Another heritage from the RNA world: self-excision of intron sequences from nuclear pre-tRNAs
Another heritage from the RNA world: self-excision of intron sequences from nuclear pre-tRNAs
Ute
Weber
,
Hildburg
Beier
and
Hans J.
Gross*
Institut für Biochemie, Bayerische Julius-Maximilians-Universität, Biozentrum, Am Hubland, D-97074
Würzburg
,
Germany
Received April 9, 1996;
Revised and Accepted May 6, 1996
ABSTRACT
The intervening sequences of nuclear tRNA precursors are known to be excised by tRNA splicing endonuclease. We show here that a T7
transcript corresponding to a pre-tRNA
Tyr
from
Arabidopsis thaliana
has a highly specific activity for autolytic intron excision. Self-cleavage occurs precisely at the authentic 3
'
-splice site and at the phosphodiester bond one nucleotide downstream of the authentic 5
'
-splice site. The reaction results in fragments with 2
'
,3
'
-cyclic phosphate and 5
'
-OH termini. It is resistant to proteinase K and/or SDS treatment and is
not inhibited by added tRNA. The self-cleavage depends on Mg
2+
and is stimulated by spermine and Triton X-100. A set of sequence variants at the cleavage sites has been analysed
for autolytic intron excision and, in parallel, for enzymatic
in vitro
splicing in wheat germ S23 extract. Single-stranded loops are a prerequisite for both reactions. Self-cleavage not only occurs at pyrimidine-A but also at U-U bonds. Since intron self-excision is only about five times slower than
the enzymatic intron excision in a wheat germ S23 extract, we propose that the
splicing endonuclease may function by improving the preciseness and efficiency
of an inherent pre-tRNA self-cleavage activity.
introduction
The intervening sequences (IVS) of nuclear tRNA precursors differ from introns
of any other RNA class in several features. They are comparatively small,
ranging in size from 14 to 60 and from 12 to 25 nucleotides (nt) in yeast and
plant intron-containing pre-tRNAs, respectively (
1
-
3
). All introns in nuclear-encoded tRNA genes are located at a conserved position 1 nt downstream of
the anticodon, but beyond this fact differ considerably in their primary
sequences, even within one tRNA family, and apparently exhibit no consensus
sequences at the intron-exon junctions. The use of chemical and enzymatic structure-specific probes has provided evidence that all pre-tRNAs adopt a tRNA-like L-shaped conformation in the mature domain and form
a helical, extended anticodon stem by base pairing between parts of the intron
and a portion of the anticodon loop (
4
). tRNA introns are excised by a unique mechanism (
5
). The enzymatic activity responsible for intron excision is thought to be the
tRNA splicing endonuclease. This enzyme cleaves the pre-tRNA precisely at the 5'- and 3'-boundaries of the intervening sequence, thus
producing a 5' tRNA half, a linear intron and a 3' tRNA half, leaving 2',3'-cyclic phosphate and 5'-hydroxyl ends. The joining of the
tRNA halves is accomplished by an ATP-dependent RNA ligase activity, resulting in a 2'-phosphomonoester, 3',5'-phosphodiester linkage followed by the
removal of the 2'-phosphate in wheat and yeast. An alternative mechanism has been
found in HeLa cell extracts and in
Xenopus
oocytes, where no 2'-phosphate is generated (
6
).
It is generally accepted that catalytic RNA plays a major role in group I and group II self-splicing introns (
7
). Even spliceosomal splicing seems to be RNA catalysed, as it becomes plausible that the small
nuclear RNAs may assemble to provide a ribozyme active site (
8
). In contrast to this, the possible role of RNA catalysis in nuclear pre-tRNA splicing has been widely neglected. Yet pre-tRNAs seem to be perfect candidates for catalytic RNAs. They adopt a
compact structure and therefore can place functional groups that are directly
or indirectly involved in catalysis in close proximity. In addition to this,
pre-tRNAs are known to have a number of binding sites for different factors,
for example high affinity sites for divalent ions and polyamines (
9
), which could act as cofactors in catalysis. Beyond that, there is another good
argument for pre-tRNAs having catalytic activity. According to the `genomic tag' hypothesis
tRNAs started off in the `RNA world' with a completely different role: the
first tRNA-like structures served as markers for RNA replication (
10
). In order to adopt their role in the evolving translational apparatus these
pre-tRNAs had to mature by themselves, at least during the early stages of
molecular evolution, without the help of protein enzymes. It is well known that
pre-tRNA 5'-processing involves catalytic RNA as a component of
ribonuclease P (
7
), at least in eubacteria. We have previously shown that a human intron-containing pre-tRNA
Tyr
has a self-cleavage activity resulting in self-excision of the intron (
11
). Using a plant pre-tRNA of different structure and completely different intron sequence we
provide new evidence that there are still more remnants of ancient catalytic
activities to be found in modern tRNA precursors.
MATERIALS AND METHODS
Enzymes and reagents
T7 RNA polymerase was prepared from an overproducing strain of
Escherichia coli
kindly provided by Dr W.Studier (
12
). RNase T1 was purchased from Calbiochem. All other enzymes were obtained from
commercial suppliers. [[alpha]-
32
P]dATP, [[alpha]-
32
P]ATP, [[alpha]-
32
P]CTP, [[alpha]-
32
P]GTP and [[alpha]-
32
P]UTP were purchased from Hartmann Analytic (Braunschweig, Germany). Triton X-100 was from Pierce (Rockford), magnesium chloride hexahydrate, 99.995%
pure, and ammonium acetate, 99.999% pure, were from Aldrich. All reagents were
ultrapure and autoclaved. Distilled water was treated with diethylpyrocarbonate
(DEPC).
Bacterial strains and plasmids
Escherichia coli
JM109 was used as host for the propagation of plasmid pUC19 and its
derivatives. The recombinant plasmid used for site-directed mutagenesis was pAtY3II, which carries a nuclear
Arabidopsis thaliana
tRNA
Tyr
gene on a 1398 bp
Rsa
I fragment in pUC19 (
13
).
Construction of tDNA clones
A
Bam
HI-
Sal
I fragment of 195 bp harbouring an intron-containing tRNA
Tyr
gene from
A.thaliana
was excised from the original pAtY3II plasmid DNA and ligated into M13mp19 RF-DNA. Subsequent oligonucleotide-directed mutagenesis (
14
) resulted in a tRNA
Tyr
gene flanked by a T7 promoter directly at the 5'-side and a
Bst
NI restriction site at the 3'-side of the tDNA. The exchange of the first base pair in the
aminoacyl stem of the tRNA from C1:G72 to G:C for efficient transcription by T7
RNA polymerase was obtained in the same way. The mutated DNA fragment was
recloned into the
Bam
HI-
Sal
I sites of pUC19 DNA. This clone was named pAtY3II*-T7. All derivatives of pAtY3II*-T7 (DS5, DS3 and M1-M7) were constructed using appropriate synthetic
oligonucleotides as primers for megaprimer PCR mutagenesis (
15
). The sequences of all constructs were confirmed by dideoxy sequencing.
Preparation and purification of pre-tRNAs
Transcription of pAtY3II*-T7 and its derivatives by T7 RNA polymerase was carried out in 20 [mu]l volumes using 0.6 [mu]g
Bst
NI-linearized plasmid DNA and 0.6 [mu]g T7 RNA polymerase (400 U/[mu]g). Incubation was for 1 h at 37oC in 40 mM Tris-HCl, pH 8.1, 12 mM MgCl
2
, 5 mM DTT, 1 mM spermidine, 20 [mu]Ci of one [[alpha]-
32
P]NTP, 0.2 mM of the same unlabelled NTP and the other NTPs at 1 mM each. The
reaction was terminated by the addition of 1 [mu]l 0.5 M EDTA. Less than 10% of the full-length product self-cleaves during transcription. The transcripts were purified by repeated phenol/CHCl
3
extractions and electrophoresis in a 10% polyacrylamide-8 M urea-TBE gel, eluted in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.5) and were further purified with
Nucleobond AX 5 columns (Macherey & Nagel, Düren, Germany) to remove acrylamide and metal ion trace contamination.
In vitro
cleavage of pre-tRNAs
The purified
32
P-labelled pre-tRNAs (4 * 10
3
c.p.m, ~10 ng or 0.3 pmol) were incubated in 100 mM NH
4
OAc, pH 7, 10 mM MgCl
2
, 0.5 mM spermine and 0.4% Triton X-100. Standard incubation was for 4 h at 37oC. Analytical reactions were carried out in 10 [mu]l volumes, whereas preparative reactions were performed with 1 * 10
6
c.p.m pre-tRNA in 400 [mu]l. The samples were analysed by electrophoresis on 10% polyacrylamide-8 M urea-TBE gels. Elution of pre-tRNA fragments for subsequent analyses was
performed as described for transcripts. Autoradiograms were evaluated by densitometry
using an LKB UltroScan XL laser densitometer for quantification of the self-cleavage products.
In vitro
splicing of pre-tRNAs in wheat germ S23 extract
In vitro
splicing of the purified
32
P-labelled pre-tRNAs (4 * 10
3
c.p.m) was performed in a total volume of 10 [mu]l, containing 2 [mu]l cell-free wheat germ S23 extract (
16
), 20 mM Tris-acetate, pH 7.4, 100 mM KOAc, 6 mM Mg(OAc)
2
, 0.8% Triton X-100, 80 [mu]M spermine, 0.1 mM ATP and 0.1 mM CTP. Incubation was for 120 min at
30oC. Quantification of the splicing products was as described for self-cleavage products.
Analysis of cleavage products
Pre-tRNA fragments were digested with RNase T1 and analysed by RNA
fingerprinting (
17
). Quantification of the labelled oligonucleotides was by scintillation counting
after scraping the corresponding spots from the DEAE-cellulose plates. For analysis of the 3'-end produced during self-cleavage, RNA fingerprinting was with chromatography
in 0.3 mM ammonium formate, 1 mM EDTA and 9 M urea, pH 5.5 (
18
). The labelled UMP was recovered by scraping the corresponding spot from the DEAE-cellulose plate, followed by elution with triethylammonium bicarbonate buffer, pH 8.5 (
19
). Analysis of the labelled UMP was done by thin layer cochromatography of
unlabelled 2'-UMP, 3'-UMP and 2',3'-cyclic UMP in saturated (NH
4
)
2
SO
4
/1 M NaOAc/isopropanol, 80:18:2 (v/v) (solvent A).
RESULTS
Pre-tRNA
Tyr
from
A.thaliana
shows self-cleavage activity
We have previously shown that a human intron-containing tRNA
Tyr
precursor which was produced by
in vitro
transcription in HeLa cell nuclear extract has a self-cleavage activity (
11
). Since this particular pre-tRNA is a poor substrate for HeLa 5'- and 3' processing enzymes it contained, in addition to the
intervening sequence, a 5' leader and 3' trailer, which impeded interpretation and analysis of the
cleavage products. Recently we have isolated a number of intron-containing tRNA
Tyr
genes from plants which are all efficiently transcribed in HeLa cell nuclear
extract. The processing of the flanking sequences of these pre-tRNAs
Tyr
is very efficient and precedes intron excision, thus generating intron-containing precursors without flanking sequences as major intermediates (
13
). Preliminary experiments with such a pre-tRNA
Tyr
transcribed from the
A.thaliana
tRNA
Tyr
gene AtY3II in HeLa cell nuclear extract revealed a pronounced autolytic intron
excision activity under conditions similar to those established for the self-cleavage of the human tRNA
Tyr
precursor.
To unambiguously rule out the involvement of any nuclease activity from the HeLa
cell nuclear extract which might co-purify with the substrate and in order to synthesize large quantities of
the desired pre-tRNA, we constructed a tRNA
Tyr
gene under the control of the bacteriophage T7 promoter. The resulting pre-tRNA
Tyr
AtY3II*-T7 with a length of 88 nt contains a 12 nt intron, no flanking sequences
and a mature 3'-CCA end (Fig.
1
a). A minor change in sequence was introduced into the aminoacyl stem of AtY3II*-T7 to facilitate transcription by T7 RNA polymerase: the first base pair,
C1:G72, was exchanged for G:C. The corresponding pre-tRNA
Tyr
had the same self-cleavage activity as the transcript produced from the original AtY3II gene
in HeLa cell nuclear extract (not shown).
Autolytic cleavage at or next to authentic splice sites
The nature of the fragments produced in the self-cleavage reaction of pre-tRNA AtY3II*-T7 (Fig.
1
b) was established by RNA fingerprint analyses. The [[alpha]-
32
P]ATP-labelled fragments were recovered from preparative gels and digested with
RNase T1. Figure
2
shows the fingerprints corresponding to the fragments shown in Figure
1
b. Self-cleavage at the 5' splice site takes place at the U-A phosphodiester bond 1 nt downstream of the G-U splice site. This is documented by the fingerprint
pattern B, which comprises all oligonucleotides specific for the 3'-half and for the intron with a reduced molar yield of the UAGp
oligonucleotide and consequently an additional AGp, as well as by fingerprint
pattern E, showing the corresponding 5'-half without the intron and an additional Up spot. This mononucleotide furthermore showed that self-cleavage leaves the phosphate at the 3'-end and produces a 5'-hydroxyl end, since the labelled
phosphate was transferred from A to U. The second cleavage necessary for intron
self-excision corresponds exactly to the iU12-A38 phosphodiester bond at the authentic 3' splice site (Fig.
1
). The AUUp oligonucleotide in fingerprint C, deriving from the 5'-half of the tRNA and from the intron, can only be explained by self-cleavage at this phosphodiester bond, with the labelled
phosphate being transferred to the 3'-end as shown for self-cleavage at the 5' splice site. The corresponding pattern of the 3'-half is shown in fingerprint D, with the
oligonucleotide AUCCUUAGp directly resulting from autolytic cleavage at the
authentic 3'-splice site.
We furthermore addressed the question whether the phosphate ends produced in
intron self-excision are authentic 2',3'-cyclic phosphates as described for enzymatic intron
excision and for the self-cleavage of human pre-tRNA
Tyr
(
11
). The fragments produced in the self-cleavage reaction of pre-tRNA AtY3II*-T7 are substrates for wheat germ RNA ligase, which accepts
only 2',3'-cyclic phosphate ends and is not specific for tRNA halves (
6
). The reaction leads to a tRNA with an 8 base anticodon loop. Apart from the
successful ligation of self-cleaved fragments, the excised U>p spot from fingerprint E co-migrates with authentic 2',3'-cyclic UMP upon thin layer chromatography (Fig.
2
F).
Optimized conditions for intron self-excision
The autolytic intron excision of plant pre-tRNA
Tyr
AtY3II*-T7 requires the same optimal reaction conditions as human pre-tRNA
Tyr
(
11
) and the initial substrate of this study, pre-tRNA
Tyr
AtY3II: 100 mM NH
4
OAc, 0.5 mM spermine, 10 mM MgCl
2
and 0.4% Triton X-100. In Figure
3
A (preliminary experiments with pre-tRNA
Tyr
AtY3II) it is shown that Triton X-100 has a pronounced stimulating effect on the cleavage rate while
reducing non-specific cleavage. The presence of spermine in the reaction mixture
contributes to both effects, albeit less efficiently. An increase in the Mg
2+
concentration also leads to the reduction of non-specific cleavage (Fig.
3
A, lane d: 1 mM MgCl
2
, lane c: 10 mM MgCl
2
). There is a broad pH optimum (not shown), ranging from slightly acidic (pH 6)
to alkaline (pH 8.5), as well as a broad temperature range (15-45oC), with optimal intron self-excision activity at 37oC (Fig.
3
B) with minimal non-specific and minor cleavage. Traces of fragments between the pre-tRNA and the tRNA halves with the intron are due to cleavage at
labile bonds in the D loop of the pre-tRNA.
Sequence requirements for autolytic intron excision of pre-tRNA
Tyr
AtY3II*-T7
A number of mutants have been produced to determine the sequence requirements of this self-cleavage reaction. The pre-tRNAs have been labelled during T7 transcription with the
appropriate [[alpha]-
32
P]NTP according to the nucleotide following the putative self-cleavage phosphodiester bond. In each case the pre-tRNA fragments resulting from intron self-excision have been analysed by RNA fingerprinting.
First of all we addressed the question whether self-cleavage sites for autolytic intron excision have to be located in single-stranded regions of the extended anticodon stem. We constructed two
mutants which separately placed the 5' and 3' splice sites in a double-stranded region. The AtY3II*-T7-derivatives DS5 and DS3 were compared to the
wild-type pre-tRNA (WT) with respect to the structure of the extended anticodon
stems, autolytic intron excision and splicing in a wheat germ S23 extract (Fig.
5
). The self-cleavage activity at the double-stranded sites of pre-tRNAs DS5 and DS3 completely vanished and only products
derived from autolytic cleavage at the single-stranded positions appeared (Fig.
5
B). Enzymatic cleavage is also abolished at the double-stranded 5' splice site and not affected at the 3' splice site in DS5, but is impaired at the 3' site and imprecise at the 5' splice site in DS3 (Fig.
5
C).
Furthermore, we have exchanged the nucleotides at the cleavage sites. The
sequence variants and the cleavage patterns of selected mutants are shown in
Figure
6
. The results of autolytic and enzymatic intron excision for all sequence
variants derived from pre-tRNA
Tyr
AtY3II*-T7 are summarized in Table
2
. At the 5' site of intron self-excision the first nucleotide of the intron, iU1 was exchanged for
C (M1) or G (M2). While the pyrimidine C is able to support the self-cleavage reaction, the purine G completely blocks any cleavage at this position (Fig.
6
B). The role of the nucleotide preceding the autolytically cleaved phosphodiester
bond was also analysed at the 3' cleavage site. In this case the sequence iU12-A38 was changed to iA12-A38 (M6), which extinguished self-cleavage activity at this site. However, since there
is another U at position i11, the newly created iU11-iA12 site was cleaved. To eliminate this shift of the cleavage site, iU11
was exchanged for another A (M7), thus creating an all-adenosine bulge loop around the 3' splice site. In this case no self-cleavage activity was detected. To analyse the role of the
nucleoside following the site of self-cleavage, A38 was exchanged for G, U or C (M3-M5). The phosphodiester bonds at iU12-G38 (M3) and iU12-C38 (M5) showed no self-cleavage activity. However, if A38 was
substituted by the pyrimidine U (M4), pre-tRNA fragments corresponding to products of intron self-excision were detected, which was verified by the presence of the
oligonucleotide AUU>p in fingerprints of the resulting 5'-half with intron (not shown). This demonstrates that the self-cleavage reaction is not strictly dependent on the sequence
pyrimidine-A.
.
Autolytic and enzymatic (wheat germ S23 extract) intron excision of sequence
variants of pre-tRNA
Tyr
AtY3II*-T7
Clone
Pre-tRNA
Cleavage at 5' site
Cleavage at 3' site
Autolytic
a
Enzymatic
Autolytic
Enzymatic
AtY3II*-DS5-T7
DS5
-
b
-
++
c
++
AtY3II*-DS3-T7
DS3
++
+/-
d
-
-
AtY3II*-iC1-T7
M1
++
+/-
++
++
AtY3II*-iG1-T7
M2
-
++
++
++
AtY3II*-G38-T7
M3
++
+/-
-
+/-
AtY3II*-U38-T7
M4
++
++
+/-
++
AtY3II*-C38-T7
M5
++
+/-
-
+/-
AtY3II*-iA12-T7
M6
++
++
-
++
AtY3II*-iA11/iA12-T7
M7
++
++
-
++
a
Cleavage between intron positions i1 and i2.
b
No cleavage.
c
Cleavage pattern and efficiency of cleavage as wild-type.
d
Impaired cleavage.
The sequence variants derived from pre-tRNA
Tyr
AtY3II*-T7 have also been assayed for their ability to be cleaved in wheat germ
S23 extract. The pre-tRNAs M2, M4, M6 and M7 showed no functional difference from the wild-type pre-tRNA. In contrast, enzymatic cleavage at the 3' splice site was impaired in pre-tRNA M1, whereas cleavage at both sites was less
efficient in pre-tRNA M3 and was severely reduced in pre-tRNA M5 (Fig.
6
C).
DISCUSSION
We have shown that pre-tRNAs from sources as diverse as human and plant and with completely
different intron sequences are able to undergo intron self-excision under conditions which are comparatively physiological as
compared with other metal ion induced tRNA-cleavages (
22
). The
A.thaliana
pre-tRNA
Tyr
transcript AtY3II*-T7 has three important advantages for studying self-cleavage as compared to the previously examined human pre-tRNA
Tyr
substrate (
11
): (i) the RNA is synthesized in a well-defined
in vitro
system with reliable exclusion of contamination; (ii) the sites of self-cleavage can be interpreted more easily due to the lack of additional
minor cleavage sites in the flanking sequences; (iii) The extended anticodon
stem formed by base pairing between nucleotides in the intron and in the
anticodon loop adopts a defined secondary structure in which both splice sites
are located in single-stranded regions (Fig.
1
a). Chemical structure probing with Pb(OAc)
2
has proven this structure (
23
).
Beyond doubt it is a point of major importance to exclude the possibility of any
contamination with traces of ribonucleases, which was accomplished by the use
of ultrapure, autoclaved and DEPC-treated reagents. In addition to the fact that intron self-excision is not influenced by proteinase K and/or SDS pre-treatment or even by addition of these reagents to the
reaction assay, supplementation with calf liver tRNA or with homologous pre-tRNA does not show any effect either. A non-specific or even specific ribonuclease would be saturated by tRNA
added in 1000-fold excess and reaction rates should decrease if RNase contamination were
present in the assay.
