Published online June 1, 2004
Nucleic Acids Research, 2004, Vol. 32, No. 10 2987±2994
DOI: 10.1093/nar/gkh619
3¢-Box-dependent processing of human pre-U1
snRNA requires a combination of RNA and protein
co-factors
Patricia Uguen and Shona Murphy*
Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK
Received February 26, 2004; Revised and Accepted May 4, 2004
ABSTRACT
Using an in vitro system we have recently shown
that the 3¢ ends of human pre-snRNAs synthesized
by RNA polymerase II are produced by RNA
processing directed by the snRNA gene-speci®c 3¢
box. Towards a complete characterization of this
processing reaction we have further investigated
the in vitro requirements for proper 3¢ end formation
of pre-U1 snRNA. Here we show that the 5¢ cap
plays a stimulatory role and processing requires
creatine phosphate. Our results also indicate that
the pre-U1 processing activity is heat sensitive and
that an RNA component is required. In addition, the
exact sequence adjacent to the 3¢ box in¯uences the
position of the pre-U1 3¢ end produced in vitro.
Interestingly, the processing extract active for 3¢box-dependent processing also contains an activity
that converts the 3¢ end of RNA containing the U1
Sm protein binding site and the 3¢ terminal stem±
loop into the mature form.
INTRODUCTION
The majority of vertebrate U snRNA genes (e.g. U1±U5) are
transcribed by RNA polymerase II (pol II) to yield short nonpolyadenylated 3¢-elongated pre-snRNAs. The 3¢ ends of these
RNAs are produced by a processing reaction dependent on the
13±16 nucleotide (nt) cis-acting 3¢ box element, located 9±19
nt downstream of the 3¢ end of the RNA-encoding region (1).
Most of the 3¢ extension is removed by further processing in
the cytoplasm before nuclear re-import (2,3). A compatible
snRNA promoter is also required for proper 3¢ end formation
of pre-snRNAs (4,5), highlighting the tight link between
transcription and the function of the 3¢ box. Relevant to this we
have recently demonstrated that the C-terminal domain (CTD)
of pol II is required for 3¢-box-dependent RNA processing
in vivo and that processing in vitro is activated by phosphoCTD (1,6).
3¢ End formation of pre-snRNAs is likely to involve
divalent-cation-dependent endonucleolytic activity, in common with 3¢ end formation of vertebrate pre-mRNAs or yeast
pre-snRNAs (1,7). However, neither subunits of the cleavage±
polyadenylation complex factors cleavage/polyadenylationspeci®city factor (CPSF), cleavage-stimulation factor (CstF)
and cleavage factor I (CFI), nor the human homologues of
yeast pre-snRNA processing factors hRNase III and PMScl100 appear to co-purify with the human pre-snRNA 3¢
processing activity (1). This suggests that there is little overlap
between known RNA 3¢ end processing factors and those used
for 3¢ end formation of human pre-snRNAs. Two cis-acting
elements, the U2 3¢ box and the upstream sequence element
(USE) act together to ef®ciently direct accurate pre-U2
snRNA 3¢ end formation both in vitro and in vivo (1), whereas
3¢ end formation of pre-U1 snRNA appears to depend largely
on the 3¢ box. Thus, there may also be differences in the cisacting sequences and trans-acting factors involved in 3¢ end
formation of different pre-snRNAs.
Further investigation of the biochemical requirements of
pre-U1 snRNA 3¢ end formation indicates that, in addition to
the phospho-CTD of pol II, creatine phosphate and a 5¢ cap
activate processing. We also demonstrate that 3¢ processing
requires an RNA component and that the activity is heat labile.
Thus, 3¢-box-dependent processing has some characteristics in
common with both AAUAAA-dependent processing (8) and
3¢ end formation of the replication-activated histone mRNAs
(9) as well as signi®cant differences. Furthermore, we show
that the ef®ciency of in vitro pre-U1 snRNA 3¢ end formation
is not affected by additional cis elements although the exact
sequence around the cleavage site in¯uences the site of the
®nal 3¢ end formed in vitro. The S100 fraction active for 3¢
end formation of pre-U1 snRNA is also active for 3¢ end
maturation and we show that these processes can occur
independently in vitro.
MATERIALS AND METHODS
DNA constructs
In 10end U1 3¢ box, 17end U1 3¢ box and 59end U1 3¢ box
templates, the 10, 17 and 59 nt, respectively, located upstream
of the 3¢ box of U1 3¢ box template described by Uguen and
Murphy (1) were replaced by the 10, 17 and 59 nt of the
endogenous U1 snRNA gene (GenBank accession number
J00318). The U1 3¢ box sequence, GTTTCAAAAGTAGA, is
deleted from the 59end D3¢ box template. DNA constructs
*To whom correspondence should be addressed. Tel: +44 1865 275616; Fax: +44 1865 275556; Email: [email protected]
Correspondence may also be addressed to Patricia Uguen at present address: Signalisation, DeÂveloppement et Cancer, BaÃtiment 442 bis, Universite Paris XI,
91405 Orsay ceÂdex, France. Tel: +33 1 69 15 65 93; Fax: +33 1 69 15 68 02; Email: [email protected]
Nucleic Acids Research, Vol. 32 No. 10 ã Oxford University Press 2004; all rights reserved
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used to produce synthetic RNA were obtained by deleting the
U1 promoter region by EcoRI/BglII digestion as described
previously (1).
Ef®cient U1 3¢-box-dependent processing requires
creatine phosphate as a co-factor and is ATP
independent
S100 fraction preparation
Since creatine phosphate (CP) (usually used to transfer Pi to
ADP, creating ATP) improves the ef®ciency of some mRNA
processing steps (11), we tested its effect on U1 3¢-boxdependent processing (Fig. 1). Little processing (1%) is
detected in the absence of added CP after incubation for 2.5 h
at 30°C (Fig. 1A, lanes 2 and 4), whereas 7% of the input RNA
is processed in the presence of 20 mM CP (Fig. 1A, lanes 3 and
5). Processing ef®ciency is only slightly improved by
increasing the concentration of CP further (8% of input
RNA is processed in the presence of 40 or 60 mM CP; Fig. 1A,
lanes 6 and 7). Furthermore, nucleoside triphosphates,
including ATP, do not support U1 3¢-box-dependent processing in the absence of CP (Fig. 1A, lane 4, and B, lanes 3±6),
indicating that CP does not act simply to regenerate the energy
source. Serine phosphate (SP) can also activate processing
although not as effectively as CP (Fig. 1C, lanes 3±5)
suggesting that the effect of CP is not entirely due to the
presence of phosphate groups.
It has been proposed that CP activates pre-mRNA 3¢
cleavage by mimicking the effect of the pol II CTD rather than
affecting the reservoir of energy (11,12) and may facilitate a
conformational change of some factors. In support of this
notion, the phosphorylated form of the CTD of pol II activates
3¢-box-dependent processing in vitro better than the unphosphorylated form (1). However, no processing is detected in the
absence of CP when phospho-CTD is added (Fig. 1D). This
indicates that CP does not simply substitute for phospho-CTD
in our system.
To eliminate the ATP present in the S100 fraction, we
fractionated the processing activity by binding to heparin±
sepharose at 100 mM KCl and eluting by 1 M KCl. Although
this fractionation procedure should markedly reduce the level
of ATP in the active fraction it does not result in a requirement
for added ATP (Fig. 1E, lanes 2 and 6). Furthermore, addition
of both glucose and hexokinase (Fig. 1E, lane 5), which
together deplete the available pool of ATP (11), has no greater
effect than the addition of glucose or hexokinase alone
(Fig. 1E, lanes 3 and 4). These results indicate that 3¢-boxdependent processing is ATP independent. Interestingly,
AAUAAA-dependent 3¢ cleavage is also CP dependent and
ATP independent in vitro (11).
HeLa cell S100 extract was prepared using a slight modi®cation of the procedure of Shapiro et al. (10). After cell lysis in
buffer A (10 mM HEPES pH 7.9, 0.75 mM spermidine,
0.15 mM spermine, 10 mM KCl, 0.5 mM DTT) sucrose was
added to a ®nal concentration of 10% before spinning.
In vitro RNA synthesis and in vitro processing
Synthetic, capped RNA was produced as described previously
(1). To produce uncapped RNA, m7G(5¢)ppp(5¢)G was
omitted from the reaction. Processing was carried out as
described in (1) for 2.5 h at 30°C in the presence of 3 mM
MnCl2 unless stated otherwise in the ®gure legends. To reduce
the level of endogenous ATP, the S100 fraction was bound to
heparin±sepharose at 100 mM KCl and eluted by 1 M KCl.
This fraction was then incubated for 15 min at 30°C with 3 mM
MgCl2, 2 mM glucose and 0.6 U of hexokinase (Boehringer
Mannheim) to deplete any residual ATP.
S1 mapping analysis
After incubation with S100, RNA was extracted, annealed to
the S1 probe, digested and analysed on a 6% polyacrylamide±
8 M urea gel as described previously (1). The S1 probes used
for each construct (U1 3¢ box, 10end U1 3¢ box, 17end U1 3¢
box, 59end U1 3¢ box and 59end D3¢ box) were prepared as
described previously (1).
Western blotting
Western blotting was carried out as described by Medlin et al.
(6). Anti-stem±loop-binding protein (SLBP) antibody was
kindly provided by Berndt Mueller (University of Aberdeen)
and used diluted at 1/100.
RESULTS
Improving U1 3¢-box-dependent in vitro processing
We recently described an in vitro system where 3¢-boxdependent processing of substrate RNAs was detected for the
®rst time (1). However, long incubation times (>5 h) are
required to detect any accurate 3¢ end processing directed by
the U1 3¢ box in unpuri®ed S100. We therefore undertook to
improve the ef®ciency of the processing activity by modifying
the extract preparation procedure (see Materials and Methods)
as a prelude to characterization of the requirements for 3¢
processing. Modi®cation of the extract preparation method
described by Shapiro et al. (10) led to the production of S100
that yields up to 8% properly processed RNA after 2.5 h
incubation rather than the 13 h needed with S100 produced by
our previously described protocol (1). The increased sucrose
concentration used in production of this extract (see Materials
and Methods) may reduce the release of nucleases from the
nuclei upon spinning and consequently reduce turnover of
substrate RNA, which proved to be a major problem for in vitro
processing (1). Relevant to this, the amount of pol II and
factors involved in mRNA 3¢ end formation is reduced in this
S100 fraction (data not shown and see Fig. 4B).
U1 3¢-box-dependent processing is activated by a
7-methyl-G cap
To examine whether 5¢ cap binding proteins activate 3¢-boxdependent RNA processing in addition to splicing, 3¢
processing, transport and translation of mRNA (13), we
compared the ef®ciency of 3¢ processing of capped and
uncapped U1 3¢ box RNA. In the absence of a 5¢ cap structure,
U1 3¢-box-dependent processing is slower (Fig. 2A), suggesting that either the 5¢ cap itself, or more likely, cap binding
proteins present in the S100, activate processing. The decrease
of proper 3¢ end formation of uncapped RNA is not due simply
to RNA instability since input RNA is still detected after 3 h
incubation (Fig. 2A). Addition of 7-methyl-G cap analogue
[m7G(5¢)ppp(5¢)G] to the in vitro reaction inhibits processing
and the highest concentration of cap analogue tested
Nucleic Acids Research, 2004, Vol. 32, No. 10
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Figure 1. Ef®cient U1 3¢-box-dependent processing requires CP as a co-factor and is ATP independent. (A) U1 3¢ box RNA was incubated with S100 in the
presence of ATP (1 mM) and different concentrations of CP as indicated. The percentage of input RNA processed (% proc.) is indicated below this and
subsequent gels. The scheme represents the position and size of the S1 probe, input (IP) and processed (proc.) RNA. The diagram on the left side of this and
subsequent ®gures indicates the position of the U1 3¢ box in the input RNA. (B) U1 3¢ box RNA was incubated with S100 in the presence of 20 mM CP or
1 mM of the nucleosides triphosphates indicated. (C) U1 3¢ box RNA was incubated with S100 in the presence of 20 mM CP or different concentrations of
SP as indicated. (D) U1 3¢ box RNA was incubated with S100 in the presence or absence of 20 mM CP and in the presence of phospho-CTD as indicated.
(E) U1 3¢ box RNA was incubated with S100 fractionated on heparin±sepharose in the presence of 20 mM CP, and with or without ATP, hexokinase and
glucose. The processing reaction was carried out in the presence of 2 mM MgCl2.
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Figure 2. U1 3¢-box-dependent processing is activated by a 7-methyl-G cap. (A) Capped (empty square) or uncapped (®lled diamond) U1 3¢ box RNA was
incubated with S100 for 0±3 h. The graph below represents the percentage relative processing activity calculated by comparison with the amount of
processing obtained after incubating capped RNA for 3 h. (B) Capped U1 3¢ box RNA was incubated with S100 in the presence of different concentrations of
unmethylated cap analogue [G(5¢)ppp(5¢)G; Epicenter] (lanes 3±7) or methylated cap analogue [m7G(5¢)ppp(5¢)G; Amersham Pharmacia] (lanes 8±12) as
indicated. The graph below represents the average percentage relative processing activity obtained in the presence of increasing concentrations of
unmethylated (®lled triangle) or methylated (empty square) cap analogue in two independent experiments.
effectively abolished processing (Fig. 2B, lanes 8±12). This
suggests that the cap analogue can sequester cap binding
proteins and interacting 3¢ processing factors. Consistent with
this, the effect of the cap structure of the RNA on processing
ef®ciency is lost after partial puri®cation of the 3¢ processing
activity as described by Uguen and Murphy (1) (data not
shown), indicating that the 5¢ cap per se is not suf®cient.
Moreover, addition of unmethylated cap analogue
G(5¢)ppp(5¢)G to the reaction has little effect on processing
(Fig. 2B, lanes 3±7). Taken together, these results suggest that
the nuclear cap binding complex (14) may be involved in the
recruitment of pre-snRNA 3¢ processing factor(s) in vivo.
U1 3¢-box-dependent processing requires an RNA
co-factor
Small non-coding RNAs function as co-factors in both
splicing and 3¢ processing of pre-mRNAs (8,9). To determine
whether 3¢ processing of U1 3¢ box RNA substrate is
dependent on a trans-acting nucleic acid component, the
S100 was treated with micrococcal nuclease (MN) before
incubation with U1 3¢ box RNA (Fig. 3). U1 3¢-box-dependent
processing is completely abolished by this treatment (lane 3).
However, when EGTA, which chelates the Ca2+ ions essential
for this nuclease to function, is added to the S100 before MN,
no reduction in processing is observed (lane 4). This suggests
the involvement of an RNA or DNA co-factor in U1 3¢-boxdependent processing. To distinguish between these two
possibilities, we treated the S100 with pancreatic DNase I
before incubation with U1 3¢ box RNA (lane 8). This has no
effect on 3¢ processing, indicating that the nucleic acid cofactor is composed of RNA. In order to test whether this RNA
component is the sole catalytic component of the 3¢ processing
activity, we extracted ribonucleic acid from the S100 by acidic
phenol/chloroform treatment and incubated it with U1 3¢ box
RNA. This fraction does not support 3¢ processing on its own
(lane 5). In addition, 3¢ processing is not recovered when the
MN-treated S100 is complemented with this RNA fraction
(lane 6). These data suggest that U1 3¢-box-dependent
processing involves both RNA and protein factor(s) and
leads us to propose that processing is carried out by an RNA±
protein complex.
U7 snRNA is required for 3¢ end formation of the mRNAs
for replication-activated histones and has a region of
complementarity to the substrate RNA (9). Since mammalian
U7 snRNAs contain no conserved sequences complementary
to 3¢ box sequences it is unlikely that U7 snRNA participates
in 3¢ end formation of U1 snRNA. Unfortunately, experiments
to test the effect of depletion of U7 snRNA and other
U snRNAs from the processing extract using antibodies to
the trimethylguanosine cap present at the 5¢ end were
inconclusive.
U1 3¢-box-dependent processing activity is heat labile
To analyse the sensitivity of the 3¢-box-dependent processing
activity to heat, the S100 fraction was heated for 10 min at
temperatures from 30 to 55°C before incubation with U1 3¢
Nucleic Acids Research, 2004, Vol. 32, No. 10
Figure 3. U1 3¢-box-dependent processing requires an RNA co-factor. U1
3¢ box RNA was incubated with S100 in the presence of 2 mM of MgCl2
before or after pretreatment with MN or DNase I and/or complemented with
the extracted RNA. MN (lane 3), S100 pretreated for 15 min at 25°C with
MN (5 U/ml of S100; Roche) in the presence of 1 mM CaCl2. The reaction
was stopped by adding 2 mM EGTA. MN in. (lane 4), S100 pretreated for
15 min at 25°C with MN inactivated by the addition of 2 mM EGTA; RNA
extracted (lane 5), S100 extracted by acidic phenol/chloroform treatment
and ethanol precipitated before resuspension in buffer D (15); RNA
extracted + MN (lane 6), S100 pretreated for 15 min at 25°C with MN in
the presence of 1 mM CaCl2 and complemented by the extracted RNA.
RNA extracted + MN in. (lane 7), S100 pretreated for 15 min at 25°C with
MN inactivated by the presence of 2 mM EGTA and complemented by the
extracted RNA; DNase I (lane 8), S100 pretreated for 15 min at 30°C with
pancreatic DNase I (10 U/ml of S100).
box RNA (Fig. 4). The ef®ciency of 3¢ processing is slightly
reduced at 45°C (inhibition of 33%; Fig. 4A, lane 4).
However, 3¢ processing is inhibited by 83% after preheating
the S100 fraction at 50°C (lane 5) and is lost completely after
treatment at 55°C (lane 6), suggesting that U1 3¢-boxdependent processing involves heat-sensitive factor(s).
3¢ Processing of replication-activated histone mRNAs is
similarly heat labile (16), raising the possibility of shared
factors with U1 3¢-box-dependent processing. A wellcharacterized SLBP is required for histone 3¢ end formation
(9) and we have used an anti-SLBP antibody to analyse our
processing extract by western blot (Fig. 4B). SLBP is not
readily detected in the S100 used for the in vitro processing
reactions shown in Figure 4A (Fig. 4B, lane 1) although it is
relatively abundant in our previously described S100 preparation (1) (Fig. 4B, lane 2). In addition, no SLBP is detected in
the partially puri®ed 0.4 M KCl SP fraction active for presnRNA 3¢ end formation (1) (Fig. 4C, lane 6). These results
suggest that SLBP is not involved in 3¢-box-dependent
processing.
Sequences upstream from the U1 3¢ box can in¯uence 3¢
processing
We have shown that accurate pre-U2 snRNA 3¢ end formation
requires two cis-acting elements, USE and U2, acting
synergically. Although the U1 3¢ box is suf®cient to direct
accurate formation of RNA 3¢ ends in vitro and in vivo
(1,4,17), the last 5±6 nt of the mature 3¢ end of U1 snRNA can
affect the in vivo accuracy of pre-U1 snRNA 3¢ end formation
(17). We therefore analysed the in¯uence of the natural
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Figure 4. U1 3¢-box-dependent processing activity is heat labile. (A) U1 3¢
box RNA was incubated with S100 preheated for 10 min at the indicated
temperature (temp.). The graph represents the percentage processing compared with the level obtained when S100 is preheated at 30°C. (B) Western
blot with SLBP antibody on the S100 used for processing in Figures 1±5
(lane 1) and on the S100 described by Uguen and Murphy (1), which is less
active for 3¢ processing (lane 2). SLBP is 45 kDa. (C) Western blot with
anti-SLBP antibody on the different fractions resulting from puri®cation of
the processing activity on different sepharose resins as described by Uguen
and Murphy (1). B-heparin and B-Q represent proteins bound to heparin±
and Q-sepharose, respectively; UB-SP represents proteins that did not bind
to SP-sepharose.
sequences preceding the U1 3¢ box on the ef®ciency and
accuracy of 3¢ processing in vitro. When RNA containing the
10 nt just upstream of U1 3¢ box (10end U1 3¢ box RNA) is
incubated with S100, no properly processed RNA is detected
(Fig. 5A, lanes 6±10, and C), even after 3 h incubation. This
may be due to instability of processed RNA rather than to a
complete loss of processing. When the 17 nt upstream of the
U1 3¢ box are present (17end U1 3¢ box), processing is
detected (Fig. 5A, lanes 11±15, and C) although the ef®ciency
is reduced by 45% with respect to U1 3¢ box RNA (Fig. 5A,
lanes 1±5, and C). A small amount of the 3¢ end processed
RNA maps to the same nucleotides seen with the U1 3¢ box
RNA but most map a few nucleotides upstream, corresponding
to the most highly represented 3¢ ends detected in vivo, which
end 6±8 nt upstream of the 3¢ box (17,18) (Fig. 5A, compare
lanes 1±5 and 11±15, and C). These results may indicate that
the addition of 17 nt of the natural U1 sequence in¯uences the
position of 3¢ endonucleolytic cleavage. Alternatively, 3¢
exonucleolytic trimming of the RNA occurs after cleavage
3±5 nt upstream from the 3¢ box.
We have also tested the effect of placing a longer
endogenous sequence (59 nt) upstream of the U1 3¢ box.
This substrate RNA contains both the Sm protein binding site
and the 3¢ terminal stem±loop, which are not required in vivo
for pre-U1 snRNA 3¢ end formation but are required for
ef®cient nuclear export (19). When 59end U1 3¢ box RNA
(Fig. 5B, lanes 3 and 4) is incubated with S100, RNA with the
mature 3¢ end mapping 10±12 nt upstream of the 3¢ box (17) is
produced. This indicates that the activities required for
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Figure 5. Sequences upstream from the U1 3¢ box can in¯uence 3¢ processing. (A) U1 3¢ box, 10end U1 3¢ box and 17end U1 3¢ box RNA substrates were
incubated with S100 for 0±3 h. The scheme below indicates the position and size of the S1 probes, input (IP) and processed (proc.) RNA. (B) U1 3¢ box,
59end U1 3¢ box and 59end D3¢ box RNA substrates were incubated with S100. The scheme below indicates the position and size of the S1 probes, input
(IP), processed (proc.) and mature RNA. (C) The sequences of the 3¢ ends of the substrate RNAs in (A) and (B) are shown with the position of in vivo and
in vitro processing sites and mature ends. The endogenous U1 snRNA sequence is highlighted in grey. The U1 3¢ box is boxed. The vertical line indicates the
boundary between the end of the mature snRNA and the ®rst nucleotide of the non-coding RNA. The bold nucleotides represent the processing sites observed
in vivo (17,18). The arrows above the sequence indicate the pre-snRNA processing sites observed in vitro. The mature ends observed in vitro are indicated by
un®lled arrows above the sequence.
production of mature U1 3¢ ends (20) are present in the S100,
in addition to factors required for pre-U1 3¢-box-dependent
processing. Some accurately processed pre-U1 RNA 3¢ ends
are still detected and these map to the same sites as are
observed when only 17 nt of natural U1 sequence are present
upstream of the 3¢ box (17end U1 3¢ box RNA; Fig. 5A, lanes
11±15, and C). This may suggest that processing proceeds in a
stepwise manner as it does in vivo and the precise pre-snRNA
3¢ ends are required for the next step, which matures the 3¢ end
of the RNA, as has been shown for 3¢ end formation of U2
snRNA (21). 3¢ Ends mapping 6±8 nt upstream from the 3¢ box
are no longer detected when the 3¢ box is removed (Fig. 5B,
Nucleic Acids Research, 2004, Vol. 32, No. 10
lanes 5 and 6, and C). However, production of the mature U1
3¢ ends is unaffected (Fig. 5B, lane 6), indicating that these
two processing events occur independently in our in vitro
system. This suggests that any RNAs containing the Sm
protein binding site and the 3¢ terminal stem±loop can be 3¢
end matured, presumably by exonucleolytic trimming. Since
the 59end D3¢box RNA has 27±29 nt downstream from the
sites of the mature 3¢ ends, these results also indicate that
RNAs longer than pre-U1 at the 3¢ end are readily 3¢ matured
in vitro. In contrast, only pre-U1 snRNAs with a short 3¢
extension (no more than 10 nt) are further processed after
injection into Xenopus oocytes (20). This may highlight
differences between species in the 3¢ end maturation of
snRNAs. Alternatively, the exact sequence of the 3¢ extension
may in¯uence trimming and/or some in vivo constraints may
be lost in our in vitro system.
DISCUSSION
The ®rst step of expression of the vertebrate U1 snRNA genes
involves the production of a 3¢ extended pre-U1 snRNA,
which occurs by a co-transcriptional 3¢-box-dependent mechanism (4,5). Proper 3¢ end formation of pre-U1 snRNA is
important for the stability of the snRNA in the nucleus, in
addition to nucleocytoplasmic export (19,20). The exact 3¢
ends of U1 snRNA precursors can also in¯uence the
cytoplasmic 3¢ trimming of the RNA required for the nuclear
translocation of U1 snRNPs and subsequent maturation steps
(20). Towards the dissection of the molecular mechanism of 3¢
end formation of human pre-U1 snRNA we have determined
several of the co-factor requirements of U1 3¢-box-dependent
in vitro processing. We have also investigated the in¯uence of
sequences upstream of the U1 3¢ box on 3¢ end formation. The
results of these studies highlight similarities and differences
between 3¢ end formation of pre-snRNAs and other vertebrate
pol II transcripts. In addition, we show that 3¢ extended RNAs
containing the U1 Sm protein binding site and the 3¢ terminal
stem±loop can be processed to the mature U1 3¢ end in vitro.
Comparing U1 3¢-box-dependent processing and
formation of mRNA 3¢ ends
Some co-factor requirements for 3¢-box-dependent processing
are shared with AAUAAA-dependent processing. Both processing events require divalent cations and CP, are ATP
independent and are activated by 5¢ cap binding proteins
(8,11,13) (Fig. 6). In addition, both require the CTD of pol II
in vivo (6,13) and are activated by phospho-CTD in vitro
(1,12) (Fig. 6). So far there is no indication that any subunits of
CPSF, CstF and CFI factors required for AAUAAA-dependent processing participate in pre-snRNA 3¢ end formation (1).
However, it has been shown that CFI participates in 3¢
processing of some yeast snoRNAs and snRNAs (22).
There are also some similarities between 3¢ end formation
of U1 snRNA and replication-activated histone mRNAs. In
both cases a stem±loop structure is present just upstream of the
mature 3¢ end, the processing activities are heat labile and a
trans-acting RNA component is required (9). Our results
therefore point to mechanistic similarities in 3¢ end formation
of non-polyadenylated histone mRNA and snRNA. However,
our data indicate that the SLBP is not involved in U1 snRNA
3¢ end formation. In addition, the lack of complementarity
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Figure 6. Requirements for U1 3¢-box-dependent RNA processing. 3¢-Boxdependent 3¢ end formation of pre-U1 snRNA requires an RNA±protein
complex, CP and divalent cations (div. ion). The reaction is also activated
by 5¢ cap binding proteins (diamond) and the CTD of pol II. In addition, the
processing activity is abolished by relatively mild heat treatment, which
suggests the involvement of a heat-labile factor (HLF).
between U7 snRNA and 3¢ box sequences makes it likely that
a distinct RNA is required.
Divalent cations are also necessary for ef®cient activity of
the yeast RNase III homologues, Pac1p and Rnt1p (23,24)
which are involved in yeast snRNA 3¢ end formation (25).
However, human RNase III does not co-purify with the 3¢box-dependent processing activity and the cis-acting elements
do not correspond to RNase III recognition sequences (1).
Divalent cations may be required for the function of a 3¢-boxspeci®c processing factor or play a more direct catalytic role.
The currently known requirements for 3¢ end processing of
pre-U1 snRNA are summarized in Figure 6. Taken together
our results indicate that there is little overlap in the factors
required for 3¢-box-dependent processing and mRNA 3¢ end
formation apart from the shared co-factors noted above. This is
consistent with the differences in cis-acting sequences and the
differential promoter dependence of these signals in vivo (4,5).
Sequences immediately upstream of the U1 3¢ box
in¯uence 3¢ end formation
The ef®cient production of accurately 3¢ processed pre-U2
snRNA requires two distinct sequences: the U2 3¢ box and the
USE, which contains a `minihelix' stem±loop structure (1).
The U2 3¢ box alone directs formation of RNA a few
nucleotides longer than cellular pre-U2 snRNA while RNA
containing the USE is processed to the authentic pre-U2
snRNA 3¢ end. In addition, the amount of processed product
detected in vitro is increased when both the U2 3¢ box and the
USE are present. The minihelix may therefore in¯uence
processing by interacting with a processing factor and/or
prevent RNA cleaved by a 3¢-box-dependent endonuclease
from being rapidly turned over by exonucleases (1). Addition
of 17 nt of the natural U1 gene-encoded sequence upstream of
the U1 3¢ box also causes an apparent shift in the position of
the ®nal 3¢ ends, some of which now map to the most abundant
pre-U1 3¢ ends detected in cellular RNA (17,18). The
sequences directly upstream from the U1 3¢ box may therefore
play a similar role to the U2 USE to determine the ®nal
position of the pre-U1 3¢ end. In both cases the site of
endonucleolytic cleavage may be in¯uenced by the exact
sequence of the target RNA or changes in processing±factor
RNA interaction. Alternatively, the natural sequences may be
preferential substrates for limited trimming by exonucleases
following 3¢ box-directed cleavage. However, unlike the U2
USE, the 17 nt upstream from the U1 3¢ box do not cause an
2994
Nucleic Acids Research, 2004, Vol. 32, No. 10
increase in the amount of processed RNA detected but rather a
decrease. In addition, unlike the U2 USE (1), the 59 nt of U1
sequence upstream of the 3¢ box do not direct formation of preU1 3¢ ends, suggesting functional differences. In agreement
with this, little authentic pre-U1 RNA is produced from
transfected U1 templates lacking the 3¢ box (17).
A precise pre-U1 3¢ end is not required for RNA
maturation in vitro
Although pre-U2 snRNA can be further 3¢ processed in vitro
(2,21), maturation of the 3¢ ends of pre-U1 snRNA in vitro has
not previously been reported. However, in our in vitro system
the inclusion of U1 snRNA sequences containing the Sm
protein binding site and the 3¢ terminal stem±loop (19) in the
RNA substrate results in the production of properly processed
mature U1 3¢ ends. Interaction of Sm protein with its binding
site is required for the cytoplasmic 5¢ cap modi®cation of U1
snRNA, which is necessary for nuclear re-import (26) and it
also plays a role in maturation of the 3¢ ends of yeast U1
snRNA (27, reviewed in 3,28). Surprisingly, in vitro 3¢ end
maturation occurs independently of 3¢-box-dependent processing, suggesting that no intermediate processing step is
required even though the substrate U1 RNAs have relatively
long 3¢ extensions (27±29 nt). Instead, 3¢ end extensions longer
than 10 nt are not processed to the mature form when human
pre-U1 RNAs are injected into Xenopus oocytes (20). In
addition, specialized sequences distinct from the Sm protein
binding site are required for cytoplasmic trimming of the 3¢
end of human U2 snRNA (29), and pre-U2 RNA with the
natural 3¢ ends are preferential substrates for the reaction
in vitro (2,21). There may therefore be differences in 3¢ end
maturation of Xenopus and human U1 snRNAs, and between
human U1 and U2 snRNAs. The availability of in vitro systems
for both 3¢-box-dependent processing and maturation of 3¢
extended pre-U1 snRNAs will facilitate the molecular analysis
of all steps leading to accurate formation of U1 3¢ ends.
ACKNOWLEDGEMENTS
We thank Alice Taylor for excellent technical assistance. We
are grateful to Berndt Mueller for the kind gift of the antiSLBP antibody. We also thank Zbigniew Dominski, Nick
Proudfoot and Andre Furger for helpful suggestions and
comments on the manuscript. P.U. was supported by MRC Cooperative Component grant No. G9900343 and the Edward
Penley Abraham Trust. S.M. was supported by MRC Senior
Fellowship No. G117/309.
REFERENCES
1. Uguen,P. and Murphy,S. (2003) The 3¢ ends of human pre-snRNAs are
produced by RNA polymerase II CTD-dependent RNA processing.
EMBO J., 22, 4544±4554.
2. Huang,Q., Jacobson,M.R. and Pederson,T. (1997) 3¢ Processing of
human pre-U2 small nuclear RNA: a base-pairing interaction between
the 3¢ extension of the precursor and an internal region. Mol. Cell. Biol.,
17, 7178±7185.
3. Will,C.L. and LuÈhrmann,R. (2001) Spliceosomal UsnRNP biogenesis,
structure and function. Curr. Opin. Cell Biol., 13, 290±301.
4. Hernandez,N. and Weiner,A.M. (1986) Formation of the 3¢ end of U1
snRNA requires compatible snRNA promoter elements. Cell, 47,
249±258.
5. Neuman de Vegvar,H.E., Lund,E. and Dahlberg,J.E. (1986) 3¢ End
formation of U1 snRNA precursors is coupled to transcription from
snRNA promoters. Cell, 47, 259±266.
6. Medlin,J.E., Uguen,P., Taylor,A., Bentley,D. and Murphy,S. (2003) The
C-terminal domain of pol II and a DRB-sensitive kinase are required for
3¢ processing of U2 snRNA. EMBO J., 22, 925±934.
7. Ryan,K., Calvo,O. and Manley,J.L. (2004) Evidence that
polyadenylation factor CPSF-73 is the mRNA 3¢ processing
endonuclease. RNA, 10, 565±573.
8. Wahle,E. and RuÈegsegger,U. (1999) 3¢-End processing of pre-mRNA in
eukaryotes. FEMS Microbiol. Rev., 23, 277±295.
9. Dominski,Z. and Marzluff,W.F. (1999) Formation of the 3¢ end of
histone mRNA. Gene, 239, 1±14.
10. Shapiro,D.J., Sharp,P.A., Wahli,W.W and Keller,M.J. (1988) A highef®ciency HeLa cell nuclear transcription extract. DNA, 7, 47±55.
11. Hirose,Y. and Manley,J.L. (1997) Creatine phosphate, not ATP, is
required for 3¢ end cleavage of mammalian pre-mRNA in vitro. J. Biol.
Chem., 272, 29636±29642.
12. Hirose,Y. and Manley,J.L. (1998) RNA polymerase II is an essential
mRNA polyadenylation factor. Nature, 395, 93±96.
13. McCracken,S., Fong,N., Rosonina,E., Yankulov,K., Brothers,G.,
Siderovski,D., Hessel,A., Foster,S., Shuman,S. and Bentley,D.L. (1997)
5¢-Capping enzymes are targeted to pre-mRNA by binding to the
phosphorylated carboxy-terminal domain of RNA polymerase II. Genes
Dev., 11, 3306±3318.
14. Flaherty,S.M., Fortes,P., Izaurralde,E., Mattaj,I.W. and Gilmartin,G.M.
(1997) Participation of the nuclear cap binding complex in pre-mRNA 3¢
processing. Proc. Natl Acad. Sci. USA, 94, 11893±11898.
15. Dignam,J.D., Lebovitz,R.M. and Roeder,R.G. (1983) Accurate
transcription initiation by RNA polymerase II in a soluble extract from
isolated mammalian nuclei. Nucleic Acids Res., 11, 1475±1489.
16. Gick,O., Kramer,A., Vasserot,A. and Birnstiel,M.L. (1987) Generation of
histone mRNA 3¢ ends by endonucleolytic cleavage of the pre-mRNA in
a snRNP-dependent in vitro reaction. Proc. Natl Acad. Sci. USA, 84,
8937±8940.
17. Hernandez,N. (1985) Formation of the 3¢ end of U1 snRNA is directed by
a conserved sequence located downstream of the coding region.
EMBO J., 4, 1827±1837.
18. Patton,J. and Wieben,E. (1987) U1 precursors: variant 3¢ ¯anking
sequences are transcribed in human cells. J. Cell Biol., 104, 175±182.
19. Terns,M.P., Dahlberg,J.E. and Lund,E. (1993) Multiple cis-acting signals
for export of pre-U1 snRNA from the nucleus. Genes Dev., 7,
1898±1908.
20. Neuman de Vegvar,H.E. and Dahlberg,J.E. (1990) Nucleocytoplasmic
transport and processing of small nuclear RNA precursors. Mol. Cell.
Biol., 10, 3365±3375.
21. Huang,Q. and Pederson,T. (1999) A human U2 RNA mutant stalled in 3¢
end processing is impaired in nuclear import. Nucleic Acids Res., 15,
1025±1031.
22. Morlando,M., Greco,P., Dichtl, B., Fatica,A., Keller,W. and Bozzoni,I.
(2002) Functional analysis of yeast snoRNA and snRNA 3¢-end
formation mediated by uncoupling of cleavage and polyadenylation. Mol.
Cell. Biol., 22, 1379±1389.
23. Rotondo,G. and Frendewey,D. (1996) Puri®cation and characterization
of the Pac1 ribonuclease of Schizosaccharomyces pombe. Nucleic Acids
Res., 24, 2377±2386.
24. Lamontagne,B. and Elela,S.A. (2001) Puri®cation and characterization of
Schizosaccharomyces pombe Rnt1p nuclease. Methods Enzymol., 342,
159±167.
25. Allmang,C., Kufel,J., Chanfreau,G., Mitchell,P., Petfalski,E. and
Tollervey,D. (1999) Functions of the exosome in rRNA, snoRNA and
snRNA synthesis. EMBO J., 18, 5399±5410.
26. Mattaj,I.W. (1986) Cap trimethylation of U snRNA is cytoplasmic and
dependent on U snRNP protein binding. Cell, 46, 905±911.
27. Seipelt,R.L., Zheng,B., Asuru,A. and Rymond,B.C. (1999) U1 snRNA is
cleaved by RNase III and processed through an Sm site-dependent
pathway. Nucleic Acids Res., 27, 587±595.
28. Yong,J., Pellizzoni,L. and Dreyfuss,G. (2002) Sequence-speci®c
interaction of U1 snRNA with the SMN complex. EMBO J., 21,
1188±1196.
29. Jacobson,M.R., Rhoadhouse,M. and Pederson,T. (1993) U2 small nuclear
RNA 3¢ end formation is directed by a critical internal structure distinct
from the processing site. Mol. Cell. Biol., 13, 1119±1129.