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Molecular and Cellular Biology, February 2001, p. 1111-1120, Vol. 21, No. 4
Department of Biochemistry and Molecular
Genetics, University of Colorado Health Sciences Center, Denver,
Colorado 80262
Received 17 October 2000/Returned for modification 3 November
2000/Accepted 8 November 2000
In Caenorhabditis elegans, polycistronic pre-mRNAs
are processed by cleavage and polyadenylation at the 3' ends of
the upstream genes and trans splicing, generally to the
specialized spliced leader SL2, at the 5' ends of the downstream genes.
Previous studies have indicated a relationship between these two events
in the processing of a heat shock-induced gpd-2-gpd-3
polycistronic pre-mRNA. Here, we report mutational analysis of the
intercistronic region of this operon by linker scan analysis.
Surprisingly, no sequences downstream of the 3' end were important for
3'-end formation. In contrast, a U-rich (Ur) element located 29 bp
downstream of the site of 3'-end formation was shown to be important
for downstream mRNA biosynthesis. This ~20-bp element is sufficient
for SL2 trans splicing and mRNA accumulation when
transplanted to a heterologous context. Furthermore, when the
downstream gene was replaced by a gene from another organism, no loss
of trans-splicing specificity was observed, suggesting that
the Ur element may be the primary signal required for downstream mRNA processing.
Two characteristic features of
Caenorhabditis elegans make it a unique model system
among eukaryotes for studying RNA processing. First, approximately 70%
of the genes in C. elegans undergo trans splicing during processing of the pre-mRNA. trans
splicing involves the transfer of a 22-nucleotide (nt) spliced
leader (SL) sequence from the SL snRNP to the 5' ends of the mRNAs
(2). The majority of trans splicing utilizes
SL1 RNA and most SL1 trans splicing occurs near the 5' ends
of pre-mRNAs that begin with an outron, an AU-rich intron-like sequence
containing a functional 3' splice site (UUUUCAG/R) but
lacking a 5' splice site (5-7). Second, many C. elegans genes are arranged in operons (16,
21). These genes are found in closely linked gene clusters that
are cotranscribed to produce polycistronic pre-mRNAs. Processing of
these polycistronic precursors into mature monocistronic transcripts
involves a combination of cleavage and polyadenylation at the 3' end of
the upstream mRNA and trans splicing at the 5' end of
the downstream mRNA. A second type of SL snRNP, called SL2, is
used exclusively for trans splicing to the downstream genes
in these polycistronic transcripts (16, 21), although
mRNAs from some downstream genes in operons are
trans spliced to both SL1 and SL2 (2). Since
the discovery of operons and SL2 trans splicing in
C. elegans, they have been found in other
nematodes, including Caenorhabditis briggsae
(13) and Dolichorhabditis (9). We
do not know how widespread operons are in eukaryotes,
although polycistronic transcripts have been identified in a variety of
organisms, including Drosophila melanogaster and mammals
(1).
Although the general splicing machinery is conserved in
C. elegans (2), the existence of
operons and trans splicing suggests there could be
some machinery specific for them. Part of the trans splicing
machinery, the SL snRNP, has been analyzed in Ascaris, another nematode (8). However, we know little about the
unique machinery involved in operon processing and
trans splicing in C. elegans. Since the
two genes in an operon are separated by only 100 to 400 bp, it
is possible that 3'-end formation at the upstream gene and
trans splicing at the 5' end of the downstream gene are
functionally coupled. Indeed, our laboratory previously showed that
mutation of the AAUAAA, the putative cleavage and polyadenylation specificity factor (CPSF) binding site of the upstream
gene required for 3'-end formation, resulted in a reduction of the
percentage of SL2 trans splicing to the trans
splice site 110 nt downstream (12). In this case, however,
even though 3'-end formation was completely prevented, some SL2
trans splicing still occurred. Thus, there must be
additional signals that specify the use of SL2.
There are three possible sources that could contain such cis
elements: the upstream gene, the intercistronic sequence, and the
downstream gene. The only element in the intercistronic region found in
all operons is the trans splice site, but this
sequence is not different from the general 3' splice site consensus
(2), so it is not a candidate for an SL2-specific signal.
Here we show that sequences in the downstream gene also play no
obligatory role in SL2 trans splicing. In addition, we have
performed a linker scan analysis of the intercistronic region. This
analysis revealed a short U-rich element required for SL2
trans splicing more than 50 bp upstream of the
trans splice site. In contrast, we found no sequences in the
intercistronic region required for 3'-end end formation of the upstream gene.
Worm strains and RNA preparation.
Maintenance and growth of
worms was as described by Brenner (3) and Sulston and
Hodgkin (17). Transgenic worm strains carrying
extrachromosomal arrays were generated as described previously (15, 16). Worms were heat shocked at 30°C for 1 to
2 h on floating petri plates spread with bacteria. Total RNA was
prepared from heat-shocked or non-heat-shocked mixed-stage populations of transgenic worms (5). The RNA was treated with DNase I
prior to analysis.
Plasmid construction.
The plasmid WT, containing the
wild-type operon, was constructed by deleting 75 bp of the
plasmid HS1496 (16) upstream of the heat shock promoter to
make the SalI site in gpd-3 unique. The linker
scan mutations (LS1 to LS9) were made from WT by replacing 10-bp
sections of the wild-type intercistronic region with linker GCTTAATTAA via recombinant PCR (11). The
primers at the ends were gpd2CLus,
5'-CAACAGAGTTGTTGATCTCATCTCG-3', and vit6pr2,
5'-AACTTGTCGCACTTCTGGTC-3'. The following pairs of primers
were used to introduce the mutations: LS1-3
(5'-ATTCATTAATTAAGCTAAATATACAACCTTTATTG-3') and LS1-5
(5'-ATTTAGCTTAATTAATGAATCTGCCATTTCCTCGT-3'), LS2-3
(5'-GAAATTTAATTAAGCATAAGATGAATAAATATACA-3') and LS2-5
(5'-CTTATGCTTAATTAAATTTCCTCGTTTTTGCGAGT-3'), LS3-3
(5'-CAAAATTAATTAAGCGGCAGATTCAATAAGATGAA-3') and LS3-5
(5'-CTGCCGCTTAATTAATTTTGCGAGTTTATATACCT-3'), LS4-3
(5'-TATAATTAATTAAGCACGAGGAAATGGCAGATTCA-3') and LS4-5
(5'-CTCGTGCTTAATTAATTATATACCTTCCAATTTTC-3'), LS5-3
(5'-TTGGATTAATTAAGCACTCGCAAAAACGAGGAAAT-3') and LS5-5
(5'-CGAGTGCTTAATTAATCCAATTTTCTTTCTATTGT-3'), LS6-3
(5'-AGAAATTAATTAAGCAGGTATATAAACTCGCAAAAAC-3') and LS6-5 (5'-TACCTGCTTAATTAATTTCTATTGTATTTTCAACT-3'), LS7-3
(5'-AAAATTTAATTAAGCGAAAATTGGAAGGTATATAAAC-3') and LS7-5
(5'-TTTTCGCTTAATTAAATTTTCAACTTCTAATTTTAATTC-3'), LS8-3 (5'-TTAGATTAATTAAGCACAATAGAAAGAAAATTGGA-3') and LS8-5
(5'-ATTGTGCTTAATTAATCTAATTTTAATTCAGGGAA-3'), and LS9-3
(5'-TGAATTTAATTAAGCAGTTGAAAATACAATAGAAAG-3') and LS9-5 (5'-CAACTGCTTAATTAAATTCAGGGAAACTGCTTCAA-3'). The last eight
bases of the linker are the recognition site for PacI. This
enzyme was used to facilitate cloning and ensure correct identification
of the different LS mutations.
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.4.1111-1120.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Intercistronic Region Required for Polycistronic
Pre-mRNA Processing in Caenorhabditis elegans
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Ur and Ur/pA, which were derived from WT and pA
(12), respectively, were constructed using an
oligonucleotide-directed in vitro mutagenesis kit (Amersham). Plasmids
pA/LS4, pA/LS21, and pA/LS26 were derived from pA by
recombinant PCR using the corresponding oligonucleotides described above.
The plasmid SUF was constructed as follows: the intercistronic region
of WT was deleted by replacing the PacI/NcoI
fragment of LS1 with the corresponding fragment from LS9 to make
pGPDPacI. To replace the intercistronic region with an unrelated
sequence, two oligonucleotides, UrSUFUS
(5'-GCTTAATTAATGTTTAAACTTCATCGATGTTTTTGCGAGTTTATATACCTATCG-3') and UrSUFDS
(5'-GAATTAATTAAGAGTTTAAACAAGAGAAAGATCTATAAATCGATAGGTATATAAACTCG-3') were added to a PCR mixture containing 1× PCR buffer (Sigma), 0.2 mM deoxynucleoside triphosphates, and 2.5 U of Taq
polymerase (Gibco BRL). The product was denatured at 92°C for 2 min,
followed by PCR as follows: 92°C for 1 min, 45°C for 1 min, and
72°C for 1 min. After 30 cycles, the products were extended at 72°C
for 7 min. The PCR product was cloned into pGEM-T easy vector (Promega) to make pTIC according to the instructions from the manufacturer. The
structure of the resulting plasmid was confirmed by sequencing, and the
intercistronic region was released with PacI and cloned into
the PacI site of pGPDPacI to make SUF. SUFR was made by
reversing the U-rich (Ur) sequence in SUF by digestion and religation
at the two ClaI sites flanking the Ur element.
Plasmids pHSWTGFP and pHSUrGFP were constructed using recombinant
PCR to replace the HpaI/SalI fragment containing
the gpd-3 gene present in the WT plasmid with the gene for
the green fluorescent protein (GFP) from pPD94.81 (kindly provided by
A. Fire). In pHSWTGFP the intercistronic region contains the wild-type
Ur region, whereas in the pHSUrGFP plasmid it contains the Ur
mutation. Plasmids used as templates for amplifying the intercistronic
region were either WT or Ur. Primers used to amplify the
intercistronic region between the gpd-2 and gpd-3
genes included gpd2Clus and ICNLS-Apa I (5'-GTACCCTCCAAGGGCCCTCCTGAATTAAAATTAGAAG-3'). The ICNLS-Apa I primer introduces an ApaI site adjacent to the
trans splice site. The gfp gene containing the
simian virus 40 nuclear localization signal and five introns was
amplified using the plasmid pPD94.81 as template. Primers used to
amplify the GFP gene included NLS-Apa I
(5'-GAGGGGCCTTGGAGGGTACCGAGC-3') and IVS-Sal I
(5'-CGATTATATGTCGACTGAAAATTTAAATATTAC-3'). Amplified
fragments obtained after the recombinant PCR were digested with
HpaI and SalI and cloned into WT plasmid digested
with HpaI and SalI to create either pHSWTGFP or
pHSUrGFP. All plasmids were sequenced and checked by restriction
enzyme analysis.
RNase protection analysis (RPA) and primer extension analysis. RNase protection assays were carried out as described previously (12). The RNA and probe were heated for 5 min at 65°C and hybridized at 45°C for 16 h. Primer extension in the presence of dideoxynucleotide was done as described previously (12). The mixtures of RNA and primer were heated at 65°C for 5 min before hybridization, and reverse transcription was carried out in the presence of 5% acetamide. Quantitation was performed on a Molecular Dynamics PhosphorImager using ImageQuant software. All constructs were assayed in at least two independent transgenic strains, most in three or four strains. Multiple assays of each strain were averaged, and error bars were calculated from all assays of each construct. In general, variations between strains carrying the same construct were equivalent to variations from multiple assays of the same strain.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Linker scan analysis of the intercistronic region between the
gpd-2 and gpd-3 genes.
Spieth et al.
(16) described an in vivo system to study operon
processing using a construct, HS1496, derived from a three-gene operon (mai-1-gpd-2-gpd-3). This synthetic
operon contains the gpd-2 and the gpd-3
genes under the control of the hsp-16-41 heat shock
promoter. Using this construct in transgenic worms, it was shown by
Kuersten et al. (12) that the gpd-2 AAUAAA
required for 3'-end formation was required for correct
SL2-specific trans splicing of gpd-3 but that the
gpd-3 trans splice site was not important for
gpd-2 3' end formation. However, the intercistronic sequence, only about 100 bp long, has never been tested for signals required for either 3'-end formation or trans-splicing
specificity. Comparison of intercistronic regions of different
operons does not reveal the presence of conserved sequences (T. Blumenthal, unpublished observation). On the other hand, comparison of
the intercistronic regions from 15 operons that have been
sequenced in both C. elegans and C. briggsae does in each case reveal a sequence, present in similar
locations, that is conserved (Table 1).
In contrast, most of the remaining intercistronic sequence is highly
diverged between the two closely related species (unpublished observations). The conserved sequences from these different
operons do not resemble each other in any way other than being
rich in uridylate.
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Effect of linker scan mutations on 3'-end formation of
gpd-2.
To determine the effect of the various LS
mutations, we examined polycistronic pre-mRNA processing by RPA.
Expression and processing of the wild-type operon resulted in
protection of a 322-nt fragment, indicative of correct 3'-end formation
(Fig. 2, top, lane 1). The downstream
gene, gpd-3, was processed by trans splicing to
give a protected fragment of 259 nt. The amount of
trans-spliced gpd-3 mRNA was approximately
40% of the amount of gpd-2 mRNA. As observed previously
(12), polycistronic pre-mRNA processing was complete
as judged by the absence of polycistronic pre-mRNA, which would
produce a fragment of 679 nt. In all of the linker scan mutants, 3'-end
formation of the upstream gene product was also complete (Fig. 2),
since we could not detect accumulation of unprocessed RNA and since
there was no obvious change in the amount of the upstream gene
mRNA. These data indicate that in this operon there are no
nonredundant sequences following the gpd-2 3' end required
for 3'-end formation. Although we would expect this region to contain a
cleavage stimulatory factor (CstF) binding site, needed for 3'-end
formation in higher eukaryotes, we cannot rule out the possibility that
there are redundant elements in the intercistronic region that are
involved in the processing of the upstream mRNA.
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Ur sequence required for downstream mRNA accumulation. In most of the mutants (LS1, LS2, and LS6 through LS9), accumulation of gpd-3 mRNA is indistinguishable from that of the wild type (Fig. 2). However, in three mutants, LS3, LS4, and LS5, the amount of protected fragment representing the 5' end of gpd-3 RNA was significantly reduced. In LS3 and LS5, gpd-3 RNA accumulation was about 50% of the wild type, whereas in LS4, only 25% of the wild-type level accumulated. These three mutations cover 30 bp of the intercistronic region that is especially rich in uridylate residues, so we call it the Ur element. It is essentially the same sequence found to be homologous between C. elegans and C. briggsae intercistronic regions (Table 1).
Effect of the linker scan mutations on gpd-3
trans-splicing specificity.
We tested all of the LS mutant
strains for alteration of their relative levels of SL2 and SL1
trans splicing by primer extension with ddGTP, which
distinguishes between SL1 and SL2 trans-spliced gpd-3 mRNA. Reverse transcription from the splice site
stops at the first C in the template, giving products that extend the
primer 9 nt with SL1-spliced mRNA, 3 nt with SL2-spliced mRNA,
and 2 nt with pre-mRNA or other unspliced intermediates. With the
wild-type construct, most trans-spliced product received SL2
(Fig. 3, top, lane 1) (12).
Most of the linker scan mutations resulted in no significant change in
the percentage of SL2 trans splicing. However, LS4, the
mutation that had the most drastic effect on gpd-3 mRNA
accumulation, also reduced the level of SL2 and increased the level of
SL1 trans splicing (Fig. 3, top, lane 5). In all cases, we
confirmed the results by primer extension in the presence of ddCTP,
which also gives differences in sizes of the products for SL1 and SL2
trans-spliced and unspliced RNA (data not shown). We
conclude that sequences mutated in LS4 play a key role in determining gpd-3 trans-splicing specificity and in the accumulation of
trans-spliced gpd-3 mRNA.
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1 to +17 from the gpd-3 trans splice site. The
presence of ddGTP results in termination of extension after 2 nt on
unspliced RNA and after either 3 or 9 nt from RNA resulting from
trans splicing to SL2 or SL1, respectively. Following primer
extension, the products were electrophoresed on a 20% polyacrylamide
denaturing gel. (Top) The positions of expected products are indicated.
(Bottom) The RPA was quantitated, and the data was plotted as the
percent of total trans-spliced gpd-3 that is
trans spliced to SL2.
Establishing the boundaries of the Ur element.
The three
linker scan mutations that affected gpd-3 mRNA
accumulation span 30 bp. To establish the boundaries more precisely, we
created additional mutations. In LS10, only the 5'-most 8 bp of
sequence covered by LS3 was mutated (Fig. 1). Unlike LS3 (Fig. 2, top,
lane 4), the phenotype was the wild type, indicating that only the
3'-most 2 bp of the sequence covered by LS3 are required for Ur
function (Fig. 4, lane 2). In the second
mutation, LS11, only the 3'-most 5 bp of the sequence covered by the
LS5 mutation was mutated (Fig. 1). This mutation resulted in a mutant
phenotype (Fig. 4, top, lane 3) even stronger than that shown by LS5
(Fig. 2, lane 6), indicating that the Ur element includes at least some of the 3'-most 5 bp of the sequence covered by the LS5 mutation. Neither LS10 nor LS11 altered trans-splicing specificity
(data not shown), similar to the behavior of the LS3 and LS5 mutations (Fig. 3, top, lanes 4 and 6). These data indicate that the Ur element
spans no more than 22 nt, from position 29 to 50 (Fig. 1), and possibly
as few as 18 nt.
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Fine-structure analysis of the Ur element.
To further dissect
the Ur element and test the idea that the U richness of the element
contributes to its function, we constructed several smaller replacement
mutations, LS21 to -26, and Ur (Fig. 1). None of these mutations
affected 3'-end formation (Fig. 4). However, all of them except LS24
and LS26 resulted in significant reductions in gpd-3
accumulation. Even LS21, in which a single G at position 29 was changed
to a C, reduced gpd-3 mRNA accumulation quite
substantially (Fig. 4, top lane 4). These results indicate that most of
the sequences between positions 29 and 50 play a role in Ur function.
Only the bases from positions 41 to 44 could be replaced without
significant reduction in gpd-3 mRNA. Thus the Ur element
has critical nucleotides between positions 29 to 40 and 45 to 50.
Sufficiency of the Ur element.
The above results showed that
the Ur element is essential for both maximal accumulation of
gpd-3 mRNA and correct trans-splicing specificity. To determine whether it is sufficient, we created the SUF
construct in which all of the intercistronic sequences except the Ur
element were replaced with unrelated polylinker sequences (Fig. 1). As
a control, SUFR was constructed by reversing Ur in the same context as
SUF (Fig. 1). In both constructs, 3'-end formation of gpd-2
was normal, again suggesting that the region downstream of the cleavage
site plays no role in 3'-end formation (Fig.
5). Remarkably, accumulation of
gpd-3 mRNA was nearly normal in SUF, while virtually no
gpd-3 mRNA accumulated in the SUFR control. This
demonstrates that the Ur element can function in a heterologous context
with no sequence from the intercistronic region except the
trans splice site. Furthermore, trans-splicing specificity was also normal, demonstrating that the Ur element is
sufficient to allow SL2 trans splicing in the absence of
other sequences from the gpd-2-gpd-3 intercistronic region
(Fig. 5).
|
Effect of Ur mutations in the absence of 3'-end formation.
It
was shown previously that deletion of the gpd-2 AAUAAA
signal for 3'-end formation completely prevents 3'-end formation but still allows gpd-3 trans splicing (12; also see Fig.
6A, top, lane 2). However, this
trans splicing is no longer predominantly to SL2; instead
about half of the trans splicing is to SL1 (12; also see
Fig. 6B, lane 2). In order to determine whether the Ur mutations still
had an effect in the absence of 3'-end formation, we created double
mutations in which both the AAUAAA signal and the Ur element
were mutated. We found that most of these double mutants were
indistinguishable from the AAUAAA mutant alone (Fig. 6). As
expected, gpd-2 3'-end formation was completely abrogated in
all of the double mutants; however, there was a substantial amount of
accumulation of gpd-3, much more than seen with the single
Ur mutations (compare Fig. 2, top, lane 5, with Fig. 6A, lane 4, for
instance). This suggests that the reduction in the levels of
gpd-3 accumulation in the single Ur mutant strains is dependent on 3'-end formation. Furthermore, the ratio of SL2 to SL1
trans splicing in most double mutant strains is
indistinguishable from that in the AAUAAA mutation alone, so
it appears that the AAUAAA mutation is epistatic to the Ur
mutations. However, the double mutations with LS4 or Ur do show a
further increase in SL1 and decrease in SL2 trans splicing
(Fig. 6B, lanes 3 and 4), so the Ur mutations may have a direct effect
on trans-splicing specificity (also see below).
|
Replacement of the downstream gene with the gfp
gene.
In order to find out if there are any sequences in the
downstream gene that play a role in defining its
trans-splicing specificity, we replaced gpd-3
with the gfp gene in our transgenic construct and measured
mRNA accumulation and trans-splicing specificity (Fig.
7). RPA showed that the gfp
gene was expressed and processed properly (Fig. 7B, lane 1). Primer
extension in the presence of ddGTP indicated that this gene was
trans spliced predominantly to SL2 (Fig. 7C, lane 2). These
results suggest that sequences in gpd-3 are not required for
SL2-specific trans splicing. We also tested the effect of
the Ur mutation in this operon. Both RPA and primer
extension with and without ddGTP demonstrate clearly that in this novel
context the Ur element is important for the accumulation of the
downstream mRNA. Interestingly, it appears that, when
gfp is the downstream gene, SL2 trans splicing is
more dramatically inhibited by the lack of the Ur element than when gpd-3 is the downstream gene (compare Fig. 7C, lanes 3 and
4, with Fig. 3, top, lane 5).
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
Accumulation of downstream mRNA. Since the genes in C. elegans operons are typically separated by approximately 100 to 400 bp of intercistronic sequence, Kuersten et al. (12) and Spieth et al. (16) proposed that 3'-end formation of the upstream mRNA is mechanistically coupled to trans splicing of the downstream mRNA. They demonstrated that a functional AAUAAA does play a role in SL2 trans splicing, but SL2 trans splicing could still occur at reduced levels in the absence of AAUAAA. This suggested that the AAUAAA is not directly involved in SL2 trans splicing to gpd-3 and that other signals must be present that are responsible for SL2 specificity.
Our evidence suggests that the trans splicing of the downstream gene is determined by sequences within the intercistronic region. When the intercistronic sequence was shortened to 30 bp or lengthened to 300 bp with a heterologous sequence, trans splicing of the downstream mRNA was virtually eliminated (J. Spieth, K. Lea, S. Kuersten, M. MacMorris, and T. Blumenthal, unpublished). Based on this observation, we undertook the detailed mutational analysis reported here to screen the entire intercistronic sequence. This resulted in the discovery of what we term the Ur element. When the Ur element was mutated, accumulation of downstream mRNA was severely reduced, enabling us to define the element with some precision. Based on the data reported here, we can conclude that the 5' end of the element is at position 29 or 30, counting from the site of 3'-end formation, and that its 3' end is between positions 45 and 50. Furthermore, not all sequences between these positions are important, based on the fact that positions 40 to 43 can be mutated without loss of activity. We suggest that Ur is likely to be a protein binding site, since a single nucleotide change at position 27 eliminated activity. The linker scan analysis along with the sufficiency experiment makes it clear that only sequences contained within the ~22 nt of the Ur element are required for accumulation of downstream mRNA. Furthermore, the experiment in which the downstream gene itself was replaced by a gfp gene makes it clear that the sequence of gpd-3 does not play a critical role. What does the Ur element do? Because it appears to also affect the choice between SL2 and SL1 trans splicing, we presume it has its effect by allowing or directly promoting trans splicing. The following model can explain its function. If gpd-2 3'-end formation occurs before gpd-3 trans splicing, then the cleavage reaction will leave a free 5' phosphate on the downstream portion of the pre-mRNA. Since this RNA is not capped, it will likely be subject to rapid degradation by an exonuclease. We hypothesize that a protein binds to the Ur element to impede the progress of the exonuclease and thereby allow SL2 trans splicing to occur. The SL2 trans-splicing event could be a default mode that occurs whenever sufficient time is allowed by the protein bound to Ur. Alternatively, the protein bound to the Ur element could interact directly or indirectly with the SL2 snRNP to facilitate correct trans splicing. The trans splicing provides a cap that will prevent degradation of the downstream mRNA and hence allow its accumulation.Involvement of the Ur region in the trans-splicing
specificity of the downstream gene.
If failure to accumulate
downstream mRNA in the Ur mutants is primarily a defect in
trans-splicing efficiency, then following 3'-end formation,
the downstream product would be inefficiently processed, and the
majority of the gpd-3 RNA would be degraded. An alternative
possibility is that the Ur element is more directly involved in SL2
trans splicing. To test this possibility, we mutated the Ur
element in the context of the gpd-2 AAUAAA
mutation (pA), which eliminates upstream 3'-end formation.
Since cleavage or transcription termination couldn't occur due to the
AAUAAA mutation, we predicted that the downstream product
would now be able to accumulate. And indeed, both polycistronic RNA
(resulting from the failure of 3'-end formation and trans
splicing) and trans-spliced gpd-3 mRNA
accumulated to the same levels whether or not the Ur element was
mutated in the pA background (Fig. 6). Since the pA mutation by
itself reduced SL2 trans splicing, it was difficult to
determine for certain whether there was an additional reduction due to
the Ur mutations. However, it appears that both Ur and the LS4
mutation did increase SL1 trans splicing at the expense of SL2.
Ur mutation was seen in the
context of the gfp operon construct (Fig. 7). Here,
SL2 trans splicing was eliminated by the Ur mutation
without any apparent effect on SL1 trans splicing. We
conclude that there is a direct effect by the Ur element on SL2
specificity, as opposed to just on trans-splicing
efficiency, but that it is more difficult to demonstrate when the
AAUAAA is also mutated. We suggest that the Ur element is
directly involved in SL2 trans splicing but that that
involvement is facilitated by CPSF bound to the AAUAAA of the upstream gene or by 3'-end formation itself. It has previously been
shown that inactivation of the gpd-2 CPSF binding site
reduces the level of trans splicing to gpd-3 but
doesn't eliminate SL2 trans splicing. Since vertebrate CPSF
and CstF are known to interact cooperatively (10, 20), the
AAUAAA mutation might exert its effect by lowering the
affinity of CstF for its binding site.
Implications for the mechanism of 3'-end formation. It is clear from earlier work that C. elegans uses the same recognition sequence for CPSF binding as vertebrates do, AAUAAA, although many variants of this sequence are known to function in C. elegans (2). In contrast, no CstF binding site has yet been identified in worms, although all of the CstF subunits are encoded in the C. elegans genome. This paper reports the first mutational analysis of a region downstream of a 3' end in C. elegans, where the CstF binding site would be expected to be located. Nonetheless, our findings indicate that all of the sequence downstream of the 3' end can be replaced without any apparent effect on 3'-end formation. The possibility that CstF does bind in this region to facilitate 3'-end formation but that the binding sites are redundant is made unlikely by the experiment showing that 3'-end formation was also complete in both the SUF and SUFR constructs. Since the latter contains no sequences normally present in the gpd-2 intercistronic region, we conclude that no sequences downstream of the 3' end are required for correct 3'-end formation in this operon. Thus, all of the sequences required for 3'-end formation of gpd-2 are present within the gene, presumably in the 3' untranslated region. Whether this is generally true of C. elegans 3'-end formation remains to be determined. It is possible that this conclusion is valid only for upstream genes in operons or even only for gpd-2.
Another possibility is that CstF could play an obligatory role but that it could interact with the 3'-end formation machinery by another means such as by forming a tight complex with CPSF (19). CstF may bind to the intercistronic region, but its binding there may not be required for 3'-end formation. Perhaps binding of CstF to a downstream region only facilitates 3'-end formation without being required for it to occur in a CstF-dependent fashion. This idea leaves open the possibility that CstF could be the protein that binds to the Ur region and that binding is required to allow production of downstream mRNA. We suggest this possibility only because the Ur element has a sequence consistent with a CstF binding site and is in a location where CstF binding sites generally reside (4, 14, 18), in this case, 29 nt downstream of the site of 3'-end formation. This idea is also consistent with the observation that each operon listed in Table 1 has a conserved sequence in this approximate location, but these sequences resemble each other only by virtue of the fact that they are all rich in U residues.| |
ACKNOWLEDGMENTS |
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We are grateful to Devin Leake for discussions and help with the figures.
This work was supported by research grant GM42432 from the National Institute of General Medical Sciences.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Biochemistry and Molecular Genetics, University of Colorado Health Sciences Center, Box B-121, 4200 E. 9th Ave., Denver CO 80262. Phone: (303) 315-8181. Fax: (303) 315-8215. E-mail: tom.blumenthal{at}uchsc.edu.
Present address: Department of Molecular Biology, Princeton
University, Princeton, NJ 08544.
Present address: Gene Expression Program, EMBL, D-69117
Heidelberg, Germany.
§ Present address: Genome Sequencing Center, Washington University School of Medicine, St. Louis, MO 63108.
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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, February 2001, p. 1111-1120, Vol. 21, No. 4
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.4.1111-1120.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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