RNA, tRNAProM, could be expressed in H. volcanii on the expression plasmid pWL302A1 (22). This gene
encoded a single stable transcript that represented the primary
transcript from this gene. The production of a single RNA species was
the result of two processing defects in this RNA, a
U6-U72 pair preventing 5
and 3
termini
processing and an intron that is not recognized by the H. volcanii endonuclease (24). The yeast tRNAProM DNA fragment also
carried a RNA polymerase III termination element that functioned as a
strong terminator in H. volcanii.2 We reasoned
that introduction of the tRNATrp
13115
gene into the 5
leader
region of the yeast tRNAProM construct would lead to the production of
a stable RNA hybrid. This hybrid gene and its expected transcript are
show in Fig. 1.
Processing
of the tRNATrp13115
-tRNAProM hybrid transcript was followed by
Northern analysis. When an oligonucleotide specific to the yeast
tRNAProM RNA exon 2 was used as a probe, three RNA species were
detected (Fig. 2A). The
approximated sizes of these RNAs were 285, 180, and 140 nucleotides.
The largest RNA species corresponded in size to the expected primary
transcript, and the smallest RNA species, 140 nucleotides, corresponded
in size to the predicted RNA resulting from cleavage at the
tRNATrp
13115
RNA intron-3
exon boundary. The intermediate species,
180 nucleotides, was similar in size to the product predicted for
cleavage at both exon-intron boundaries, followed by exon ligation
(Fig. 2A, left panel). The other expected
intermediate of the reaction, exon 1-intron RNA, was also detected when
an exon 1-specific probe was used (data not shown).
Since correct cleavage and ligation of tRNATrp1315
-tRNAProM RNA
would generate a structure having sequences identical to the mature
tRNATrp anticodon loop, these RNAs were also probed with an
oligonucleotide that corresponds to the mature anticodon stem and loop
sequence (Fig. 2A, right panel). This probe
hybridized to both the mature, chromosome-encoded tRNATrp RNA and the
180-nucleotide species, suggesting that correct cleavage and ligation
had occurred with the hybrid RNA. As an independent test that the
cleavage reactions observed in vivo were the result of the
tRNA intron endonuclease activity, and not a general ribonuclease, the
processing pattern of a cleavage-defective form of the tRNATrp RNA,
tRNATrp
13115
T3-ProM, was also examined. This RNA contains a single
point mutation at the exon 1-intron boundary, A3 to T3 at position 41 (see Fig. 1B), which leads to an 80% decrease in in
vitro cleavage when compared with the wild-type RNA (Fig.
2B, left panel). Northern analysis of RNAs from
cells carrying the tRNATrp
13115
T3-ProM gene show that this RNA
remains predominately as the primary transcript (Fig. 2B,
right panel). A minor species (<10% of the total), which corresponded in size to an RNA resulting from cleavage at the exon
1-intron boundary, was also detected. The inability of this RNA to
process in vivo is consistent with its cleavage properties in vitro and suggests that the in vivo processing
of the tRNATrp
13115
-ProM RNA was the result of endonuclease
activity.
To further verify that the 180-nucleotide RNA species produced from the
tRNATrp13115
-ProM gene was the product of both cleavage and
ligation, cDNAs were synthesized from this RNA and used as template
for PCR amplification. The cDNA was synthesized using a primer
specific for the yeast tRNAProM RNA, and PCR amplification was carried
out with oligonucleotides specific for sequences 5
of the
tRNATrp
13115
encoding region and a sequence internal to the yeast
tRNAProM RNA. This prevented cDNA synthesis and amplification of
chromosome-encoded tRNATrp RNA. Five DNA products were sequenced, and all had the predicted sequence for accurate cleavage and ligation of the tRNATrp
13115
RNA (Fig. 3).
Based on in vitro processing studies of the H. volcanii tRNATrp RNA we predicted that in vivo cleavage
of the intron from this pretRNA would be dependent on exon-intron
boundary sequences and structures and independent of mature tRNA
structure and sequences beyond those of the anticodon stem and loop (8,
9). To determine if the requirements observed in vitro
reflected the requirements for in vivo processing we
introduced the H. volcanii tRNATrp13115
gene into the 5
unprocessed leader region of the yeast tRNAProM RNA. As anticipated,
Northern analysis confirmed that the predicted hybrid RNA was produced
and that this RNA underwent cleavage in the absence of a complete
mature tRNA structure (Fig. 2A). RNA species consistent with
cleavage at both 5
and 3
exon-intron boundaries were detected
indicating that both cleavage sites were recognized. The inability of
the in vitro processing-defective tRNATrp
13115
T3-ProM
RNA to undergo processing in vivo supported the proposal
that the hybrid RNA was cleaved by endonuclease rather than a general
ribonuclease. Unexpectedly, an additional 180 nucleotide species was
detected in cells that carried the tRNATrp
13115
-ProM RNA gene. This
RNA corresponded in size to an RNA that had undergone cleavage at both
sites and exon ligation (Fig. 2B). Sequence analysis of
cDNAs derived from this 180-nucleotide RNA by reverse transcription and PCR amplification verified that this RNA resulted from both accurate cleavage and exon ligation (Fig. 3). These data confirm earlier in vitro observations that the halobacterial tRNA
processing system is capable of acting on non-tRNA substrates (8) and show for the first time that the tRNA ligation reaction is independent of mature tRNA structure. These results also provide an explanation for
how a single endonuclease could act on a population of
intron-containing pretRNAs where all introns are not located in the
same relative position in the mature tRNA. In this case the primary
criteria for recognition would be the presence of the bulge-helix-bulge motif. Indeed, most archaeal intron-containing tRNAs possess this or a
closely related structure at their exon-intron boundaries (8, 25,
26).
A processing system that is directed toward sequences and structures at the exon-intron boundaries could in theory cleave any RNA, regardless of its origin or final structure. Structural analysis of archaeal intron-containing 16 S and 23 S rRNA precursors has shown that these RNAs have the tRNA bulge-helix-bulge motif or closely related structures at their exon-intron boundaries (27). Some rRNA introns also encode homing endonucleases (28-31) similar to those found in some group I introns; however, the characteristic core group I RNA structures and sequences are absent in these introns. This has led to the proposal that these intron-containing rRNAs are processed by the same enzyme system as the pretRNAs (8, 15, 25, 26). In support of this proposal a partially purified Desulfurococcus mobilis 23 S rRNA intron endonuclease was found to cleave an intron-containing tRNA from this same organism (15). The ability of this endonuclease to act on tRNA and rRNA substrates raises the question of whether this enzyme could act as a general RNA endonuclease. One possible candidate is the primary transcript from the rRNA operon. Sequence analysis of archaeal rRNA operons has shown that the 16 S and 23 S rRNA coding regions are flanked by large inverted repeats. As in bacterial cells, these helices are though to be sites for RNaseIII cleavage (32). However, we and others (25, 33) have noted that the archaeal helices possess the characteristic bulge-helix-bulge motif of the tRNA exon-intron boundaries. It is possible that these structures are recognized by the tRNA endonuclease rather than a RNaseIII-like enzyme. Interestingly, a survey of the Methanococcus jannaschii genome (34) did not reveal the presence of an RNaseIII-like gene in this organism.
The results of this study also show that ligation can take place in the absence of mature tRNA structure. A ligase capable of joining exon ends held in close juxtaposition would be expected to act on those tRNAs where the intron was excised from a position other than the anticodon loop. This ligase could also act on the rRNA exons produced by endonuclease cleavage of introns from these RNAs. The lack of an in vitro assay for ligase has prevented us from determining if the archaeal ligase is similar to the ATP-GTP-requiring ligase of yeast and HeLa cells (35) or the ATP-independent ligase of vertebrates (36). Intron circularization has been observed during D. mobilis rRNA cleavage; however, the exons remain unligated in this reaction (37). Halophile extracts also lack ligase activity (8). A survey of the M. jannaschii genome (34) did not reveal genes related to the eucaryal tRNA ligase.
Similarities in the exon-intron boundaries of archaeal rRNAs and tRNAs and the finding that the halobacterial tRNA intron endonuclease and ligase enzymes act on non-tRNA RNAs provide further support for the proposal that the archaeal tRNA and rRNA introns are processed by the same enzyme system. In addition, the recently discovered similarities between the archaeal and eucaryal tRNA intron endonucleases (3, 4) strongly suggests that the tRNA introns of these two domains and the rRNA introns of the Archaea represent a single class of introns. The molecular mechanisms that have led to changes in the substrate recognition properties of the archaeal and eucaryal endonucleases is not yet understood. It is likely that the these differences are in part due to the divergence in the two yeast endonuclease subunits that are related to the halobacterial endonuclease protein. These two subunits are not identical as they are in the archaeal enzyme, and as a consequence they may have different RNA binding characteristics. The presence of other subunits in the yeast endonuclease may also influence the interaction of this enzyme with its substrate. Finally, no intron-containing mRNAs have been detected in the Archaea to date; however, the properties of the archaeal intron processing system described in this report indicate that a tRNA-like intron could exist in a mRNA.