Báo cáo khoa học: Biochemical characterization of a U6 small nuclear RNA-specific terminal uridylyltransferase potx

Biochemical 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 m
M
KCl) and depended on the presence of 5 m
M
MgCl
2
.
In vitro synthesized 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 (ÔlikeÕ 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,withitshigh
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:
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)
Eur. J. Biochem. 270, 971–980 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03466.x
Materials 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 m
M
KCl) was dialyzed against phosphate buffer
[25 m
M
K
2
HPO
4
/KH

2
PO
4
(pH 7.9); 0.2 m
M
EDTA; 20%
glycerol (v/v)] and applied to a 10-mL hydroxyapatite (Bio-
Rad) column. The HA2 fraction was obtained by step
elution with 150 m
M
potassium phosphate in the buffer
system described above and was further fractionated in a
26/60 Superdex G200 column (Pharmacia). For affinity
chromatography, 3 lgoligoA/U6-3RNAwereheatedto
70 °C, cooled down slowly, and incubated for 20 min at
4 °C with 100 mg washed oligo(dT)–cellulose (Roche
Molecular Biochemicals) in buffer D [20 m
M
Hepes/KOH
(pH 7.9); 100 m
M
KCl; 5 m
M
MgCl
2
;0.2m
M
EDTA;
2m
M

dithiothreitol, 20% glycerol (v/v)]. This suspension
waspackedintoacolumn,loadedwith10mLfraction
HA2, washed with buffer D, and eluted with the same
buffer containing 600 m
M
KCl. 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 DraIenzyme,T7RNA
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 m
M
KCl, 12 m
M
Hepes/KOH (pH 7.9),
5m
M
MgCl
2
,2m
M
dithiothreitol, 0.1 m
M
EDTA, 12%
(v/v) glycerol and 5 lCi [a-
32
P]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 30 °C. Phenol-extracted
RNAs were analysed in 6% polyacrylamide gels in TEB
buffer [90 m
M
Tris base, 90 m
M
boric acid, 2 m
M
EDTA
(pH 8.3)] containing 6
M

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)
ForEMSAs,U6-3RNAsweresynthesizedin vitro with
T7 RNA polymerase (Fermentas), in the presence of
[a-
32
P]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 4 °Cin12m
M
Hepes/
KOH (pH 7.9), containing 60 m
M
KCl, 5 m
M
MgCl
2
,
2m
M
dithiothreitol, 0.1 m

M
EDTA, 1 lgyeasttRNA
(Roche Molecular Diagnostics) and 12% (v/v) glycerol.
Electrophoresis in nondenaturing 6% polyacrylamide gels
with 0.25 · TEB buffer was at 6 VÆcm
)1
for 4 h at 4 °C.
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
)2
Æmin
)1
for
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
972 R. Trippe et al.(Eur. J. Biochem. 270) Ó FEBS 2003
enzyme 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 lgtotalRNAfrom
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 m
M
MgCl
2
(lanes 2 and 6), with no activity
detectable in the absence of Mg
2+
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 m
M
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 m
M
), 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 m
M
KCl; lanes 3 and 8) and
showed no detectable activity at and above 200 m
M
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 Mg
2+
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-
32
P]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 (ÔmÕ 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 unspecific (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 ÔmÕ
represents labeled marker DNA.
Ó FEBS 2003 Characterization of U6 terminal uridylyltransferase (Eur. J. Biochem. 270) 973
lane 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-
32
P]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 of
32
P-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 Ôtemplate-strandÕ 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)Thesameenzymeasin(A)wastestedwith[a-
32
P]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).
974 R. Trippe et al.(Eur. J. Biochem. 270) Ó FEBS 2003
that, 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
(m
1
,m
2
) 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 (m
1
)andU6-2/ARNA(m
2
). 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 m
2
band (Fig. 3C). These data provide evidence that the
enzyme was not able to ÔreadÕ 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
U6 snRNA. (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-
32
P]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).
Ó FEBS 2003 Characterization of U6 terminal uridylyltransferase (Eur. J. Biochem. 270) 975
A 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 vitro transcription 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 U6 snRNA. (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 ÔlÕ 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).
976 R. Trippe et al.(Eur. J. Biochem. 270) Ó FEBS 2003
This 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 m
M
KCl (lane 3) and
2
M
KCl (lane 4). Before being analysed for TUTase

activity, these fractions were dialysed against 100 m
M
KCl.
This confirmed that most TUTase molecules were eluted
from the affinity column at 600 m
M
KCl(lane3).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 (Ô–Õ RNA column) and a second

one loaded with poly(A)/U6-3 RNA (Ô+Õ RNA column).
The TUTase assay performed with the material eluted from
both columns at 600 m
M
KCl 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 Ô+Õ column. In
contrast, virtually no enzyme activity was detectable in lane
1, representing the elution fraction of the mock column (Ô–Õ).
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 Ô+Õ 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 (Ô–Õ)
or an oligo(dT) column coupled with oligoA/U6-3 RNA (Ô+Õ). 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 Ô–Õ
column (lane 1) and of the Ô+RNAÕ column (lane 2) were analysed as
before. DNA marker fragments are shown on the left (m). (B) Affinity-
purifiedTUTasewasincubatedwithlabeledU6-3RNAundershift
conditions, followed by UV cross-linking. Half of the material was

analysed directly (without nuclease digestion, Ô–Õ, lane 1) and half after
RNase A treatment (Ô+Õ, lane 2) in SDS/polyacrylamide gels (see
Materials and methods). Numbers on the right indicate the positions
of unlabeled marker proteins (kDa).
Ó FEBS 2003 Characterization of U6 terminal uridylyltransferase (Eur. J. Biochem. 270) 977
numbers of proteins. Electrophoretic analysis of proteins
obtained from the Ô+Õ and Ô–Õ 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 Ô+Õ (lane
1) and the Ô–Õ (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 Ô+Õ 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 m
M
Mg
2+
, 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 Ôopposite strandÕ
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.
978 R. Trippe et al.(Eur. J. Biochem. 270) Ó FEBS 2003
extensive 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 ÔownÕ 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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