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From the
Received for publication, March 7, 2002, and in revised form, April 10, 2002
U2 RNA is one of five small nuclear
RNAs that participate in the majority of mRNA splicing. In addition
to its role in mRNA splicing, the biosynthesis of U2 RNA and three
of the other spliceosomal RNAs is itself an intriguing process
involving nuclear export followed by 5'-cap hypermethylation, assembly
with specific proteins, 3' end processing, and then nuclear import.
Previous work has identified sequences near the 3' end of pre-U2 RNA
that are required for accurate and efficient processing. In this study,
we have investigated the structural basis of U2 RNA 3' end processing by chemical and enzymatic probing methods. Our results demonstrate that
the 3' end of pre-U2 RNA is a minihelix with an estimated stabilization
free energy of The major spliceosome that operates on most eukaryotic
pre-mRNAs contains five small RNAs, U1, U2, U4, U5, and U6. Beyond their role in mRNA splicing, the biosynthesis of these small
spliceosomal RNAs is itself an interesting process. U6 small
nuclear is transcribed by RNA polymerase III (1, 2) and associates with
spliceosomes without extensive 3'-processing or nucleocytoplasmic
transit (3). In contrast, the other four major spliceosomal RNAs, U1,
U2, U4, and U5, are transcribed by RNA polymerase II in mammalian cells as precursor molecules extended at their 3' ends that are then exported
to the cytoplasm where they undergo cap hypermethylation and
ribonucleoprotein assembly followed by 3' end processing and nuclear
import (4-13).
The mammalian precursor molecules of U1, U2, U4, and U5 RNAs have been
defined in several studies (4, 6, 8-11, 14), and their 3'-processing
has been well characterized particularly for U2 RNA (11, 13, 15-17). A
distinct region of pre-U2 RNA lying near the 3' end of mature RNA has
been shown to be critical for accurate and efficient 3'-processing
(16). It was also found that sequences in the 5'-half of pre-U2 RNA
play no role in the 3'-processing reaction (16), suggesting that in the
folded structure of pre-U2 RNA, the 5'-half of the molecule is not
interactive with the 3' end, at least not in a way that influences the
3'-processing reaction. The 3' end of pre-U2 RNA also has been
implicated in the nucleocytoplasmic traffic of U2 RNA. Non-processed 3'
end variants of pre-U2 RNAs display impaired nuclear import in both Xenopus oocytes (18) and human cells (19), indicating that cytoplasmic 3'-processing is a key step in the nuclear import pathway.
The 3' end of the precursor of human U2 RNA consists of an
11-nucleotide element (11, 14, 20, 21), but the structure of this 3'
tail is not known. The secondary structures of the processed mature
forms of U2 RNA from human and other organisms as well as that of U1,
U4, and U5 RNAs have been experimentally determined in several previous
investigations (22-32). In this study, we have investigated the
structure of the 3' end of human pre-U2 RNA by chemical and enzymatic
probing methods and find that it exists as a stably folded minihelix.
Additional results suggest that this 3' end minihelix of pre-U2 RNA may
be more relevant to the initial cleavage of pre-U2 RNA from its primary
transcript or its intracellular traffic rather than its 3'-processing reaction.
Full-length wild-type human pre-U2 RNA was transcribed from
plasmid pMRG3U2 (16). Human pre-U2 RNA with a wild-type 3' tail and
lacking nucleotides 1-104 (non-essential for 3'-processing) was
transcribed from the plasmid pMRG3U2-54 (16). Pre-U2 RNAs with mutant
3' tails, U2-75 and U2-80, were transcribed from the plasmids
described previously (17). The RNAs were labeled at their 5' ends with
g-[32P]ATP and polynucleotide kinase or at their 3' ends
with [32P]pCp and RNA ligase. The RNA samples containing
10-100 ng/µl reaction (2 × 104 dpm/µl) were
subjected to Pb2+-mediated cleavage in 50 mM
potassium acetate, 10 mM magnesium acetate, 50 mM HEPES-KOH, pH 7.5, containing 4 or 10 mM
PbCl2. The reactions were carried out at 20 °C for 6 min. Nuclease VI and RNase T2 and T1 digestions were done in 50 mM KCl, 10 mM MgCl2, 50 mM Tris-HCl, pH 7.5, for 6 min at 20 °C. Nuclease VI was
used at 5 or 10 units/ml, RNase T2 was used at 10 or 25 units/ml, and RNase T1 was used at 5 or 25 units/ml. In all of the experiments, the
RNA was preincubated for 5 min at 70 °C followed by 15 min at
20 °C in the absence of PbCl2 or enzyme to renature the
RNA. Yeast tRNA (Roche Molecular Biochemicals) was present at 200 mg/ml in all enzymatic digestions and chemical probing with the exception of
RNase T1 digestions where it was present at 20 mg/ml. After chemical
cleavage, the reactions were stopped by the addition of EDTA (20 mM), and the RNAs were recovered by ethanol precipitation. After enzymatic cleavages, the RNAs were recovered by phenol/chloroform extraction in the presence of EDTA (25 mM final
concentration). In the case of RNase T1, the reactions were also
stopped by the addition of an excess of tRNA (2 mg/ml). The RNAs
were dissolved in deionized formamide containing 20 mM
EDTA, 0.05% (w/v) cyanol blue, and 0.05% (w/v) bromphenol blue and
were displayed by electrophoresis on 15% polyacrylamide gels
containing 8 M urea followed by autoradiography. Sequencing
ladders were obtained by alkaline hydrolysis of labeled RNA (90 min at
96 °C in 20 ml of deionized H2O) followed by ethanol precipitation. RNase T1 sequencing was performed by the digestion of
heat-denatured labeled RNA with 1000 units/ml RNase T1 in the presence
of 2.5 mg/ml tRNA for 30 min at 55 °C in 7 M urea, 1 mM EDTA, 20 mM sodium citrate, pH 5.0. Diethylpyrocarbonate sequencing was performed for 9 min at 96 °C in
500 mM sodium acetate, 1 mM EDTA, pH 4.5, in
the presence of tRNA (50 mg/ml) followed by ethanol precipitation and
aniline treatment (33).
Fig. 1A shows both the
full-length human pre-U2 RNA, and Fig. 1B shows the
miniature pre-U2-54 RNA used in this investigation. We have previously
shown that the 3'-processing of pre-U2-54 RNA is identical to that of
full-length pre-U2 RNA (17). To determine the structure of the 3' end
of both pre-U2 RNAs, the pre-U2 RNAs were labeled at their 3' (both
RNAs) or 5' (pre-U2-54) ends with 32P and subjected to
chemical (Pb2+) or enzymatic digestion methods that produce
RNA structure-dependent phosphodiester bond cleavages (34).
Fig. 2 shows a representative RNA
sequencing gel of the pre-U2-54 RNA cleavage products. Fig. 2,
A and B, shows Pb2+ and enzymatic
cleavages obtained with 5' end-labeled pre-U2-54 RNA, and Fig.
2C shows the probing of 3' end-labeled pre-U2-54 RNA. Fig.
3 presents the RNA structure deduced from
the experimental data. The results indicate that the 3' extension of
the pre-U2-54 RNA has a minihelix structure with an estimated
stability (change in free energy upon its formation) of approximately
It is noted that the conditions employed for both Pb2+ and
enzymatic cleavage are ones that have been calibrated in numerous previous studies to generate a very low frequency of hits (a maximum one cut per molecule and often less than one). The expected low yield
of cleaved molecules in the present experiments is indicated by the
large amounts of uncut RNA remaining at the origins of the sequencing
gels (Fig. 2). The deliberate use of these low frequency cleavage
conditions minimizes or eliminates intermolecular interference by
fragments or cleavage-induced alterations of the intramolecular folded structure.
To determine whether stem-loop V, as detected in pre-U2-54, is also
present in wild-type full-length pre-U2 RNA, similar Pb2+
and enzymatic-probing experiments were carried out. As shown in Fig.
4, the results indicated a structure
virtually identical to that obtained for pre-U2-54. In addition to
demonstrating that stem-loop V is a feature of the normal cellular
pre-U2 RNA, these results show that the overall secondary structure of
the mature region of the molecule is congruent with the
previously determined secondary structure of mature U2 small nuclear
RNA itself (23). To our knowledge, this is the first experimentally
based description of the secondary structure of the precursor form of
any of the spliceosomal small RNAs.
A 3'-Terminal Minihelix in the Precursor of Human Spliceosomal U2
Small Nuclear RNA*
§,,,
,
Unité Mixte Recherche 7567 CNRS-Université Henri Poincaré Nancy I, Maturation des ARN
et Enzymologie Moléculaire, Université H. Poincaré,
54506 Vandoeuvre-les Nancy, France and the ¶ Department of
Biochemistry and Molecular Pharmacology, University of Massachusetts
Medical School, Worcester, Massachusetts 01605
ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
6.9 kcal/mol. Parallel RNA structure mapping
experiments with mutant pre-U2 RNAs revealed that the presence of this
3' minihelix is itself not required for in vitro 3'-processing of pre-U2 RNA, in support of earlier studies implicating internal regions of pre-U2 RNA. Other considerations raise the possibility that this distinctive structural motif at the 3' end of
pre-U2 RNA plays a role in the cleavage of the precursor from its
longer primary transcript or in its nucleocytoplasmic traffic.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
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EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
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RESULTS AND DISCUSSION
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6.9 kcal/mol (35). In keeping with the Roman numeral-based
nomenclature used for the previously determined secondary structures of
mature spliceosomal small nuclear RNAs, we term the 3' minihelix
"stem-loop V." We noted in particular that there was no indication
in the structure probing results for the previously entertained
pseudoknot interaction between stem-loop V and an internal region at
the base of stem-loop IV (see Fig. 10 in Ref. 17). Indeed, our results
indicate that the internal sequence previously considered as the
partner for the contemplated pseudoknot is actually a part of stem-loop
III itself and thus is unavailable for pseudoknot formation.
View larger version (15K):
[in a new window]
Fig. 1.
A, human pre-U2 RNA. The proposed
secondary structure is that of Keller and Noon (39). Arrows
indicate the 3' ends of mature U2 RNA. B, pre-U2-54
RNA. In both RNAs, the 3' extension is deliberately drawn in
single-stranded form, because its structure was unknown at the outset
of this study.
View larger version (83K):
[in a new window]
Fig. 2.
Chemical and enzymatic probing of pre-U2-54
RNA. Electrophoresis of the RNA fragments obtained after chemical
or enzymatic treatment is shown. In vitro transcripts of the
plasmid pMRG3U2-54 were either 5' end-labeled (panels A and
B) or 3' end-labeled (panel C) and subjected to
Pb2+ or enzymatic cleavage under the conditions described
under "Experimental Procedures." Lead (Pb) and enzymes
RNase T1 (T1), RNase T2 (T2), and RNase V1
(V1) used are indicated at the top of the lanes.
Adjacent lanes marked 1 and 2 correspond to two concentrations of Pb2+ or the enzyme (see
"Experimental Procedures"). Lanes marked 0 refer to the reaction with neither Pb2+ nor enzyme.
Sequencing lanes C, H, T,
and D are control (C), alkaline hydrolysis ladder
(H), T1-sequencing ladder (T), and
diethylpyrocarbonate-sequencing ladder (D), respectively.
The gel positions of RNA fragments corresponding to the stem-loops in
pre-U2-54 RNA are indicated.
View larger version (18K):
[in a new window]
Fig. 3.
Schematic compilation of the data obtained
from chemical and enzymatic probing of pre-U2-54 RNA.
A, the sites of nuclease V1 cleavage. Arrows with
open boxes denote weak cleavages, and arrows with
shaded boxes indicate sites of moderate cleavages.
B, site of cleavage by Pb2+
(arrowheads), RNase T1 (arrows with
diamonds), and RNase T2 (arrows with
circles). Open symbols denote weak cleavage
sites, shaded symbols denote moderate cleavage sites, and
black symbols denote strong cleavage sites.
View larger version (15K):
[in a new window]
Fig. 4.
Structure probing of the 3' end of
full-length human pre-U2 RNA. The full-length wild-type
human pre-U2 RNA was transcribed from plasmid pMRG3U2, 3' end-labeled,
and subjected to Pb2+ and nuclease cleavage.
Arrows with boxes indicate sites of nuclease VI
cleavage, arrows with diamonds indicate RNase T1,
arrows with circles indicate RNase T2, and
arrowheads indicate Pb2+. Open symbols
denote weak cleavage sites, shaded symbols denote moderate
cleavage sites, and black symbols denote strong cleavage
sites.
We next investigated the 3' end structure of a previously described
mutant of pre-U2 RNA (pre-U2-75) that is deficient in 3'-processing
(17). U2-75 has an altered 3' end in which the previously contemplated
pseudoknot would not form. As can be seen in Fig.
5, even though U2-75 is not processed
efficiently (17), its 3' end nevertheless has a minihelical structure.
Based on this finding, we went on and determined the 3' end of another mutant, pre-U2-80, which has a compensatory base-pairing mutation designed to restore the aforementioned hypothetical pseudoknot and
which is processed nearly as efficiently as wild-type pre-U2 RNA (17).
As can be seen in Fig. 6, the 3' end of
pre-U2-80 has a structure very different from the 3' end of
pre-U2-54. Instead of stem-loop V, the 3' end forms a base-paired
configuration with an internal region near the 5' base of stem-loop
III.
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The results of this study provide an important step in defining the molecular structure of the 3' end of human pre-U2 RNA and also offer new insights into its 3'-processing reaction. The major conclusion is that the 3'-terminal nucleotides of human pre-U2 RNA form a stable minihelix. Such a structure had been suggested previously by computational RNA-folding algorithms1 but could not be singularly considered because of a then-plausible alternative structure in which the pre-U2 RNA 3' tail was potentially engaged in a pseudoknot with a single-stranded region lying just upstream from the 5' edge of stem-loop III (see Fig. 10 in Ref. 17). The second conclusion from this study is that the stem-loop V minihelix is not essential for 3'-processing, because it is present in a processing-defective mutant, viz. pre-U2-75, and is absent from a mutant that does process well, viz. pre-U2-80. However, in the present study, the structures of the 3' end of pre-U2 RNA were studied in the absence of cellular proteins including the processing activity itself, and it is possible that in vivo the 3' ends of pre-U2-55 and pre-U2-80 would adopt different structures. This notwithstanding, the results of this study are compatible with the idea that the 3' minihelix has some other function in U2 RNA biosynthesis of which the following examples are the two most plausible possibilities. One possibility is that this structure serves as a recognition element for the machinery that cleaves pre-U2 RNA from the initial U2 RNA primary transcript. It is known that the primary transcript of human U2 RNA extends 250 nucleotides or more beyond the 3' end of the penultimate pre-U2 RNA molecule (36). It is not known whether the sequence that forms stem-loop V in the penultimate pre-U2 RNA has the same structure in the primary transcript as opposed to being single-stranded because of other dominant secondary structure or by being itself tied up in a longer range structure. Nonetheless, it is conceivable that the stem-loop V sequence does adopt the same minihelical structure and thus potentially plays a role in the 3' cleavage of the primary transcript to form pre-U2 RNA.
A second and not mutually exclusive possibility is that the 3' minihelix of pre-U2 RNA plays a role in its nuclear export. This idea is based on the indication in an earlier study that a mutant pre-U2 RNA, which could not have possibly formed the presently defined 3' minihelix, was exported less efficiently from the nucleus to the cytoplasm (17). Moreover, a recent investigation has revealed that 3' minihelix structures are essential for the nuclear export of other small RNAs (37), and 3' minihelix extensions built onto chimeric tRNA-ribozyme RNAs have also been shown to enhance export from the nucleus (38).
The next step in understanding the structure of pre-U2 RNA will
probably come from crystallography, because pre-U2 RNA (~200 nucleotides) is considerably beyond the size of RNA that can be studied
by NMR at present. Crystallization efforts using mature-length U-small
nuclear RNAs have not yielded diffracting crystals so far, but such
efforts should certainly be pursued and encouraged. Meanwhile, our
present study reinforces the continuing important role of chemical and
nuclease cleavage based methods for RNA structure determination
(34).
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FOOTNOTES |
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* This work was supported by grants from the French Centre National de la Recherche Scientifique and the French Ministère de la Recherche et de l'Enseignement Supérieure (to A. M. and C. B.) and National Institutes of Health Grant GM-21595 (to T. P.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Present address: Unité Mixte Recherche 5099 CNRS-Université Paul Sabatier Toulouse III, Laboratoire de Biologie Moleculaire Eucaryote du CNRS, 31062 Toulouse, France.
Present address: Impact Biosciences, Boulder, CO 80303.
** Present address: Whitehead Institute for Biomedical Research, Cambridge, MA 02142.
To whom correspondence should be addressed. Tel.: 508-856-8667;
Fax: 508-856-8668; E-mail: thoru.pederson@umassmed.edu.
Published, JBC Papers in Press, April 15, 2002, DOI 10.1074/jbc.M202258200
1 M. R. Jacobson and T. Pederson, unpublished results.
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Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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