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Molecular and Cellular Biology, December 2000, p. 9225-9235, Vol. 20, No. 24
Verna and Marrs McLean Department of
Biochemistry and Molecular Biology, Baylor College of Medicine,
Houston, Texas 77030
Received 19 April 2000/Returned for modification 7 June
2000/Accepted 19 September 2000
Intronic G triplets are frequently located adjacent to 5' splice
sites in vertebrate pre-mRNAs and have been correlated with splicing
efficiency and specificity via a mechanism that activates upstream 5'
splice sites in exons containing duplicated sites (26).
Using an intron dependent upon G triplets for maximal activity and 5'
splice site specificity, we determined that these elements bind U1
snRNPs via base pairing with U1 RNA. This interaction is
novel in that it uses nucleotides 8 to 10 of U1 RNA and is independent
of nucleotides 1 to 7. In vivo functionality of base pairing was
documented by restoring activity and specificity to mutated G triplets
through compensating U1 RNA mutations. We suggest that the G-rich
region near vertebrate 5' splice sites promotes accurate splice site
recognition by recruiting the U1 snRNP.
Most eukaryotic genes contain
introns which are cotranscriptionally removed from precursor
transcripts by RNA splicing (32). Splicing occurs in a
macromolecular ribonucleoprotein complex termed the spliceosome, the
ordered assembly of which has been characterized in vitro. The initial
metazoan complex, the commitment complex, contains the 5' splice site
recognition factor U1 snRNP, the 3' splice site recognition
factor U2AF, and at least one member of the arginine-serine-rich (SR)
family of splicing factors (14, 28, 36). The second complex,
the prespliceosome, or A complex, is defined by the ATP-dependent
addition of the U2 snRNP (20). As the spliceosome
matures, U1 snRNP at the 5' splice site is replaced by U5 and
U6 snRNPs (31, 41).
Studies investigating the mechanism through which mammalian 5' splice
sites are recognized have implicated SR proteins, hnRNP A1, the U1
snRNP, and the U6 snRNP as important (5, 12,
21, 22, 24, 31, 38). In studies using precursor RNAs containing exons with multiple 5' splice sites, the binding of any one of these
may be determinative for usage. Most studies examining the mechanism
through which duplicated 5' splice sites are recognized have
concentrated on the involvement of SR proteins via their capacity to
bind to either 5' splice site sequences or exon enhancer sequences,
which in turn influence the usage of nearby 5' splice sites (12,
19, 43). These studies have led to models suggesting that the
choice of splice site is dictated by the affinity of SR proteins and
snRNPs for the site. In sites with low affinity, only a
single site is bound, and this is the site used to direct splicing. In
sites with high affinity, double occupation occurs, and the site
proximal to the 3' splice site across the intron predominates
(12).
Intron enhancer sequences can also regulate 5' splice site usage. In
general, when intron enhancer sequences are placed between competing 5'
splice sites, they direct usage to the upstream, distal 5' splice site
(7, 11, 19, 26). In at least one of these cases, in the gene
coding for hnRNP A1, the enhancer element directs upstream 5' splice
site usage without altering the ability of U1 snRNPs to bind
to the competing 5' splice sites, suggesting that 5' splice site usage
is not dominantly determined by the occupancy of splice sites by U1
snRNPs (7). In this same system, the addition of
SR proteins caused a reversal of phenotype such that usage of the
proximal 5' splice site predominated, indicating the possibility of
alternate competing mechanisms of 5' splice site selection. Indeed, it
has been demonstrated in a number of test cases that SR proteins and
hnRNP A1 antagonistically affect 5' splice site recognition and that
subtle changes in the relative concentrations of the two types of
factors can be determinative for 5' splice site recognition (5,
24).
One sequence element frequently found in close proximity to mammalian
5' splice sites is the sequence GGG (26, 30, 35). The
presence of a G triplet in the vicinity of a 5' splice site has been
demonstrated to be of predictive value for identification of exons in
sequenced genomic DNA, which suggests that the recognition of such
sequences by splicing factors may play an important role in 5' splice
site selection in a large number of pre-mRNAs. Indeed, several intron
enhancer sequences that function to activate an upstream exon contain G
triplets (2, 6, 18, 25, 34). We have modeled the function of
G triplets in 5' splice site recognition using an intron containing
multiple G triplets (26). This intron, the second intron of
the human alpha-globin gene, contains multiple G triplets, which we
arbitrarily grouped into three elements designated G1, G2, and G4 (Fig.
1). Maximal in vitro and in vivo activity of the intron is dependent upon the presence of these G triplets, and
we therefore refer to these elements as intronic splicing enhancers.
When the G1, G2, and G4 elements were mutated, in vivo splicing of the
alpha-globin intron was reduced from near 100% to about 45%
(26).
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
An Intronic Splicing Enhancer Binds U1
snRNPs To Enhance Splicing and Select 5' Splice
Sites
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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FIG. 1.
Sequence of the second intron of the human alpha-globin
gene. G elements are denoted, as well as mutants used in this and
previous studies (26).
The alpha-globin G triplet elements also affect splice site selection. In precursor RNAs containing two identical 5' splice sites separated by wild-type G1 and G2 elements, the distal or 5'-most splice site was used almost exclusively. Progressive mutation of the G triplets in these elements resulted in a switch from the distal to the proximal 5' splice site (26). The elements act additively to increase activity and define 5' splice sites, suggesting that only a single element need be occupied by a trans-acting factor (26).
In the present study, we provide evidence that the alpha-globin G-rich elements interact directly with the RNA component of the U1 snRNP by base pairing with nucleotides 8 to 10 of U1 RNA. This interaction is novel in that it does not require nucleotides 2 to 7 of U1 RNA, which are generally believed to interact with 5' splice site sequences. Replacement of one or more G triplets in alpha-globin substrates containing duplicated 5' splice sites with mutant sequences complementary to altered U1 RNAs permitted substantial, but not total, rescue of the 5' splice site selection phenotype, suggesting that the ability of the G triplets to bind U1 RNA can account for most, but not all, of the selectivity afforded by G triplets. Interestingly, increased concentrations of SR proteins were unable to modulate 5' splice site choice in alpha-globin substrates containing duplicated 5' splice sites. Given the prevalence of G triplets in the region immediately downstream of vertebrate 5' splice sites, it is likely that interaction between G-rich elements and U1 RNA represents a common mechanism for recognizing 5' splice sites in vertebrate pre-mRNAs.
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MATERIALS AND METHODS |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Plasmids, in vitro transcription, and HeLa cell transfection. Alpha-globin sequences were inserted into pCDNA3 (Invitrogen) for in vitro and in vivo transcription as described previously (26). The wild-type and mutant G1-G2 RNA expression plasmids used to generate psoralen cross-linking probes were constructed by ligating annealed complementary oligonucleotides into pUC19. The T7 promoter used for in vitro transcription of these RNAs was positioned so that transcription initiated at the first G nucleotide of the G1 element. The wild-type U1 RNA expression vector was kindly provided by Alan Weiner (University of Washington) and has been previously described (42). Mutations were introduced into this U1 plasmid by PCR-mediated mutagenesis. Mammalian expression vectors coding for human ASF-SF2 and SC35 were kindly provided by James Manley (Columbia University) and have been described previously (38). HeLa cell maintenance, transfection, RNA isolation, and reverse transcription (RT)-PCR analysis were done as described previously (26). Cloned RT-PCR products were sequenced to confirm splice site usage. Capped [32P]GTP-labeled in vitro transcripts were prepared from linearized templates as described previously (26).
Psoralen cross-linking. For psoralen cross-linking, 150,000 cpm of 32P-labeled RNA was incubated for 5 min at 30°C under splicing conditions described previously (26) with the addition of psoralen (HRI Associates) to a final concentration of 0.02 µg/µl. Reactions were illuminated at 365 nm for 15 min to generate RNA-RNA cross-links, deproteinated, and analyzed by electrophoresis on 5 or 8% polyacrylamide-8.3 M urea gels as indicated in the figure legends. For immunoprecipitation, cross-linking reaction mixtures were added to beads bound by an antibody to the U1 70,000-molecular-weight protein (U1 70K) in 500 µl of cold NET buffer (150 mM NaCl, 50 mM Tris[pH 7.5], 0.05% NP-40), rocked at 4°C for 12 h, and washed extensively with NET buffer. The bound RNAs were purified from the washed beads and analyzed by electrophoresis on 5% polyacrylamide-8.3 M urea gels.
Oligonucleotide-mediated cleavage and blocking of U1 snRNA. The 5' end of U1 snRNA was removed by preincubating reaction mixtures with 2 µg of a DNA oligonucleotide complementary to U1 snRNA nucleotides 1 to 11 and 2 U of RNase H (Promega) at 30°C for 15 min prior to the addition of substrate RNA. Loop 2 of U1 snRNA was removed after cross-linking and deproteinization by incubating the cross-linked RNAs with 2 U of RNase H and 2 µg of a DNA oligonucleotide complementary to U1 RNA nucleotides 64 to 75 for 30 min at 37°C. RNAs were purified by proteinase K digestion and phenol-chloroform extraction, precipitated with ethanol, and analyzed by denaturing gel electrophoresis and primer extension.
The 5' end of U1 snRNA was blocked with a 2'-O-methyl RNA oligonucleotide (Oligos Etc.) complementary to U1 positions 1 to 11 which was added to cross-linking reactions just prior to substrate addition. A similar oligonucleotide complementary to U3 RNA was used as a control.Primer extension mapping of psoralen cross-links. To generate enough cross-linked RNA for primer extension analysis, psoralen cross-linking reactions were scaled up to 200 µl (80 µl of nuclear extract) and the labeled G1-G2 substrate was supplemented with unlabeled substrate. The cross-linked RNAs were purified from 5% polyacrylamide denaturing gels and used for primer extension as described previously (16). The sequence of the U1 loop 2 primer used in this analysis was 5' CGGAGTGCAATG 3'. The primer extension products were separated on 7% polyacrylamide-8.3 M urea DNA sequencing gels and visualized by autoradiography.
Glycerol gradient fractionation. Psoralen cross-linking reactions were performed in a 200-µl volume using 80 µl of nuclear extract. The reaction mixtures were layered onto 10 to 30% glycerol gradients prepared in a buffer which contained 2 mM MgCl2, 20 mM Tris (pH 7.9), and 80 mM KCl. The gradients were centrifuged for 16 h at 33,000 rpm in an SW40Ti rotor. Fractions (0.5 ml) were collected, treated with proteinase K, and phenol-chloroform extracted. RNAs were recovered by ethanol precipitation and separated on 8% polyacrylamide-8.3 M urea gels. The distribution of nuclear RNAs across the gradients was monitored by silver staining, and the free and cross-linked 32P-labeled G1-G2 RNAs were visualized by autoradiography.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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SR proteins are ineffective in regulating 5' splice site usage in
human alpha globin.
As shown in Fig. 1, the second intron of the
human alpha-globin gene contains multiple G triplets, four of which are
close to the 5' splice site and which we have grouped into two elements termed G1 and G2. Introduction of a single mutant G into either of
these elements both reduces splicing efficiency in vivo and alters 5'
splice site usage when substrates are created in which the region
around the 5' splice site (including the G1 and G2 elements) is
duplicated (the duplication construct is diagramed in Fig.
2B). When the elements are wild type,
over 90% of the spliced RNA product arises from use of the upstream
distal 5' splice site. Mutation of both G1 and G2 drops usage of this
site to 25%, indicating the importance of these sequences for splice site specificity (26). The observed additivity of the
ability of the elements to dictate distal usage suggested recognition of individual G triplets by a trans-acting factor.
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An RNA containing the G1 and G2 elements interacts with the U1 snRNP. Previous experiments had indicated that the G-rich enhancer sequences operated prior to the formation of the spliceosomal A complex, suggesting that they might interact with a factor important for initial complex formation and which could participate in 5' splice site specificity. This possibility led us to ask if these elements interact directly with U1 snRNA. The base pairing of U1 RNA to substrate RNAs can be monitored by RNA-RNA psoralen cross-linking (39). In this assay, 32P-labeled substrate RNAs are incubated with nuclear extract under splicing conditions in the presence of the RNA cross-linking agent psoralen. Samples are illuminated with 365-nm-wavelength light to induce RNA-RNA cross-linking and analyzed on denaturing polyacrylamide RNA gels. Cross-linked RNAs appear as radiolabeled species migrating more slowly than substrate RNA.
We applied this assay to short RNAs containing either wild-type or mutant G1 and G2 elements, but no splice sites (referred to here as the G1-G2 RNA, representing nucleotides 10 to 36 of the second intron of the human alpha-globin gene [Fig. 3]). We noticed a prominent band migrating above wild-type but not mutant substrate after cross-linking (Fig. 3B, lanes 2 and 3). This band was both psoralen and extract dependent (data not shown), suggesting that it represented cross-linking of the wild-type G1-G2 RNA to a nuclear RNA and not inter- or intramolecular cross-linking of substrate RNA. It was also ATP dependent (not shown). The gel migration of the band was severely retarded in the gel compared to the migration of free substrate, immediately suggesting that it represented an adduct between substrate RNA and a nuclear RNA. The migration of the band was also retarded with respect to the position of free U1 snRNA (Fig. 3B, compare the cross-link position in lane 2 to the stained U1 RNA position in lane 1), suggesting that it could have resulted from cross-linking between substrate and U1 RNAs.
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Interaction of U1 snRNA and G1-G2 RNA does not require
the complete 5' end of U1 snRNA.
The 5' end of U1
snRNA (nucleotides 1 to 10 [Fig.
4A]) is known to interact with 5' splice
sites through base pairing (29, 42), and removal or
sequestration of these nucleotides inhibits the in vitro splicing of
most substrates (1, 3, 37). To determine if this 5' sequence
is required for the interaction of U1 RNA with the G1-G2 RNA, we
performed the psoralen cross-linking assay using nuclear extracts in
which the 5' end of U1 had been sequestered with a
2'-O-methyl RNA oligonucleotide complementary to U1
nucleotides 1 to 11. This treatment effectively inhibited the assembly
of spliceosomal complexes on an adenovirus splicing substrate,
presumably by preventing U1 snRNA from interacting with the
5' splice site (data not shown). The same concentration of a control
oligonucleotide complementary to U3 RNA had no effect (data not shown).
The anti-U1 but not the anti-U3 2'-O-methyl RNA inhibited
formation of the psoralen cross-link between U1 RNA and G1-G2 RNA (Fig.
4B). This result supports the observation that U1 snRNA can
be psoralen cross-linked to the G1-G2 RNA. Furthermore, it implicates
U1 nucleotides 1 to 11 as critical for the interaction.
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The site of cross-linking to G triplets maps to uridine residues 10 and 22 in U1 snRNA. To exactly determine the position of the cross-link(s), we performed primer extension using purified cross-linked product RNA and an oligonucleotide primer complementary to U1 loop 2 (U1 nucleotides 64 to 75 [Fig. 4A]). This assay is based on the observation that reverse transcriptase pauses at cross-linked nucleotides. Using this approach, we identified two RT stops at uridine 10 (U10) and U22 in U1 snRNA (Fig. 4F, lanes 3 and 4, and 4A) that were not observed when total nuclear RNA was extended (Fig. 4F, lanes 1 and 2). These stops likely represent cross-linked nucleotides. The U10 and U22 cross-links were not observed when the G1-G2 RNA was omitted form the psoralen cross-linking reaction, suggesting that the stops do not result from intramolecular cross-links in the U1 snRNA (not shown). The U10 cross-link is consistent with our 2'-O-methyl and RNase H cleavage data and supports a model in which a G triplet in the substrate RNA interacts with U1 nucleotides 8 to 10 (CCU). The cross-link involving U22 might result from additional, novel interactions of the substrate with U1, or perhaps the unpaired U22 is spatially positioned so that it is cross-linked to substrate bound near nucleotides 8 to 10.
The G1-G2 RNA exists in a large complex.
The U1 70K
immunoprecipitation data (Fig. 3C) indicated that the G1-G2 RNA
interacts with U1 snRNA in a complex which contains the U1
70K protein. We were interested in knowing the size of this complex and
if it was likely to contain intact U1 snRNP. To address this
issue, cross-linking reaction products were fractionated on 10 to 30%
glycerol gradients, and the fractionated RNAs were analyzed by
electrophoresis on denaturing gels. The position of U1 snRNA
in the gradient was identified by silver staining (Fig. 5A), and the cross-link was detected in
the same gel by autoradiography (Fig. 5B). This analysis indicated that
the cross-linked species containing the G1-G2 RNA and the U1 RNA
resides in a complex large enough to contain a complete U1
snRNP. In fact, the observed complex peak is slightly larger
than the peak of bulk U1 snRNA, suggesting the involvement of
additional yet-unidentified components.
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Targeting modified U1 snRNAs to a mutant G1 element.
The experiments described above indicated that U1 snRNPs bind
to G-rich intronic elements in the alpha-globin intron. We next wanted
to determine if U1 binding to these elements accounts for their effects
on splicing. Our approach was to attempt to mimic the binding of U1 RNA
to a G element by creating a mutant G element and a mutant U1 RNA
capable of base pairing with the created element. The modified U1
snRNAs generated in this study are summarized in Table
1. A mutant alpha-globin in vivo splicing
substrate was created in which the wild-type G1 element GGGCCGGG
was altered to UGACCAGC (the G1 mutant diagrammed in
Fig. 1). This alteration depressed both activity and 5' splice site
specificity to an extent sufficient to reproducibly observe
complementation should it occur. The sequence of the 5' end of U1 was
changed to be complementary to the mutant G1 element (denoted G1
MutASU1) and coexpressed in HeLa cells along with the mutant
alpha-globin gene. We also generated a modified U1 complementary to the
wild-type G1 element as a control (denoted G1WtAs U1 snRNA).
The sequences of the 5' ends of these U1 RNAs are shown in Fig.
6A. This approach, which bypassed the
natural mechanism for bringing U1 to the enhancer, allowed us to ask if
U1 binding alone is sufficient to mimic the activity of a G1 element.
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Rescue of distal splice site selection by U1 snRNAs
complementary to mutant G elements.
Our first functional test of
the G1MutAS U1 was to determine its effect on 5' splice site
utilization using a 5' splice site duplication substrate similar to
that described in the legend to Fig. 2 in which both of the wild-type
G1 or G2 elements between the 5' splice sites were mutated as described
in the previous section. The role of U1 RNA binding to these elements
was tested by attempting to mimic distal splice site utilization in the
mutant duplication minigene by expression of complementary mutant U1 RNAs (Fig. 7). The potential base pairing
of these modified U1 snRNAs and substrate elements is shown
in Table 1. HeLa cells were cotransfected with vectors expressing the
mutant duplication minigene and either the wild-type G1WtAS, or G1MutAS
U1 snRNAs shown in Fig. 6A and Table 1. The potential
base-pairing interactions between the G1MutAS U1 RNA and the mutant G1
element in this substrate are modeled in Fig. 7B and in Table 1. As
shown in the RT-PCR assay in Fig. 7C, the proximal site was preferred
when either wild-type U1 (25%; SE, 1.9) or the G1WtAs U1 was
cotransfected with the G(1+2) mutant duplication substrate (lanes 2 and
3). This is the splicing pattern previously observed for this substrate in the absence of mutant U1 RNA expression (26). In
contrast, the distal site was activated in a dose-dependent manner when the G1MutAS U1 RNA that can bind mutant G1 elements was coexpressed (Fig. 7C, lane 4, and E). When 2 µg of G1MutAS DNA was used for transfection, the distal splice site was utilized in 44% (SE, 1.2%)
of the spliced transcripts. This is comparable to what was observed for
a substrate with a wild-type G1 element in this position (26). Thus, expression of a suppressor U1 RNA complementary to a mutant G element restored the 5' splice site specificity of the
element.
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Rescue of mutant alpha-globin splicing efficiency with a U1
snRNA complementary to the mutant G1 element.
We next
asked if the in vivo splicing efficiency of a substrate with a single
5' splice site and a mutant G1 element could be rescued by
cotransfection with the G1MutAS U1 snRNA. A previously characterized alpha-globin splicing substrate containing mutant G1 and
G4 elements (Fig. 8A), which is
incompletely spliced after transfection because of the mutated G
elements (26), was expressed in HeLa cells cotransfected
with either PUC 19 DNA or plasmids expressing wild-type or G1MutAS U1
snRNA. As shown in Fig. 8C, the percentage of accumulated
transcripts that were spliced was higher in RNA from cells expressing
the G1MutAS U1 snRNA (88%; SE, 1.2 [lane 3]) than in the
PUC- (80%; SE, 1.1 [lane 1]) or wild-type U1 (74%; SE, 0.9 [lane
2])-transfected cells. Therefore, the splicing defect attributed to
the mutant G1 element was reversed by experimentally restoring its
interaction with U1 snRNP.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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We have previously identified G-rich elements in the second intron of the human alpha-globin gene that are important for efficient splicing in vivo and in vitro. These elements also promote distal 5' splice site selection when positioned between duplicated, identical 5' splice sites and affect in vitro spliceosome assembly (26). The minimal discernible element is the simple sequence GGG, and these elements function additively (26).
Interestingly, despite their purine content, G-triplet elements appear not to be binding sites for SR proteins (Fig. 2). In repeated attempts to alter either activity or splice site specificity in the alpha-globin intron through the addition of SR proteins, we were unable to alter either by increasing the concentration of a number of SR proteins, including the SR proteins ASF-SF2 and SC35 that dominantly cause recognition of proximal 5' splice sites in a number of systems (5, 38). Given the intronic location of the elements, the preference of the G triplets for activating 5' splice sites residing upstream of the elements, and the observed preference of SR proteins for binding to exonic rather than intronic enhancer sequences, it is perhaps not surprising that G-triplet elements do not operate via SR-mediated pathways. Furthermore, the observed refractory response to SR proteins suggests a difference between recognition of G triplets and recognition of the similar sequence intron enhancer (UAGAGU) from the hnRNP A1 gene that is recognized by hnRNP A1 (7).
Because these elements affect early spliceosome assembly and 5' splice site selection, we postulated that they might bind a factor that recruits U1 snRNP to the 5' splice site to initiate commitment complex formation. Surprisingly, we found that the G-rich elements themselves interact with the U1 snRNP and that the U1 snRNA can be cross-linked to these elements in the presence of the RNA cross-linking agent psoralen (Fig. 3). The interaction of U1 snRNP with these elements is different from other U1-substrate interactions in that it does not require nucleotides 2 to 7 of U1 snRNA (Fig. 4). The nature of this interaction remains under investigation, and the contributions of additional factors are possible.
We tested the functional significance of this interaction by targeting
modified U1 snRNAs to mutant G-rich elements. Expression of
these modified U1 RNAs in transfected HeLa cells was able to rescue
both the splicing efficiency and splice site selection properties of
the G-rich element. The rescue U1 increased the splicing efficiency of
a substrate with a single 5' splice site and a mutant G-rich element
(Fig. 8) and restored distal splice site utilization in a substrate
with duplicated 5' splice sites separated by mutant G-rich elements
(Fig. 7). The degree of rescue imparted by these engineered U1 genes
was significant. Mutation of the G1 element positioned between two
alpha-globin 5' splice sites reduced distal 5' splice site use by 48%
(26), while expression of a U1 complementary to this mutant
G1 element (G1MutAS U1) restored distal splice site use by 44% (Fig.
7C). This represents a 90% rescue of G1 activity. A similar rescue
efficiency was seen when a complementary U1 RNA was targeted to a
mutant G2 element (Fig. 7C). These rescue U1s, however, had extensive
complementarity to their target elements. In contrast, the G1-1 U1 RNA
complemented only the first 3 nucleotides of a mutant G1 element.
Mutating these 3 nucleotides of the G1 element in an otherwise
wild-type alpha-globin intron reduced distal 5' splice site usage by
18% (26). The G1-1 U1 was able to rescue distal 5' splice
site usage by 8% (Fig. 7D). This represents a rescue efficiency of
about 50%. Given the low 
G associated with a 3-bp
interaction (Table 1), the degree of compensation observed with the
G1-1 U1 strongly supports the functionality of the U1 RNA-G-triplet
interaction proposed in this study.
These results indicate that the interaction of U1 with G-rich elements is functionally relevant and represents a mechanism for loading U1 snRNP onto splicing substrates. Thus, G triplets bind U1 snRNPs similarly to the SR-induced binding of U1 snRNPs to regions near 5' splice sites; in the former case, however, binding favors usage of a 5' splice site upstream of the U1 binding site, suggesting fundamental differences between the binding of U1 snRNPs to either exon enhancers or 5' splice sites and the binding to an intronic GGG-containing enhancer.
The interaction of U1 with these elements likely initiates commitment complex assembly and nucleates spliceosome formation. We do not know if the U1 snRNPs that interact with an intronic G-rich element also base pair with the 5' splice site during splicing. We do know that such base pairing is not required, because the rescue U1 RNAs used in our cotransfection experiments have modified 5' ends that are unable to base pair with the 5' splice site. It is plausible that the U1 bound to the intronic elements recruits a second U1 via protein-protein interactions, but it is more likely that a second U1 is not required. There is ample evidence indicating that U1 base pairing with the 5' splice site is not required for efficient, accurate splicing. First, U1 is not necessarily required for splicing. Its function can be replaced by high concentrations of SR proteins which subsequently recruit U4, -5, and -6 snRNPs (9, 37). In some introns, the U4, -5, and -6 tri-snRNP can recognize the 5' splice site in the absence of U1 or high concentrations of SR proteins (10). Furthermore, Cohen and coworkers have shown that U1 can act at a distance from the 5' splice site to promote recognition of the 5' splice site in the absence of base pairing (8). In their experiments, a weakened 5' splice site unable to interact with U1 was rescued by targeting U1 snRNPs (termed shift U1) to introduced sequences upstream or downstream of the mutant 5' splice site. This effect was dependent upon the ability of U6 snRNA to base pair with the remaining nonmutated sequences of the 5' splice site (21). It is unclear if U1 snRNPs affect splicing by a similar mechanism in the natural alpha-globin pre-mRNA. There are, however, fundamental differences between alpha globin and the shift U1 system. Most prominently, U1 binding to G elements in alpha globin affects the activities of strong, wild-type splice sites while the shift U1s activated incapacitated sites via binding of U6 snRNA.
We suggest that our observations with alpha globin can be extended to other introns with abundant G triplets (26, 30, 35). In small introns, G-rich elements are proposed to interact with U1 snRNPs as an initial step in intron definition, maximizing mRNA production. In large introns, we propose that G-rich elements downstream of 5' splice sites serve to load U1 snRNP onto the 5' end of the intron and promote recognition of the 5' splice site. Such a mechanism would increase the information content of authentic 5' splice sites and help the splicing machinery accurately identify legitimate exons. The G-rich sequences at the 5' and 3' ends of large introns have also been reported to bind hnRNP A1 (4). Blanchette and Chabot have proposed that interactions between hnRNP A1 molecules bound at these sites function to bring the ends of the intron together for splicing (4). The interaction of G-rich elements with U1 and hnRNP A1 are most likely temporally separated and compatible.
The binding of U1 snRNPs to G-rich sequences is not fully understood. It is unclear if the base-pairing interaction detected by psoralen cross-linking is the basis of the interaction or if it represents a footprint that allowed us to visualize the interaction. Our observation that U1 remains bound to the G1-G2 RNA throughout lengthy immunoprecipitation experiments (precipitated, free substrate in Fig. 3C, lane 1) in the absence of cross-linking demonstrates that the interaction of U1 snRNP and the G1-G2 RNA is quite stable. This suggests that interactions in addition to the brief base pairing documented here may be involved. If an additional factor is involved, however, such a factor may be U1 snRNP associated, because purified U1 snRNP recognizes and binds to the G1-G2 RNA (unpublished results). The involvement of an additional component is supported by the fact the cross-linking reported here is ATP dependent. It is reasonable to speculate that this interaction is mediated by a phosphorylation event, or perhaps a helicase activity.
The U1 snRNP has several roles in RNA maturation beyond base pairing with the 5' splice site. The U1 snRNP is found in complexes assembled on exonic splicing enhancers (36, 40), and it is a component of a polyadenylation enhancer in the human calcitonin gene (23). U1 binding to sequences upstream of polyadenylation signals in terminal exons can inhibit polyadenylation (15, 17), and the binding of U1 to pseudo-5' splice sites contributes to tissue specific splicing of Drosophila P element transcripts (33). U1 is also a component of a complex that assembles on the negative regulator of splicing element in the Rous sarcoma virus, where it is proposed to compete for binding with U11 snRNA (27). Finally, we provide evidence that U1 snRNP interacts with intronic G-rich elements to promote efficient splicing and accurate splice site selection. Considering this ever-expanding list of functions, it is not surprising that U1 is the most abundant of the spliceosomal snRNPs.
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ACKNOWLEDGMENTS |
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We thank the members of the Berget laboratory, especially Leslie Landree and Hua Lou, for valuable discussions throughout the course of this project. We recognize Valerija Vitkauskas, an undergraduate summer student, for performing several transfection experiments, and we acknowledge the dedicated technical assistance of Wade Wilson. We thank James Manley (Columbia University) for providing us with ASF-SF2 and SC35 expression plasmids, and we acknowledge Alan Weiner (University of Washington) for providing the wild-type U1 snRNA gene used in these studies.
This work was supported by grant RO1 GM38526 to S.M.B.
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FOOTNOTES |
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* Corresponding author. Mailing address: Verna and Marrs McLean Department of Biochemistry, Baylor College of Medicine, Houston, TX 77030. Phone: (713) 798-4622. Fax: (713) 795-5487. E-mail: andrewm{at}bcm.tmc.edu.
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REFERENCES |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Molecular and Cellular Biology, December 2000, p. 9225-9235, Vol. 20, No. 24
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Copyright © 2000, American Society for Microbiology. All rights reserved.
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