After run-off transcription by T7 RNA polymerase of mutant U6 genes, in vitro synthesized U6 snRNA molecules were obtained that contained either no U6-0 or up to five U6-5 3¢-terminal UMP
Trang 1Biochemical characterization of a U6 small nuclear RNA-specific
terminal uridylyltransferase
Ralf Trippe, Holger Richly* and Bernd-Joachim Benecke
Department of Biochemistry, Ruhr University Bochum, Germany
The HeLa cell terminal uridylyltransferase (TUTase) that
specifically modifies the 3¢-end of mammalian U6 small
nuclear RNA (snRNA) was characterized with respect to
ionic dependence and substrate requirements Optimal
enzyme activity was obtained at moderate ionic strength
(60 mMKCl) and depended on the presence of 5 mMMgCl2
In vitrosynthesized U6 snRNA without a 3¢-terminal UMP
residue was not accepted as substrate In contrast, U6
snRNA molecules containing one, two or three 3¢-terminal
UMP residues were filled up efficiently, generating the
3¢-terminal structure with four UMP residues observed in
newly transcribed cellular U6 snRNA In this reaction, the
addition of more than one UMP nucleotide depended on
higher UTP concentrations The analysis of internally
mutated U6 snRNA revealed that the fill-in reaction by the U6-TUTase was not controlled by opposite-strand nucleo-tides, excluding an RNA-dependent RNA polymerase mechanism Furthermore, electrophoretic mobility-shift analyses showed that the U6-TUTase was able to form stable complexes with the U6 snRNA in vitro On the basis of these findings, a protocol was developed for affinity purifi-cation of the enzyme In agreement with indirect labeling results, PAGE of a largely purified enzyme revealed an apparent molecular mass of 115 kDa for the U6-TUTase Keywords: 3¢ uridylation; affinity chromatography; terminal uridylyltransferase; U6 snRNA
Nuclear pre-mRNA splicing is a process by which introns
are removed from primary transcripts This two-step
trans-esterification mechanism is performed by the spliceosome,
an RNP complex of five small nuclear RNA molecules
(snRNA U1, U2, U4, U5 and U6), and more than 60
proteins (reviewed in refs [1,2]) Spliceosomes are newly
formed on each intron in a well-defined manner Initially,
the 5¢ splice site of the pre-mRNA is recognized by the U1
snRNP Then, U2 snRNP binds to the branchpoint
sequence located near the 3¢-end of the intron Finally, the
spliceosome is completed by incorporation of the U4/U6/
U5 tri-snRNP The splicing reaction requires extensive
rearrangements of RNA–RNA interactions, including the
unwinding of the U4/U6 snRNA duplex and the formation
of a U2/U6/pre-mRNA structure [3–5] The precise
mech-anism by which proteins control these RNA–RNA
inter-actions within the catalytic spliceosome remains to be
elucidated However, evidence exists that intermediate
structural variants of U6 snRNA are involved in RNA–
RNA rearrangements that take place in the center of the
spliceosome [6]
U6 snRNA differs from the other spliceosomal snRNAs
in several ways Unlike other snRNAs, it is transcribed by RNA polymerase III [7,8] and also has a different cap structure [9] Furthermore, U6 snRNA was unusually well conserved during the evolution of eukaryotes [10] and has
no binding site for Sm proteins [11,12] Instead, U6 snRNPs contain LSm-proteins (like Sm) which seem to recognize the 3¢-end oligouridylic structure of the U6 snRNA and which are thought to be involved in U4/U6 snRNP formation [13–16] Finally, U6 snRNA molecules have a remarkable heterogeneity, resulting from extensive post-transcriptional modification of their respective 3¢-termini Most ( 90%) cellular U6 snRNA molecules are blocked
by a cyclic 2¢,3¢-phosphate (> p) 3¢-end group, and 10%
of the U6 snRNA has been found to contain 3¢-oligo(U) stretches up to 20 nucleotides long [17–19] Recently, two highly specific U6 snRNA-modifying enzyme activities have been identified: a 3¢-exonuclease [20] and a terminal uridylyltransferase (TUTase) [21] Both enzymes exclusively accept the 3¢-terminus of U6 snRNA as substrate for the addition or deletion of UMP residues The functional significance of these structural variants of U6 snRNA molecules remains to be elucidated It is conceivable, however, that all of these modifications together form a cyclic process of regeneration of U6 snRNA, which in turn may be essential for the assembly and catalytic function of spliceosomes
In this report, we present a detailed characterization of the reaction catalyzed by U6-TUTase in vitro, with its high selectivity for distinct structural requirements of U6 sub-strate RNA This study also includes an analysis of the U6 snRNA–TUTase interaction in vitro, which provided the basis for further purification to near-homogeneity of this low-abundancy protein
Correspondence to B.-J Benecke, Department of Biochemistry NC6,
Ruhr-University, D-44780 Bochum, Germany.
Fax: + 49 234321 4034, Tel.: + 49 234322 4233,
E-mail: bernd.benecke@ruhr-uni-bochum.de
Abbreviations: TUTase, terminal uridylyltransferase; snRNA, small
nuclear RNA; EMSA, electrophoretic mobility-shift analysis.
*Present address: Department of Molecular Cell Biology, Max Planck
Institute of Biochemistry, D-82152 Martinsried, Germany.
(Received 12 November 2002, revised 13 January 2003,
accepted 17 January 2003)
Trang 2Materials and methods
Protein fractionation
Cytoplasmic S100 extracts (15 mgÆmL)1 protein) were
isolated from HeLa cells as described [22] For purification
of the U6-TUTase, 200 mg extract proteins were separated
in a 40-mL Q-Sepharose FF (Pharmacia) column and
fractions obtained by step elution The QS3 fraction (200–
400 mM KCl) was dialyzed against phosphate buffer
[25 mMK2HPO4/KH2PO4(pH 7.9); 0.2 mMEDTA; 20%
glycerol (v/v)] and applied to a 10-mL hydroxyapatite
(Bio-Rad) column The HA2 fraction was obtained by step
elution with 150 mM potassium phosphate in the buffer
system described above and was further fractionated in a
26/60 Superdex G200 column (Pharmacia) For affinity
chromatography, 3 lg oligoA/U6-3 RNA were heated to
70C, cooled down slowly, and incubated for 20 min at
4C with 100 mg washed oligo(dT)–cellulose (Roche
Molecular Biochemicals) in buffer D [20 mMHepes/KOH
(pH 7.9); 100 mM KCl; 5 mM MgCl2; 0.2 mM EDTA;
2 mM dithiothreitol, 20% glycerol (v/v)] This suspension
was packed into a column, loaded with 10 mL fraction
HA2, washed with buffer D, and eluted with the same
buffer containing 600 mMKCl Electrophoresis of proteins
in denaturing 7.5% polyacrylamide gels in the presence of
SDS was essentially as described by Laemmli [23]
Templates
Construction of the U6-3, U6-4 and U6-5 cDNA templates
under control of the bacteriophage T7 promoter has been
described in detail previously [21] Based on the U6-5
construct, U6-0, U6-1 and U6-2 cDNA templates were
generated by PCR with 5¢-TTTAATACGACTCACTAT
AGGGTGCTCGCTTCGGCA-3¢ as upstream primer and
5¢-TATGGAACGCTTCACGAATT-3¢ (U6-0), 5¢-ATAT
GGAACGCTTCACGAATT-3¢ (U6-1) or 5¢-AATATGG
AACGCTTCACGAATT-3¢ (U6-2) as downstream primer,
respectively PCR fragments were purified by agarose gel
electrophoresis and used for in vitro transcription by T7
RNA polymerase as described previously [24] DNA
templates with internal mutations (U6-2/C and U6-1/A)
were obtained as follows: in a first PCR, U6 snRNA-coding
sequences were amplified with a 5¢-primer carrying the
desired mutation and a wild-type 3¢-primer These
frag-ments were used in a second PCR with the same 3¢-primer
but a longer 5¢-primer carrying the T7 promoter as
upstream flanking sequence element The amplified PCR
fragments were cloned blunt-end into the EcoRV site of the
Bluescript KS+vector (Stratagene) PCR fragments
suit-able for in vitro transcription and carrying different numbers
of 3¢-end UMP residues (i.e U6-1 and U6-2) were then
obtained with the T7 promoter-specific upstream primer
and one of the downstream primers described above The
oligo(A)/U6-3 RNA construct was cloned from the
previ-ously described U6-3 RNA gene [21] From this gene, an
AspHI (+5 of U6-DNA)–HindIII (downstream of coding
sequence) fragment was recovered Two synthetic
oligonu-cleotides were hybridized and gave rise to a double-stranded
element containing the oligoA(20)flanked by an upstream
SacII overhang and the first 5 bp of the U6-coding
sequence, in the form of a downstream AspHI overhang These two DNA fragments were inserted simultaneously into the KS+vector, restricted with SacII and HindIII and purified by agarose gel electrophoresis before use After linearization of this plasmid with the DraI enz yme, T7 RNA polymerase transcription from the neighboring KS+ pro-moter yielded suffciently large quantities of oligo(A)/U6-3 RNA for coupling to oligo(dT)–cellulose Primary struc-tures of all constructs were confirmed by sequencing Assay of TUTase
Standard TUTase reaction assays were performed in buffer containing 60 mM KCl, 12 mM Hepes/KOH (pH 7.9),
5 mM MgCl2, 2 mM dithiothreitol, 0.1 mM EDTA, 12% (v/v) glycerol and 5 lCi [a-32P]UTP, in a total volume of
50 lL Substrate RNAs were either 1 lg total cellular RNA
or 50 ng in vitro synthesized U6 snRNA, and incubations were performed for 60 min at 30C Phenol-extracted RNAs were analysed in 6% polyacrylamide gels in TEB buffer [90 mMTris base, 90 mM boric acid, 2 mMEDTA (pH 8.3)] containing 6M urea Electrophoresis was for
50 min at constant 30 W To discriminate between RNA molecules differing in length by only 1 nucleotide, high-resolution gels were used in some experiments These gels contained 7% polyacrylamide, were twice as long (50 cm), and were run for 210 min at constant 37.5 W Autoradio-graphy of the dried gels was at)70 C for 16 h, using a Cronex intensifier screen
Electrophoretic mobility-shift assay (EMSA) For EMSAs, U6-3 RNAs were synthesiz ed in vitro with T7 RNA polymerase (Fermentas), in the presence of [a-32P]UTP (800 CiÆmmol)1; New England Nuclear) Labe-led U6-3 RNA (100 000 c.p.m.) was incubated with Super-dex G200 fractions for 10 min at 4C in 12 mM Hepes/ KOH (pH 7.9), containing 60 mM KCl, 5 mM MgCl2,
2 mM dithiothreitol, 0.1 mM EDTA, 1 lg yeast tRNA (Roche Molecular Diagnostics) and 12% (v/v) glycerol Electrophoresis in nondenaturing 6% polyacrylamide gels with 0.25· TEB buffer was at 6 VÆcm)1for 4 h at 4C
Indirect labeling analysis Affinity-purified TUTase was incubated with labeled U6-3 RNA (100 000 c.p.m.) as described for shift analyses UV cross-linking (Hoefer UVC 500) was at 0.3 JÆcm)2Æmin)1for
10 min on ice Subsequently, half of the reaction mixture was incubated with 10 lg RNase A for 10 min at room temperature After acetone precipitation, proteins from both fractions were analysed in 7.5% polyacrylamide/SDS Laemmli gels
Results Ionic requirements of HeLa cell TUTases Earlier experiments had established that, aside from an unspecific enzyme, HeLa cells contain a highly specific TUTase that exclusively modifies the 3¢-ends of U6 snRNA molecules To separate this activity from the unspecific
Trang 3enzyme and also to remove contaminating RNases, a
protocol had been developed for the preparation of partially
purified enzyme fractions [21] This purification started with
phosphocellulose P11 chromatography of HeLa cell S100
extracts Subsequently, the unspecific TUTase was
separ-ated from the U6-TUTase by gel filtration in Superdex
G200 These two crude enzyme fractions were analysed in
parallel for magnesium and salt (KCl) dependence of their
respective transferase reactions, with 1 lg total RNA from
HeLa cells added as substrate As shown in Fig 1A,B, the
two activities separated by gel filtration were clearly
distinguishable by their acceptance of substrate RNA In
agreement with our previous results [21], the unspecific
enzyme (unspec; left panels of Fig 1A,B) modified a variety
of cellular RNA molecules, whereas the U6-specific TUTase
(spec; right panels) exclusively accepted U6 snRNA as substrate However, the two enzymes revealed similar salt optima As seen in Fig 1A, both activities showed a clear optimum at 5 mMMgCl2(lanes 2 and 6), with no activity detectable in the absence of Mg2+ ions (lanes 4 and 8) Corresponding results for the two enzymes were also observed with respect to ionic strength (Fig 1B) With both enzymes, optimal conditions for the transfer of UMP residues were obtained in the presence of 60 mM KCl (Fig 1B, lanes 1 and 6) This is the amount of KCl already provided by the protein fractions, with no further salt added
to the reaction It should be noted that lower amounts of KCl (40 mM), obtained after dialysis of protein fractions, did not further increase the enzyme activity (not shown) However, both enzymes were inhibited significantly by higher salt concentrations (150 mMKCl; lanes 3 and 8) and showed no detectable activity at and above 200 mM KCl (lanes 4 and 5 and 9 and 10) Therefore, although slight differences between the two enzymes were observed in response to higher salt conditions, these two TUTase activities basically depended on similar reaction conditions, but with clearly different substrate requirements These results on Mg2+ and KCl dependence were primarily required to establish the affinity-purification protocol for the U6-TUTase, described below Therefore, we did not analyse in more detail the suitability of other bivalent cations and/or salts such as manganese or ammonium sulfate
Restoration of authentic 3¢-ends by the U6 snRNA-specific TUTase
Next, we wanted to determine RNA substrate requirements
of the U6-TUTase For this, a U6-TUTase fraction was used that was prepurified by Q-Sepharose and hydroxy-apatite chromatography, followed by gel filtration in Superdex G200 (see Materials and methods section) U6 snRNA molecules were synthesized as substrates that differed with respect to the number of UMP residues at their 3¢-ends respectively After run-off transcription by T7 RNA polymerase of mutant U6 genes, in vitro synthesized U6 snRNA molecules were obtained that contained either
no (U6-0) or up to five (U6-5) 3¢-terminal UMP residues To ensure homogeneity of the RNA molecules applied, in vitro synthesized RNA was first separated in high-resolution polyacrylamide gels, and transcripts of the correct length were recovered, before their use as substrate in a standard TUTase reaction with [a-32P]UTP Subsequently, labeled U6 snRNA molecules were analysed again in high-resolu-tion gels In this analysis, U6-3 marker RNA, labeled by T7 RNA polymerase transcription of the corresponding tem-plate, was included as a size standard (m in Fig 2A) From the results presented in Fig 2A it is evident that U6-1, U6-2 and U6-3 RNA molecules (lanes 2–4) were efficient substrates for the U6-TUTase reaction Furthermore, the high-resolution capacity of our gel system allowed the resolution of closely related molecules, differing in length by only one nucleotide A close examination of the bands in lanes 2–4 of Fig 2A revealed that concomitant with increasing length of the UMP tails of the substrate RNA,
a decreasing number of distinguishable labeled RNA products was obtained: three bands in lane 2, two in
Fig 1 Ionic requirements of HeLa cell TUTases The U6
snRNA-specific (spec.) and the unsnRNA-specific (unspec.) TUTase of HeLa cell
extracts were separated in a Superdex G200 column [21] and analysed
with 1 lg total cellular RNA as substrate (A) Peak fractions of the
two activities were tested in the presence of various concentrations of
MgCl 2 (Mg ++ ): 10 m M (lanes 1,5), 5 m M (lanes 2,6), 2.5 m M (lanes
3,7) and 0.0 m M MgCl 2 (lanes 4,8) (B) Same analysis as in (A), except
that standard TUTase reactions (5 m M MgCl 2 ) were performed in the
presence of increasing amounts of KCl: 60 m M (lanes 1,6), 110 m M
(lanes 2,7), 150 m M (lanes 3,8), 200 m M (lanes 4,9) and 250 m M KCl
(lanes 5,10) Analysis of labeled RNA products was in 6%
poly-acrylamide gels containing 6 M urea Exposure of the dried gels to
Kodak X-ray films was for 16 h using a Cronex intensifier screen.
The position of labeled U6 snRNA is indicated by an arrow and m
represents labeled marker DNA.
Trang 4lane 3, and a single band in lane 4 In comparison with the
labeled U6-3 marker RNA, these results indicate that the
U6-TUTase has a clear preference to fill up the 3¢-ends of
U6 snRNA molecules to the four UMP residues found in
newly transcribed cellular U6 snRNA The 3¢-ends were not
elongated further, as evidenced by the absence of labeled
products observed with U6-4 (lane 5) and U6-5 (lane 6) as
substrate RNA Furthermore, the U6-TUTase did not
accept U6-0 RNA (lane 1) as substrate, indicating that at
least one pre-existing UMP residue at the 3¢-end of U6
snRNA is a prerequisite for this modification reaction to
take place In addition, the finding of intermediates of the
transferase reaction (Fig 2A, lanes 2 and 3) seemed to point
to dependence on the nucleotide concentration of the chain
elongation rate Therefore, TUTase reactions were
per-formed as before with U6-1 RNA and [a-32P]UTP, but this
time in the presence of increasing amounts of unlabeled
UTP (Fig 2B) Again, U6-3 RNA labeled by T7
transcrip-tion in vitro was included as size marker (m) The
comparison in Fig 2B of the standard reaction products,
i.e in the presence of labeled UTP only (lane 1), with those obtained in the presence of increasing amounts of unlabeled UTP (lanes 2–5) shows a clear shift to the full-length modification product (U6-4 RNA, lane 5), at the expense of the intermediate bands seen in lanes 2–4 of Fig 2B It should be noted that the significant reduction in overall signal intensity (lanes 3–5) is simply due to the competition for incorporation of labeled nucleotides by the excess of unlabeled UTP Together, we conclude that the U6 snRNA-specific TUTase reaction depends on both the 3¢-end structure of the template RNA and the concentration
of substrate nucleotides present in the reaction mixture Structural requirements for the U6 substrate RNA The finding that the U6-TUTase preferentially fills in the 3¢-end of U6 snRNA only to four UMP residues raises the question of how this elongation reaction is controlled This
is even more intriguing because the proposed secondary structure of U6 snRNA (Fig 3A [25]) has an extended 3¢–stem–loop structure with exactly four internal AMP nucleotides (+27/+30) opposing the four 3¢-terminal UMP residues (+103/106) found in newly transcribed cellular U6 snRNA Therefore, it is tempting to speculate that the U6-TUTase may be guided by the sequence of the opposite strand, establishing some sort of substrate-specific RNA-dependent RNA polymerase reaction To test this hypo-thesis, we analysed two U6 mutant genes One (U6-2/C) consisted of a U6-2 clone that contained one additional CMP nucleotide, inserted between positions +27/+28 (two
of the four AMP residues mentioned above) The corres-ponding mutant U6-2/C substrate RNA was tested either in
a standard TUTase reaction (with labeled UTP) or in the presence of32P-labeled GTP, supplemented with unlabeled UTP The analysis of the modified RNA is shown in Fig 3B, with unmodified U6-2/C RNA, labeled during T7 transcription, as size marker (lanes 1 and 4) The result shown in lane 2 of Fig 3B confirmed that, in a standard TUTase reaction, U6-2/C RNA still served as an efficient substrate for the transferase reaction Surprisingly, however, the TUTase reaction stopped after the addition of one UMP residue and did not fill-up this U6 mutant RNA to the four UMP residues observed previously (compare with lane 3 of Fig 2A) This is evident from the size comparison between the modified U6-2/C RNA (Fig 3B, lane 2) and the unmodified control RNA (lanes 1 and 4) In contrast, the TUTase reaction performed with the same substrate RNA und unlabeled UTP, but in the presence of labeled GTP as tracer, did not give rise to any labeled RNA product (Fig 3B, lane 3) This indicates that the newly introduced CMP nucleotide at position +28 of the mutant gene was not functional as a template-strand nucleotide, able to direct the incorporation of GMP residues into the opposite 3¢-end of the mutant RNA
A second mutation of the U6 substrate RNA consisted of the introduction of two additional AMP nucleotides in front
of the oligo(A) stretch, beginning at +27 of the wild-type sequence This mutation was introduced into a U6-1 construct (resulting in U6-1/A) and aimed to extend the internal oligo(A) sequence from four to six AMP residues The analysis of this mutant RNA as substrate for the U6-TUTase is shown in Fig 3C We have shown above
Fig 2 RNA substrate requirements of the U6-TUTase (A) A partially
purified U6-TUTase was tested under standard reaction conditions
with 50 ng in vitro synthesized U6 snRNA substrate molecules
containing zero (lane 1), one (lane 2), two (lane 3), three (lane 4), four
(lane 5) or five (lane 6) 3¢-terminal UMP residues A size standard
consisting of unmodified U6-3 RNA, labeled by T7 RNA polymerase
transcription in vitro, is indicated (m) Labeled RNA products were
analysed in high-resolution gels as described in Materials and methods.
(B) The same enz yme as in (A) was tested with [a-32P]UTP and U6-1
RNA as substrate under standard reaction conditions In this case,
however, increasing amounts of unlabeled UTP were added to the
reaction: 0.0 l M (standard reaction; lane 1), 0.25 l M (lane 2), 0.5 l M
(lane 3) 1.0 l M (lane 4) and 2.0 l M (lane 5) As above, unmodified
labeled U6-3 RNA was included as a size standard (m).
Trang 5that, in these TUTase reactions, the nucleotide
concentra-tions may be a limiting factor for the prolongation of U6
substrate RNA Therefore, as in Fig 2B, the TUTase
reactions were performed either under standard conditions,
i.e in the absence of unlabeled UTP (lane 1), or with
increasing amounts of unlabeled UTP added to the reaction
(lanes 2–5) As before, increasing amounts of unlabeled
nucleotides led to the disappearance of modified
inter-mediates and to an overall reduction in signal intensities
However, comparison of the longest labeled products in
lanes 2–5 with the two different unmodified marker RNAs
(m1, m2) revealed that the TUTase-catalysed reaction again
stopped exactly at a position corresponding to four
3¢-terminal UMP residues In this analysis, the unmodified
marker RNAs, labeled during T7 transcription, were U6-3
RNA (m1) and U6-2/A RNA (m2) In its unmodified form,
the length of U6-3 RNA exactly matches that of the
unmodified U6-1/A sequence The U6-2/A marker RNA
corresponds to the U6-1/A mutant analysed here, but
containing two instead of one 3¢-terminal UMP residues
Consequently, migration of this unmodified marker RNA
should correspond exactly to the smallest labeled band of
the modified U6-1/A RNA This is confirmed by
compar-ison of the lower bands of lanes 1–4 with the labeled m2
band (Fig 3C) These data provide evidence that the
enzyme was not able to read as template the two additional
AMP nucleotides of the opposite RNA strand Therefore,
we conclude that the TUTase does not act as an
RNA-dependent RNA polymerase Rather the enzyme catalyses a
strictly selective modification reaction, solely to regenerate
the authentic 3¢-structure of newly transcribed cellular U6
snRNA, constituted by four UMP residues
Complex formation of the U6-TUTase
with substrate RNA
The U6 snRNA-specific TUTase described here differs from
the unspecific enzyme of HeLa cells by its high selectivity for
its substrate RNA Therefore, we wanted to know whether
this highly specific RNA–protein interaction might provide
a useful tool for affinity purification of the enzyme To test this possibility, we first studied binding of the TUTase to U6 snRNA by EMSA For this, a partially purified U6-TUTase was run in a preparative Superdex G200 gel filtration column and individual fractions tested with 1 lg total cellular RNA (Fig 4A) As indicated by the labeled U6 snRNA products, the specific enzyme was obtained as a broad peak with fractions 27 through 51, corresponding to a size range from 70 to 130 kDa for elution from this column
Fig 3 Substrate analysis of the TUTase with structural mutants of the
U6snRNA (A) Proposed secondary structure of U6 snRNA [25].
Mutant RNAs were cloned by insertion of CMP or AMP nucleotides
into the internal oligo(A) stretch (+27/+30) of the wild-type
sequence (B) U6-2/C substrate RNA represents a U6 snRNA
con-taining two 3¢-terminal UMP residues and one additional internal
CMP nucleotide, inserted at position +28 of the wild-type sequence.
50 ng of this RNA were tested with labeled UTP under standard
reaction conditions (lane 2) or with labeled [a-32P]GTP in the presence
of 2.0 l M unlabeled UTP (lane 3) The position of unmodified U6-2/C
RNA is indicated on the right This marker RNA (lanes 1,4) was
labeled during T7 transcription in vitro and served as a size standard.
(C) U6-1/A RNA contains one 3¢-terminal UMP residue and an
insertion of two additional AMP residues at position +27 of the
wild-type sequence U6-1/A RNA was analysed either under standard
conditions (lane 1) or in the presence of increasing amounts of
unlabeled UTP: 0.25 l M (lane 2), 0.5 l M (lane 3), 1.0 l M (lane 4) and
2.0 l M (lane 5) T7 RNA polymerase-labeled marker RNAs were:
U6-3 (m 1 ) and U6-2/A (m 2 ); the latter corresponds to U6-1/A, but with
two 3¢-terminal UMP residues (see text).
Trang 6A clear maximum of enzyme activity was detected in
fractions 39–45 Subsequently, aliquots of the gel filtration
fractions were incubated with U6-3 RNA, labeled by T7
RNA polymerase transcription in vitro Complexes were
separated in 6% polyacrylamide gels, as described in
Materials and methods As shown in Fig 4B, the fractions
with maximum enzyme activity, mainly fractions 39–45,
clearly shifted the free U6 snRNA (arrowhead) to a
complex of higher molecular mass (arrow) Furthermore,
the distribution between fractions of the major shifted
complex paralleled that of the enzyme activity observed in
Fig 4A Additional minor complexes did not correspond to
the pattern of enzyme activity, and may represent other
cellular proteins capable of binding U6 snRNA, either
specifically (such as LSm proteins) or in an unspecific way
These results confirm that in vitro stable complexes may
be obtained between U6 snRNA and the corresponding
U6-TUTase
In a second step, we wanted to generate a U6
snRNA-based affinity column for purification of the enzyme As
proteins have a significant tendency to bind nonspecifically
to a variety of matrices, the choice of carrier is important in
affinity purification Previous experiments had shown that cellulose may be superior to other materials (unpublished observation) Another important point is the accessibility of the immobilized RNA TUTase is expected to primarily recognize the 3¢-terminal structure of U6 snRNA However, bulky compounds such as biotinylated nucleotides may interfere with the correct folding of the target RNA, thereby changing the structural motif recognized by the TUTase Therefore, we decided to couple U6 snRNA to oligo(dT)– cellulose via an oligo(rA) linker, fused to the 5¢-end of the wild-type sequence For this, a mutant gene was cloned with
an oligo(A)(20)linker preceding the U6-3 RNA sequence
In vitrotranscription of this template by T7 RNA poly-merase gave rise to U6-3 transcripts 149 nucleotides in length A control experiment confirmed that the presence of the oligo(rA) linker and the joining element did not interfere with the TUTase reaction This is shown in Fig 5A Comparison of lanes 2 and 3 shows that the amount of modified U6 RNA labeled in a standard TUTase reaction stayed the same, irrespective of whether U6-3 RNA (lane 2)
or oligo(A)/U6-3 RNA (lane 3) was applied In this standard U6-TUTase reaction, a control was included with
1 lg total cellular RNA (Fig 5A, lane 1) It should be noted that the slightly slower migration observed with the modified U6-3 RNA (lane 2), compared with cellular U6 snRNA (lane 1), is due to two additional 5¢-terminal GMP residues in the U6-3 RNA The resulting three 5¢-terminal GMP nucleotides of such U6 constructs are required for efficient initiation of transcription by T7 RNA polymerase
Fig 5 Affinity chromatography of U6-TUTase with immobilized 5¢-adenylated U6snRNA (A) In vitro transcribed oligo(A)/U6-3 RNA (149 nucleotides in length; 50 ng) was tested in a standard TUTase reaction (lane 3) in comparison with the authentic U6-3 sequence (lane 2) Lane 1 shows a control reaction with 1 lg total cellular RNA Labeled DNA fragments are included as marker (m) Products were analysed in 6% polyacrylamide gels containing 6 M urea (B) After dialysis against 100 m M KCl, a partially purified U6-TUTase fraction (hydroxyapatite step, see Materials and methods) was applied to an oligo(dT)–cellulose column loaded with oligoA/U6-3 RNA The load fraction (lane 1), the flow-through material (lane 2), and the fractions eluted with 600 m M KCl (lane 3) or 2000 m M KCl (lane 4) were analysed in a standard TUTase reaction with 1 lg total cellular RNA Electrophoretic analysis of labeled RNA products in 6% polyacryl-amide gels and autoradiography were as before.
Fig 4 Complex formation of U6-TUTase with substrate RNA (A)
Prepurified U6-TUTase was run in a Superdex G200 column and the
fractions indicated above each lane tested under standard conditions
with U6-3 RNA as substrate The loaded material is indicated by l and
labeled DNA used as a marker indicated (m) (B) EMSA with the same
TUTase fractions as in (A) and T7-transcribed labeled U6-3 RNA as
substrate Electrophoresis was in nondenaturing 6% polyacrylamide
gels (see Materials and methods) Lane 1 shows a minus-protein
control, with the free U6-3 RNA marked by an arrowhead The shifted
complex is indicated by an arrow (left side).
Trang 7This system was used for affinity chromatography of the
U6-TUTase For this, in vitro synthesized oligoA/U6-3
RNA was coupled to an oligo(dT)–cellulose matrix via A/T
base-pairing In an initial experiment, a fairly crude protein
fraction (hydroxyapatite step) was applied to the column
At this level of purification, residual amounts of the
nonspecific cellular TUTase are still present This is evident
from the standard TUTase reaction with total cellular RNA
as substrate (Fig 5B) As seen in lane 1 of Fig 5B, the load
fraction of the affinity column was able to transfer UMP
residues to more than just U6 snRNA, although the latter
was by far the most abundant labeled reaction product In
contrast, very little (if any) TUTase activity was associated
with the flow-through fraction (lane 2) It should be noted
that most of the minor bands seen with the load material
(lane 1) were obtained in this flow-through fraction As
these labeled bands probably reflect residual activity of the
nonspecific TUTase, it appears that the nonspecific enzyme
did not bind to the affinity matrix Subsequently, two more
fractions were step-eluted with 600 mM KCl (lane 3) and
2M KCl (lane 4) Before being analysed for TUTase
activity, these fractions were dialysed against 100 mMKCl
This confirmed that most TUTase molecules were eluted
from the affinity column at 600 mMKCl (lane 3)
Further-more, the upper section of lane 3 in Fig 5B indicates that a
small amount of the oligo(A)/U6-3 target RNA was also
eluted from the affinity column One has to keep in mind,
however, that in this analysis the TUTase reaction was
performed in the presence of 1 lg total cellular RNA as
substrate Taking into account the low concentration of U6
snRNA in total cellular RNA, even the smallest amounts of
oligo(A)/U6-3 RNA coeluted from the column would
attract considerable labeling by the U6-TUTase Such
artificial leakage of a relatively small proportion of the
immobilized target RNA, however, would not affect the
general suitability of this purification step
With this information to hand, a purification scheme was
developed for the U6 snRNA-specific TUTase Starting
with HeLa cell S100 extracts, the enzyme was prepurified by
Q-Sepharose, hydroxyapatite chromatography, and gel
filtration in Superdex G200 This partially purified
U6-TUTase fraction was subjected to affinity
chromato-graphy on the oligo(A)/U6-3 RNA column described above
As proteins tend to bind to any matrix in an unspecific way,
the combined peak fractions of the G200 column were split
and run in parallel in two affinity columns: one consisting of
oligo(dT)–cellulose only (– RNA column) and a second
one loaded with poly(A)/U6-3 RNA (+ RNA column)
The TUTase assay performed with the material eluted from
both columns at 600 mMKCl confirmed that binding of the
enzyme strictly depended on the presence of the substrate
RNA (Fig 6A) As seen in lane 2 of Fig 6A, the TUTase
activity was exclusively eluted from the + column In
contrast, virtually no enzyme activity was detectable in lane
1, representing the elution fraction of the mock column (–)
In agreement with these findings, the distribution of enzyme
activities associated with the two flow-through fractions was
exactly the other way around, i.e full activity in the case of
the mock column and less than 5% of TUTase passing
through the + column (data not shown) To determine
more precisely the molecular mass of the U6-TUTase,
indirect labeling experiments were performed with the
affinity-purified enzyme Labeled U6-3 RNA was incubated with the protein under shift conditions, followed by UV cross-linking Analysis of labeled proteins in SDS/poly-acrylamide gels was either directly (–) or after RNase A digestion (+) Figure 6B shows that a distinctly labeled RNP complex 145 kDa in size was obtained without RNase A treatment (lane 1) As expected, RNase treatment
of the cross-linked material resulted in an overall loss of radioactivity associated with proteins (lane 2) Furthermore, albeit low in intensity, now one additional new band appeared that was not observed in lane 1 This band (arrow) had an apparent molecular mass of 115 kDa All other labeled bands visible in lane 2 of Fig 6B (mainly corres-ponding to a size range of 45–60 kDa) were already detectable in the absence of RNase treatment These bands probably represent cross-linking products labeled by resid-ual amounts of free UTP
Finally, the protein composition of the affinity-purified material was analysed in silver-stained SDS/polyacrylamide gels Initial results showed that high-salt elution from the affinity matrix still resulted in a very complex spectrum of polypeptides, not allowing unambiguous identification of the TUTase (data not shown) The high-salt conditions obviously mobilized a large number of proteins unspecifi-cally bound to the matrix Therefore, a more gentle mode of elution was applied, avoiding changes in ionic strength This approach consisted of RNase A treatment of the affinity columns, and indeed resulted in recovery of vastly reduced
Fig 6 Affinity purification and indirect labeling of U6-TUTase (A) U6-TUTase prepurified by Q-Sepharose, hydroxyapatite and Super-dex G200 was loaded in parallel to an oligo(dT)–cellulose column (–)
or an oligo(dT) column coupled with oligoA/U6-3 RNA (+) After being washed with 100 m M KCl, material eluted at 600 m M KCl was tested for TUTase activity with U6-3 substrate RNA in a standard reaction Labeled products obtained with the elution fraction of the – column (lane 1) and of the + RNA column (lane 2) were analysed as before DNA marker fragments are shown on the left (m) (B) Affinity-purified TUTase was incubated with labeled U6-3 RNA under shift conditions, followed by UV cross-linking Half of the material was analysed directly (without nuclease digestion, –, lane 1) and half after RNase A treatment (+, lane 2) in SDS/polyacrylamide gels (see Materials and methods) Numbers on the right indicate the positions
of unlabeled marker proteins (kDa).
Trang 8numbers of proteins Electrophoretic analysis of proteins
obtained from the + and – RNA affinity columns is
presented in Fig 7 As is evident from lanes 1 and 2, a
simple protein spectrum was obtained after RNase A
elution (lane 3 shows a control with the RNase A alone)
Most importantly, the fractions obtained from the + (lane
1) and the – (lane 2) columns differed by one prominent
polypeptide only (arrow) Apart from a few minor
quan-titative differences, all other bands were identical between
the two fractions The prominent band selectively eluted
from the + column showed an apparent molecular mass of
115 kDa, which is in full agreement with the size obtained
previously for the U6-TUTase by indirect labeling
(Fig 6B) Together, these lines of evidence suggest that
the 115-kDa protein is probably the human U6
snRNA-specific TUTase
Discussion
By several criteria, U6 snRNA is remarkable among the
small stable RNA molecules of eukaryotic cells (see the
Introduction) Furthermore, it seems to play a major role in
the center of the active spliceosome The finding of
accompanying enzymes responsible for the highly specific
modification of this particular RNA, such as a 3¢-nuclease
and a 3¢-terminal uridylyltransferase [20,21], supports the
structural significance of U6 snRNA Consequently, one
would expect that the combined action of these two proteins
(and presumably others) is closely associated with the biological function of U6 snRNA in pre-mRNA splicing Therefore, detailed information on the reactions catalysed
by these two U6-specific enzymes may provide a valuable tool for studying internal events within the spliceosome Here, we have reported on the catalytic properties and substrate requirements of the U6 snRNA-specific TUTase With respect to its general properties, it seems to correspond closely to the nonspecific TUTase (Fig 1) [26] Both enzymes were inhibited by high salt concentrations and showed maximal stimulation by 5 mM Mg2+, but with clearly different RNA substrate specificities In contrast with the U6-TUTase, however, the reaction catalysed by the nonspecific enzyme was found to be strictly limited to the transfer of only one UMP residue [26], irrespective of whether or not higher concentrations of UTP were applied The transfer of more than one UMP residue by the U6-TUTase (Fig 2B) was not observed in our initial study [21], because it depends on the presence of higher UTP concentrations In addition, these experiments were per-formed with substrate RNAs that contained three (or more) 3¢-terminal UMP residues At first glance, the finding that the U6-TUTase is able to transfer more than one UMP may classify it as a common poly(U) polymerase However, for this class of enzyme, the length of the poly(U) product is a function of the incubation time Certainly, this was not the case here The addition of UMP residues was strictly limited
to four, thereby restoring the 3¢-terminal structure of newly transcribed cellular U6 snRNA Therefore, the mode of action of the U6-TUTase appears to be tightly controlled by the structure of the RNA substrate This notion is supported
by the observation that a synthetic U6-0 RNA, containing
no 3¢-UMP at all, was not accepted as substrate As U6-0 RNA carries an AMP nucleotide at its 3¢-end, this finding was reminiscent of results obtained with various substrate RNAs in unfractionated HeLa cell extracts and frog oocytes [27] One has to bear in mind, however, that the uridylating activity analysed in those experiments did not show any substrate RNA specificity
The four 3¢-terminal UMP residues of newly tran-scribed U6 snRNA exactly match four internal AMP residues Consequently, it is tempting to speculate that the U6-TUTase functions as an RNA replicase How-ever, analysis of constructs with internal opposite strand mutations definitely excluded such a mode of action Surprisingly, even the introduction of two additional AMP residues into the internal oligo(A) stretch did not give rise to an extended TUTase reaction product, now containing six instead of four complementary 3¢-terminal UMP residues Therefore, it appears that the underlying principle is not simply to ensure a double strand at the basis of the 3¢-terminal stem-loop structure of U6 snRNA Rather, these results suggest that a sophisticated mechanism controls a highly restrictive elongation pro-cess, only allowing restoration of the authentic 3¢-end of U6 snRNA Such a scenario attributes a special import-ance to the 3¢-terminal structure of this RNA, with four UMP residues being involved in base-pairing Apart from a more general contribution to the overall folding
of U6 snRNA, this A/U RNA duplex element may ensure the appropriate stability for melting and reasso-ciation of this spliceosomal RNA, a prerequisite to the
Fig 7 SDS/PAGE of proteins recovered from affinity
chromato-graphy U6-TUTase was affinity-purified as described in Fig 6 In this
case, however, elution of the bound material from the plus (+, lane 1)
and the minus (–, lane 2) column was by treatment with 100 lg
RNase A Lane 3 shows the control analysis of the RNase A used for
elution Protein bands were visualized by silver staining of the gel The
molecular mass (kDa) of marker proteins (m) is indicated on the left.
Trang 9extensive RNA rearrangements that occur during the
splicing procedure
Several uridylating enzyme activities have been
charac-terized [26,28–30] However, the TUTase analysed here
differs from those by its pronounced RNA substrate
specificity [21] This RNA selectivity is superimposed by
the highly specific control of the elongation reaction
described above In this context, it is interesting to note
that the molecular mass of the U6-TUTase activity obtained
under native conditions in the gel-filtration column
(Fig 4A) exactly corresponded to that of the specific
polypeptide observed in denaturing gels, after affinity
chromatography (Fig 7) This supports the notion that
the catalytic activity of the U6-TUTase, together with its
two specificities outlined above, is associated with a single
polypeptide chain Therefore, binding of the U6-TUTase to
its target RNA will certainly establish an interesting model
system for studying a very specific but transient RNA–
protein interaction For such a detailed analysis, however, a
recombinant TUTase will be required Such a recombinant
protein would also allow a structural comparison of the
enzyme with other previously cloned TUTases [30,31] In
addition, the availability of a recombinant U6-TUTase
would give access to monoclonal antibodies, which in turn
should provide a valuable tool to study the functional
significance of this U6 snRNA modification Such
func-tional studies would provide clues to why evolution allowed
this small stable U6 snRNA the unique luxury of acquiring
its own modifying enzyme
Acknowledgements
We thank Dr Andre´ Frontzek for skilful introduction into the RNA
electrophoretic mobility-shift analysis technique, and Nadine Pieda for
expert technical assistance Thanks are also due to Klaus Grabert for
the photographs This work was supported by a grant from the
Deutsche Forschungsgemeinschaft (Be 531/19-1).
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