Department of Cell Biology, Harvard Medical
School, Boston, Massachusetts 02115
Received 31 July 2000/Returned for modification 14 September
2000/Accepted 5 December 2000
Specific mechanisms must exist to ensure fidelity in selecting the
AG dinucleotide that functions as the 3' splice site during the second
transesterification step of splicing. Here we show that the optimal
location for this AG is within a narrow distance (19 to 23 nucleotides
[nt]) downstream from the branch point sequence (BPS). Contrary to
previous expectations, AGs located less than 23 nt from the BPS are
always recognized, even when a second AG located more optimally
downstream is used in the transesterification reaction. Indeed, the AG
closest to the BPS actually dictates the precise location of the AG
that engages in the reaction. This mechanism, in which the AG is
identified by a general localization step followed by a precise
localization step, may be used to achieve fidelity while allowing
flexibility in the location of 3' splice sites.
 |
INTRODUCTION |
The first AG dinucleotide located
downstream from the branch site is generally chosen as the 3'-splice
site during catalytic step II of splicing (reviewed in references
2, 18, and 26). How this AG is selected from among other
AGs in the RNA is not known. One proposal is that the AG is selected by
a linear scanning mechanism (9, 15, 21, 22). According to
this model, a spliceosome component begins at the branch site and scans
downstream to the first AG. A related model proposes that the AG is
selected via a threading mechanism, in which a 5'-to-3' search is also involved (1, 3). In Saccharomyces cerevisiae,
simple scanning is not thought to occur because distal AGs can
outcompete proximal AGs depending on the sequence context
(19). In addition, insertion of secondary structure
between the branch point sequence (BPS) and the AG does not inhibit
step II (8, 13). Finally, step II efficiency is optimal
over a narrow BPS-to-AG distance (13 to 22 nucleotides [nt])
(16).
In contrast to that in S. cerevisiae, it is still widely
believed that 3' splice site selection in metazoans involves linear scanning (2). Indeed, despite the high evolutionary
conservation of the splicing pathway, it has been proposed that the
mechanism of 3' splice site selection might diverge in yeast and
metazoans (1, 3, 21, 22, 26). Among the evidence in favor
of the scanning and threading models in metazoans are the observations that the first AG downstream of the BPS is selected in
cis-splicing assays (reviewed in reference 26)
and that, in a trans-splicing assay, the 5'-most AG on a 3'
RNA substrate is selected (1). Step II is also blocked by
hairpin loops inserted between the BPS and AG, consistent with stalling
of a scanner on the secondary structure (3, 15, 22). An
alternative model to scanning proposes that the 3' splice site is
selected by a BPS-to-AG measuring mechanism. This model is based on the
observation that both the sequence and the distance between the BPS and
AG affect the efficiency of step II (5). In addition, U5
snRNP proteins contact the intron prior to step II, spanning the
distance (~25 nt) from the BPS to the AG (5). Thus, the
AG may be specified by its location relative to the U5 snRNP binding
site (5).
The essential splicing factors Slu7 and human Slu7 (hSlu7) have been
shown to play important roles in AG selection during step II. In yeast,
Slu7 interacts genetically with U5 snRNP and is the last known factor
to associate with the 3' splice site prior to catalysis
(11). We recently used pre-mRNAs containing duplicate AGs
to analyze AG selection in nuclear extracts lacking hSlu7
(6). This study revealed that hSlu7 is required for
selection of the correct AG and defined two steps in AG selection. To
gain further insight into the mechanism for AG selection, we have now used normal nuclear extracts to analyze the pre-mRNAs containing duplicate AGs, as well as pre-mRNAs containing single AGs. These data
do not support simple scanning or measuring models for AG selection.
Instead, the data reveal a complex, multistep mechanism that may be
necessary for achieving the high levels of fidelity required for AG selection.
| |
MATERIALS AND METHODS |
Plasmids.
Plasmids encoding pre-mRNAs were constructed by
inserting oligonucleotides into the HindIII and
AccI sites of pAdML (12) to replace the
sequences from the BPS to the AG dinucleotide. Beginning with the
branch site adenosine (in lowercase), the sequences in this region are
the following: 15/18, 5'-aCUUUUUUUCUUUCAGCAG-3'; 15/19,
5'-aCUUUUUUUCUUUCAGUCAG-3'; 15/20,
5'-aCUUUUUUUCUUUCAGUUCAG-3'; 15/21,
5'-aCUUUUUUUCUUUCAGUUUCAG-3'; 16/19,
5'-aCUUUUUUUCUUUUCAGCAG-3'; 16/20,
5'-aCUUUUUUUCUUUUCAGUCAG-3'; 16/21:
5'-aCUUUUUUUCUUUUCAGUUCAG-3'; 16/22,
5'-aCUUUUUUUCUUUUCAGUUUCAG-3'; 17/20,
5'-aCUUUUUUUCUUUUUCAGCAG-3'; 17/21,
5'-aCUUUUUUUCUUUUUCAGUCAG-3'; 17/22,
5'-aCUUUUUUUCUUUUUCAGUUCAG-3'; 17/23,
5'-aCUUUUUUUCUUUUUCAGUUUCAG-3'; 18/21,
5'-aCUUUUUUUCUUUUUUCAGCAG-3'; 18/22,
5'-aCUUUUUUUCUUUUUUCAGUCAG-3'; 18/23,
5'-aCUUUUUUUCUUUUUUCAGUUCAG-3'; 18/24,
5'-aCUUUUUUUCUUUUUUCAGUUUCAG-3'; 19/22,
5'-aCUUUUUUUCUUUUUUUCAGCAG-3'; 20/23,
5'-aCUUUUUUUCUUUUUUUUCAGCAG-3'; 23/26,
5'-aCUUUUUUUCUUUUUUUUUUUCAGCAG-3'; 27/30,
5'-aCUUUUUUUCUUUUUUUUUUUUUUUCAGCAG-3'; 33/36,
5'-aCUUUUUCUUUUUUUCUUUUUUUCUUUUUUUCAGCAG-3'; 15/18/21,
5'-aCUUUUUUUCUUUCAGCAGCAG-3'; 15GG/18/21,
5'-aCUUUUUUUCUUUCGGCAGCAG-3'; 15GA/18/21,
5'-aCUUUUUUUCUUUCGACAGCAG-3'; 15AG,
5'-aCUUUUUUUCUUUCAG-3'; 18AG,
5'-aCUUUUUUUCUUUUUUCAG-3'; 21AG,
5'-aCUUUUUUUCUUUUUUUUUCAG-3'; 24AG,
5'-aCUUUUUUUCUUUUUUUCUUUUCAG-3'; 30AG,
5'-aCUUUUUUUCUUUUUUUCUUUUUUUUUUCAG-3'; and 40AG,
5'-aCUUUUUUUCUUUUUUUCUUUUUUUCUUUUUUUCUUUUUCAG-3'.
Splicing reactions.
32P-labeled pre-mRNAs (10 ng) were incubated in 25-µl splicing reaction mixtures for 1 h
unless otherwise indicated. Total RNA was prepared and fractionated on
6.5, 7.5, or 15% denaturing polyacrylamide gels. RNAs were quantitated
by phosphorimager (Quantityone; Bio-Rad). The relative use of each AG
is indicated in each figure and is also presented in Table
1. The splicing intermediates and
products were identified by comigration with known markers on the
gels with different percentages of denaturing polyacrylamide.
| |
RESULTS |
AG selection depends on the BPS-to-AG and AG-to-AG distances.
Previous analysis of a pre-mRNA containing duplicated AGs demonstrated
that an AG located 11 nt from the BPS is outcompeted by an AG located
15 nt from the BPS (5). To determine whether an AG at 15 nt is optimally located, we asked whether this AG can be outcompeted by
distal AGs at various positions downstream (see schematic in Fig.
1A). As shown in Fig. 1B, the proximal AG
at 15 nt is efficiently outcompeted by a distal AG located 3 nt
downstream (Fig. 1B, lane 1 [compare levels of distal and proximal
lariat]; see Fig. 1C, lane 1, for mRNAs). However, as the distal AG is
moved further downstream (Fig. 1A), the AG at 15 nt becomes more
competitive until it is used almost exclusively (Fig. 1B and C, lanes 2 to 6). The same pattern of AG selection is observed at late time points
(90 min) in the splicing reaction (data not shown). We conclude that
distal AGs located less than 6 nt downstream from the proximal AG can
efficiently compete with the proximal AG.
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FIG. 1.
Distal AGs can complete with a proximal AG located 15 nt
from the branch site. (A) Schematic of AdML pre-mRNA derivatives. The
proximal AG is 15 nt downstream from the BPS, and the distal AG is
located between 18 and 23 nt from the BPS. The relative use of the
competing AGs is indicated by the thickness of the underlining and is
presented in Table 1. y, pyrimidine. (B) Total RNA from splicing
reactions was isolated and fractionated on a 7.5% denaturing
polyacrylamide gel. The mRNA and lariat introns corresponding to use
of the proximal and distal AGs were identified by comigration with
known markers on gels with different percentages of polyacrylamide
(data not shown). (C) A lighter exposure of the gel in panel B shows
the mRNAs generated using proximal versus distal AGs.
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|
To test whether it is a general rule that distal AGs can compete with
proximal AGs, we examined pre-mRNAs containing a proximal AG at
increasing BPS-to-AG distances and distal AGs at increasing AG-to-AG
distances (Fig. 2). This analysis
revealed that distal AGs can indeed compete with proximal AGs located
16, 17, or 18 nt from the BPS (Fig. 2A, B, and C, respectively). Again,
the AG-to-AG distance must be less than 6 nt in order for the distal AGs to compete. Within this AG-to-AG distance, distal AGs compete better the shorter the BPS-to-AG distance. For example, a distal AG at
an AG-to-AG distance of 5 nt competes optimally when the proximal AG is
at 15 nt and does not compete well when the proximal AG is at 17 or 18 nt (Fig. 1 and 2, lanes 3).
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FIG. 2.
Competition between duplicated AGs depends on the
BPS-to-AG distance and the AG-to-AG distance. Pre-mRNAs contain a
proximal AG at 16 nt (A), 17 nt (B), 18 nt (C), and 19 or 23 nt (D)
from the BPS. In each panel, a distal AG was located from 3 to 6 nt
downstream of the proximal AG. Pre-mRNAs were spliced and analyzed on a
7.5% (A to C) or 6.5% (D) denaturing polyacrylamide gel. Schematics
of the pre-mRNAs are shown in each panel. The relative use of each AG
is indicated by the thickness of the underlining and is presented in
Table 1.
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|
Our assay using duplicated AGs shows that AG selection depends on both
the BPS-to-AG distance and the distance between the two adjacent AGs.
Moreover, the data indicate that there is probably a maximal BPS-to-AG
distance beyond which the proximal AG outcompetes any distal AG. To
find this distance, we increased the BPS-to-AG distance to 19 or 23 nt.
At a BPS-to-AG distance of 19 nt, the distal AG 3 nt away was still
preferentially used (Fig. 2D, lane 1). Efficient use of the proximal AG
was observed only when the AG-to-AG distance was further increased to 4 nt (Fig. 2D, lane 2). Significantly, however, when the BPS-to-AG
distance was 23 nt, a switch occurred. For the first time, the proximal
AG was preferred even when the AG-to-AG distance was only 3 nt (Fig. 2D, lane 3). Moreover, the proximal AG was used exclusively when the
AG-to-AG distance was 4 nt (Fig. 2D, lane 4). We conclude that distal
AGs can no longer outcompete the proximal AG at a BPS-to-AG distance of
23 nt. Together, these data define the maximal BPS-to-AG distance
beyond which a proximal AG can no longer be outcompeted by a distal AG.
This distance is between 19 and 23 nt.
An optimal BPS-to-AG distance for step II.
Our assay using
duplicated AGs indicated that distal AGs can compete when a proximal AG
is located 19 to 23 nt or less from the BPS. The observation that
distal AGs can be used at all suggests that proximal AGs are sterically
hindered by being too close to the BPS. If this is the case, then we
would predict that the efficiency of step II should be impeded when a
single AG is located 19 to 23 nt or less from the BPS. To test this
possibility, we examined pre-mRNAs containing single AGs with BPS-to-AG
distances ranging from 15 to 40 nt (Fig.
3A). As shown in Fig. 3B and C, step II efficiency is indeed impeded when a single AG is located 15 or 18 nt
from the BPS (the level of step II was quantitated on a darker exposure
of the gel [data not shown]). Significantly, however, when the AG is
moved further than 21 nt from the BPS, the efficiency of step II also
progressively decreases (Fig. 3B and C). Thus, these data indicate that
there is an optimal BPS-to-AG distance of between 19 and 23 nt for step
II. Our data with the duplicated AGs indicate that upstream of this
distance AGs are sterically hindered by being too close to the BPS. The
data with the single AGs indicate that beyond this distance AGs are not
used efficiently because they are too far from the BPS. We conclude
that step II is optimally efficient over a very narrow BPS-to-AG
distance (19 to 23 nt). We note that this distance may vary with other
pre-mRNAs due to their different pyrimidine tract sequences. However,
given the consistency of our results, it is likely that the distance range is close to what we have observed with this pre-mRNA.
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FIG. 3.
A BPS-to-AG distance of 19 to 23 nt is optimal for step
II. (A) Schematic of pre-mRNAs containing single AGs located at
increasing distances from the BPS. (B) Pre-mRNAs were spliced for 45 min and analyzed on a 15% denaturing polyacrylamide gel. (C) Step II
efficiency was calculated as the ratio of spliced products (mRNA and
lariat) to splicing intermediates (lariat-exon and exon 1).
|
|
Uncoupling two recognition steps for AG selection.
Further
insight into the mechanism for AG selection came from comparing the
data obtained with the duplicated and single AGs. The single-AG data
show that a BPS-to-AG distance between 19 and 23 nt is optimal. Yet
with the duplicated AGs, a proximal AG located closer to the BPS was
used even when there was a distal AG more optimally located. For
example, at a BPS-to-AG distance of 15 nt, the AG at 15 was selected
over an AG at 21 nt (Fig. 1). Indeed, the location of the proximal AG,
rather than the absolute distance between the BPS and distal AG,
determines whether the distal AG is used (Fig. 1 and 2). Specifically,
at any given BPS-to-AG distance, distal AGs must be located less than 6 nt from the proximal AG in order to be used. Thus, these data suggest
that the proximal AG is recognized, even when it is not used.
To further test this possibility, we asked whether inserting a third AG
upstream of two competing AGs alters the use of the two original AGs
(see schematic in Fig. 4A). As shown in
Fig. 4A (lane 1), the distal AG at 21 nt is used slightly more
efficiently than the proximal AG at 18 nt. When a third AG is inserted
into this pre-mRNA, 15 nt from the BPS, this AG is not used (Fig. 4A, lane 2). Importantly, however, the presence of the AG at 15 nt alters
the pattern of AG selection, as the AG at 18 nt is now exclusively
selected (Fig. 4A, lane 2).
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FIG. 4.
A proximal AG determines whether a distal AG is
selected. (A) An AG at 15 nt determines selection of distal AGs.
Splicing reactions were performed using pre-mRNAs containing duplicate
AGs at 18 and 21 nt from the BPS (lane 1) or containing a third AG
inserted at 15 nt from the BPS (lane 2). Lane 3 shows splicing of
pre-mRNA 15/21, which serves as a marker for the lariat. (B) Other
purine dinucleotides at 15 nt do not affect selection of distal AGs.
Pre-mRNAs are identical to that shown in panel A (lane 2) except that
GG (lane 1) or GA (lane 2) replaces the AG at 15 nt. Pre-mRNAs 15AG and
18AG are markers for the lariats (lanes 3 and 4).
|
|
The observation that AG selection can be switched depending on the
presence or absence of the AG at 15 nt suggests that the AG at 15 nt is
recognized even though it is not used. To further test this
possibility, the AG at 15 nt was replaced with either a GG or a GA.
Significantly, neither of these purine dinucleotides caused a switch to
the AG at 18 nt. We conclude that the AG at 15 nt is specifically
recognized even when it is not used. Thus, our assay using multiple AGs
suggests that there is an initial step in AG recognition that can be
uncoupled from the final selection of the AG.
| |
DISCUSSION |
Previous studies showed that the first AG downstream of the BPS is
normally used as the 3' splice site (reviewed in references 2 and
26). This observation is compatible with several models for 3'
splice site selection. In scanning models, the first AG is used because
a 5'-to-3' linear search is conducted. Our analysis does not support a
simple scanning model. We show that AGs located closer to the BPS than
19 to 23 nt are not efficiently used, most likely due to steric
hindrance from a factor bound at the BPS. Yet, the efficiency of step
II also begins to decrease when AGs are located immediately beyond this
distance. Thus, assuming that scanning does not become inefficient at
the exact point at which it is expected to start (i.e., where steric
hindrance is relieved), the AG is not likely to be selected by simple
scanning. Our data do not address the previously suggested possibility
that scanning is involved in selection of AGs when they are located
farther than the optimal distance from the BPS (4, 9, 15, 21, 22). A simple BPS-to-AG measuring mechanism also does not
explain our data because this model predicts that step II should always occur at an optimally located AG if one is present. However, our data
show that AGs optimally located 19 to 23 nt from the BPS are not always
selected if an upstream AG is present (see below).
An assay using duplicate AGs uncouples multiple AG recognition
steps.
We have used pre-mRNAs containing duplicated AGs as an
assay to gain insight into the mechanism of AG selection. Our results show that an AG located closer than the optimal BPS-to-AG distance (19 to 23 nt) is recognized even when it is not used. Indeed, the proximal
AG determines whether a distal AG will be selected. Thus, these data
reveal two distinct AG recognition events. One possible model to
explain our observations, shown in Fig.
5, is that the initial recognition of the
proximal AG functions to position the splicing machinery on the RNA
downstream of the BPS. The recognition of this positioning AG is
relatively flexible, as it can occur when an AG is suboptimally
located, only 15 nt from the BPS (Fig. 5A). After this initial
positioning of the splicing machinery, selection of the AG that
actually functions in the transesterification reaction (the
transesterification AG) occurs (Fig. 5B). Our data show that selection
of the transesterification AG is more stringent than recognition of the
positioning AG. Specifically, the transesterification AG must be
located within a narrow range of distance (<6 nt) downstream of the
positioning AG. Such a multistep mechanism, which could involve a
flexible ruler followed by a stringent ruler, may accommodate the
variability in the location of 3' splice sites while still ensuring
high fidelity.
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FIG. 5.
Model for AG selection. (A) The proximal AG positions
the splicing machinery downstream of the BPS. Recognition of this
positioning AG is flexible, as this AG can be located outside the
optimal distance for step II (in this example, 15 nt). (B) The AG used
in the transesterification reaction is selected in a second step. This
transesterification AG must be located within a narrow distance (<6
nt) downstream of the positioning AG. If a second AG is not present
within this window, the positioning AG itself is used (but
inefficiently). In the absence of hSlu7, the transesterification AG is
sequestered, and step II is blocked at this location. (C) In the
presence of hSlu7, the sequestered transesterification AG is exposed,
and catalysis of step II (arrow) occurs.
|
|
In naturally occurring pre-mRNAs, closely clustered AGs are not always
present at the 3' splice site. Nevertheless, our assay using the
duplicated AGs uncouples two distinct recognition events that most
likely occur when only a single AG is present. It is possible that the
two events involve more than one factor or a single factor that
undergoes a conformational change. The only proteins known so far to
affect AG selection during step II are Slu7 and hSlu7 (in yeast and
humans, respectively) (6, 10). We analyzed AG selection in
extracts lacking hSlu7 using all of the pre-mRNAs containing the
duplicated AGs (6; our unpublished observations). In
all cases, the AG that was used in normal extracts was specifically not
used in the absence of hSlu7 (whereas incorrect AGs were used). Thus,
the hSlu7 study shows that the transesterification AG is selected
but is specifically sequestered prior to hSlu7 function. hSlu7 is then
required for use of this AG in catalytic step II (Fig. 5C).
One candidate for a factor involved in recognition of the positioning
AG is U2AF35, which recognizes the AG early in metazoan spliceosome assembly (17, 27, 30). However, no
changes in AG selection were observed in extracts lacking U2AF35,
arguing against a role for this protein (K. Chua, R. Reed, and R. Gaur, unpublished data). Previously, it was shown that
recognition of a downstream AG was necessary for step I but that a
proximal AG was used for step II (14, 29). In this case,
it is likely that the first recognition event involved U2AF35. In
contrast to these studies, in our constructs the duplicated AGs both
have effects on step II.
A candidate factor for recognition of the positioning and/or the
transesterification AG is the highly conserved U5 snRNP protein Prp8.
In yeast, Prp8 can be UV cross-linked to the YAG, and Prp8 mutants that
suppress step II defects caused by mutations in the YAG (7, 20,
23-25, 28) have been identified. These and other observations
have led to the proposal that Prp8 may play a role in positioning the
AG for step II (7, 20). This possibility is compatible
with the observation that hPrp8 can be cross-linked on both sides of
the AG at or near the time of step II in metazoans (4).
Slu7 is also synthetically lethal with Prp8 in yeast (11). Thus, it will be interesting to determine how these factors function in
the different events involved in AG selection.
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Abstract
Introduction
