Vol. 10, No. 1
MOLECULAR AND CELLULAR BIOLOGY, Jan. 1990, p. 341-352
0270-7306/90/010341-12$02.00/0 Copyright © 1990, American Society for Microbiology
Octamer and SPH Motifs in the Ul Enhancer Cooperate To Activate Ul RNA Gene Expression KENNETH A. ROEBUCK,t DANIEL P. SZETO, KENNETH P. GREEN,t QIAN N. FAN, AND WILLIAM E. STUMPH* Department of Chemistry and Molecular Biology Institute, San Diego State University, San Diego, California 92182-0328 Received 3 July 1989/Accepted 13 October 1989 The transcriptional enhancer of a chicken Ul small nuclear RNA gene has been shown to extend over approximately 50 base pairs of DNA sequence located 180 to 230 base pairs upstream of the Ul transcription initiation site. It is composed of multiple functional motifs, including a GC box, an octamer motif, and a novel SPH motif. The contributions of these three distinct sequence motifs to enhancer function were studied with an oocyte expression assay. Under noncompetitive conditions in oocytes, the SPH motif is capable of stimulating Ul RNA transcription in the absence of the other functional motifs, whereas the octamer motif by itself lacks this ability. However, to form a transcription complex that is stable to challenge by a second competing small nuclear RNA transcription unit, both the octamer and SPH motifs are required. The GC box, although required for full enhancer activity, is not essential for stable complex formation in oocytes. Site-directed mutagenesis was used to study the DNA sequence requirements of the SPH motif. Functional activity of the SPH motif is spread throughout a 24-base-pair region 3' of the octamer but is particularly dependent upon sequences near an SphI restriction site located at the center of the SPH motif. Using embryonic chicken tissue as a source material, we identified and partially purified a factor, termed SBF, that binds sequence specifically to the SPH motif of the Ul enhancer. The ability of this factor to recognize and bind to mutant enhancer DNA fragments in vitro correlates with the functional activity of the corresponding enhancer sequences in vivo.
The small nuclear RNAs (snRNAs) of the U family are evolutionarily conserved and metabolically stable RNA molecules present in the nuclei of eucaryotic cells. Considerable evidence exists that the Ul, U2, U4, U5, and U6 snRNAs are involved in the splicing of mRNA precursors (37). With the exception of U6, the snRNAs are synthesized by RNA polymerase II (6) and have a distinctive N2,N2,N7-trimethylguanosine cap structure (34). The genes that code for the vertebrate snRNAs are usually present in multiple copies per genome and have several features that distinguish them from protein-coding genes. The promoter regions of snRNA genes lack transcription signals normally found upstream of genes transcribed by RNA polymerase II (e.g., they lack TATA and CCAAT boxes). Nevertheless, they contain two distinct and evolutionarily conserved cis-acting regulatory regions within their 5'-flanking DNA sequences that are important for snRNA gene expression: a proximal region centered near position -55 relative to the transcription initiation site and a distal region located near position -200 (reviewed in references 6 and 32). Moreover, the formation of the 3' ends of the snRNAs is dependent on the initiation of transcription from an snRNA gene promoter (4, 13, 14, 28). Because of the unique properties of snRNA transcription complexes, the promoter elements of snRNA genes are not functionally interchangeable with comparable elements of mRNA genes (5, 7, 13, 39, 41). Finally, vertebrate snRNA genes are not accurately transcribed under ordinary in vitro transcription conditions with soluble HeLa cell extracts (26, 44). *
Two general conclusions have been drawn from the snRNA gene in vivo expression studies. First, the proximal control element is essential for the efficient and accurate initiation of snRNA transcription (3, 24, 25, 27, 36); it also is required for proper 3' end formation (13, 28, 33). Second, the distal region is an enhancerlike element that is required for a high level of snRNA gene expression and for the formation of a stable transcription complex (3, 23-25, 27, 35, 41). We previously showed that the chicken Ul RNA gene distal element is composed of at least two distinct functional domains which are both required for full enhancer activity (35). The first enhancer domain contains two well-characterized sequence motifs, an octamer sequence and a GC box. These motifs are believed to be binding sites for the ubiquitous transcription factors Oct-1 and Spl, respectively (18, 38). The second domain of the chicken Ul enhancer, the SPH motif (for SphI postoctamer homology), consists of approximately 24 base pairs (bp) immediately 3' of the octamer that are highly conserved in sequence among chicken Ul RNA genes (9). Expression assays indicated that a deletion of 28 bp encompassing the SPH motif was sufficient to eliminate Ul enhancer activity even though the octamer and GC box remained intact (35). We have now further dissected the functional motifs of the chicken Ul enhancer; in particular, we have mapped at a finer level the DNA sequences important for the activity of the SPH motif. We have also detected and partially purified a factor from chicken embryos that interacts sequence specifically with the SPH motif. The ability of this factor to bind to the wild-type and mutant enhancer sequences strongly correlates with the transcriptional activity of the respective templates. Our results suggest that multiple enhancer-binding factors (Spl, Oct-1, and SBF [i.e., the SPH motif-binding factor]) interact with each other (and presumably with proteins bound to the proximal regulatory region) to form a stable snRNA transcription complex.
t Present address: Department of Medicine, University of California, San Diego, La Jolla, CA 92093. t Present address: Department of Environmental Science and Engineering, University of California, Los Angeles, Los Angeles, CA 90024.
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MATERIALS AND METHODS Construction of initial Ul mutant DNA templates. The chicken Ul RNA gene used in this study was previously described (9, 35). The pU1A-246 wild-type construction contains 246 bp of 5'-flanking DNA, the U1-52a gene, and 28 bp of 3'-flanking DNA cloned into the pUC19 vector. The derivatives pU1A-206 and pU1A-188 were described by Roebuck et al. (35). The construction designated pUlA(-211/-182) in Roebuck et al. (35) was renamed pU1A-246dl for consistency of nomenclature in this report. To create within the Ul enhancer region several unique restriction enzyme sites which could be used to facilitate site-directed mutagenesis of the SPH motif, we made the following degenerate oligonucleotide double stranded and prepared it for molecular cloning by the method of mutually primed synthesis (15, 30): 5'-acggaattCATGCAAATggtA(C/ g)(C/g)GCGCGCTGCATGCCGGGAGCtCC-3'. This 46-mer contained Ul enhancer sequences from positions -182 to -219 but contained specific base differences (shown as lowercase letters) from the wild-type sequence from positions -208 to -210 and at position -184 and potential differences at positions -205 and -206, depending upon the degeneracy incorporated. Following mutually primed synthesis, the double-stranded oligonucleotide was digested with SstI and EcoRI and cloned into the SstI and EcoRI sites of pUC19. A 380-bp Bsp1286-HindIII DNA fragment was isolated from the wild-type Ul template, pU1A-246, and cloned into the SstI-HindIII polylinker sites of the plasmids that contained different synthetic variants of the Ul enhancer sequence. From this procedure, the following Ul mutant templates were obtained: pU1A-219s2, pUlT, pUlTsl, and pU1A-219s4. Each of these constructions contained a synthetic DNA sequence from positions corresponding to -219 to -184 of the Ul enhancer region and wild-type Ul sequence downstream of position -183. To construct pUlA-219 and pUlA-219sl, we cloned a synthetic double-stranded oligonucleotide of wild-type sequence into the EcoRI and BssHII sites of pU1lA-219s2 and pUlT, respectively. Additional insertion and deletion templates (see Fig. 1) were obtained as follows: pU1PiACGT was derived from pUlP by digestion with Asp718 at the KpnI site, filling in the 3'-recessed ends with Escherichia coli DNA polymerase (Klenow enzyme), and recircularizing; pUlTsldl was derived from pUlTsl by digestion with SstI and SphI and trimming back the 3'-overhanging ends with T4 DNA polymerase; and, finally, pU1A-205 was similarly derived from pU1A-219s2 with EcoRI, KpnI, and Klenow DNA polymerase. To make the pUlTinv template, we cloned an inverted repeat (i.e., two head-to-head copies) of the synthetic 46-mer (sequence shown above) into the EcoRI site of pU1A-188 by omitting the SstI digestion of the mutually primed synthetic oligonucleotide. When the resulting plasmid was digested with SstI and AatII (which cut in the pUC19 vector), only an inverted copy of the oligo-derived enhancer sequence remained. Construction of Ul deletion DNA templates derived from pUl. A number of deletion templates (see Fig. 3) were derived from the pUlP construction. The pseudo-wild-type plasmid contains an SstI site at the 3' border of the SPH motif, a KpnI-Asp718 site between the octamer and SPH motifs, and an EcoRI site immediately 5' of the octamer. We used these restriction sites, as well as the naturally occurring BssHII and SphI sites within the SPH motif, to construct a number of different Ul deletion templates. In general, the
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parental plasmid was first digested with the desired restriction enzyme(s) and then treated with E. coli DNA polymerase (Klenow fragment), T4 DNA polymerase, or Si nuclease to produce flush ends for religation. This method generated the following internal deletion templates: pUlTdl, pUlTd2, pUlTd3, pUlTd4, and pUlTdll. This last construction removed the DNA sequences from positions -205 to -178 in the enhancer region but juxtaposed a very similar sequence that naturally occurs from positions -178 to -170. Beginning with pUlTd4, the following additional deletion templates were generated: pUlTd5, pUlId6, pUlTd7, pUlTd8, pUlTd9, pUlTdlO, and pUlTA-193. Construction of Ul substitution and insertion DNA templates. To construct several substitution templates (see Fig. 3), we cloned double-stranded synthetic oligonucleotides into plasmid templates digested at appropriate restriction enzyme sites in the Ul enhancer, generating the following substitution templates: pUlTs2 derived from pUlT with the KpnI and SphI sites, pUlTs4 derived from pUlT with the BssHII and SstI sites, pUlTs7 derived from pUlT with the KpnI and SstI sites, and pUlTs5 derived from pUlTdl with the KpnI and SphI sites. To construct the three single-nucleotide insertion templates, a degenerate oligonucleotide containing a G, C, or T residue between positions -198 and -199 was prepared for cloning by mutually primed synthesis (15). After gel isolation, it was cloned between the SphI and Asp718 sites of the pUlT construction to yield the three expected templates: pUlTiG, pUlPiC, and pUlTiT. All mutant constructions were verified by sequencing. Oocyte nuclear injection and RNA analysis. Microinjection of plasmid DNA into Xenopus laevis oocytes, extraction of RNA from pooled oocytes, and analysis of the labeled RNA in denaturing polyacrylamide gels were performed as previously described (16, 35). Relative transcription levels were estimated by densitometric scanning of autoradiographs exposed for various lengths of time. Transcriptional competition experiments between chicken Ul and U4B RNA genes were performed as previously described (25), except that the Ul gene plasmid (320 ,ug/ml) was in an approximate eightfold molar excess over the U4B gene plasmid (40 ,ug/ml). In the competition assays, different batches of oocytes expressed the Ul and U4B genes with different relative efficiencies; however, that phenomenon does not affect conclusions regarding stable complex formation on the Ul gene templates. Preparation and partial purification of the SPH motifbinding factor (SBF). (i) Preparation of nuclear extracts. Nuclei were purified and extracts were prepared by the procedure of Gorski et al. (11) with several modifications to adapt the procedure for the use of whole chicken embryos as the starting material. Heads were severed from 24 8- to 10-day-old chicken embryos, and 20 g of the remaining tissue was homogenized in 50 ml of homogenization buffer (11) containing 1.75 M sucrose. The resulting homogenate was diluted to a volume of 170 ml with 1.70 M sucrose homogenization buffer, layered in aliquots over six 10-ml cushions of 1.75 M sucrose homogenization buffer, and centrifuged for 1 h at 24,000 rpm and 2°C in an SW27 rotor (Beckman Instruments, Inc., Fullerton, Calif.). The nuclear pellets were combined and suspended in 100 ml of 1.80 M sucrose homogenization buffer, layered over four 10-ml cushions of 1.85 M sucrose homogenization buffer, and centrifuged as described above. The combined nuclear pellets were suspended in 10 ml of nuclear lysis buffer (11). Subsequent lysis of the nuclei and isolation of protein extracts were per-
COOPERATIVITY BETWEEN Ul GENE ENHANCER MOTIFS
VOL. 10, 1990
formed as described in Gorski et al. (11). The final dialyzed extract was treated with carrier-fixed a2-macroglobulin (Boehringer Mannheim Biochemicals, Indianapolis, Ind.) prior to storage. We normally obtained approximately 1 mg of nuclear protein per g of starting embryonic tissue, determined with a protein assay kit (Bio-Rad Laboratories, Richmond, Calif.) with bovine gamma globulin as a standard. (ii) Heparin-agarose chromatography. Approximately 2 ml of nuclear extract (-10 mg of protein) was routinely loaded onto a 1.5-ml heparin-agarose column equilibrated with TM100 buffer (50 mM Tris hydrochloride [pH 7.5], 12.5 mM MgCl2, 1 mM EDTA, 20% glycerol, 1 mM dithiothreitol, 100 mM KCl). The SBF activity was recovered by step elution with TM250 buffer (same as TM100 buffer, except for 250 mM KCl) containing leupeptin at 0.5 ng/ml and pepstatin at 0.7 ng/ml. (The protease inhibitors were maintained throughout the remainder of the purification procedure in all buffers containing the SBF activity.) Generally, starting with 10 mg of nuclear protein extract, approximately 0.5 mg of protein was recovered in the TM250 fraction from the heparinagarose column. (uii) Sequence-specific DNA affinity chromatography. A DNA affinity column was constructed by the procedure of Kadonaga and Tjian (19) with the following oligonucleotides: 5'-GATCCGCGCGCTGCATGCCGGGAG-3' and 3'-GCGC GCGACGTACGGCCCTCCTAG-5'. When annealed and tandemly ligated, the Ul enhancer sequences of the SPH motif between positions -206 and -186 were constituted. The TM250 fraction from the heparin-agarose column was adjusted to a buffer concentration of TM100, 0.1% Nonidet P-40 (NP-40), and 10 ,ug of either poly(dI-dC) poly(dI-dC) or poly(dA-dT) poly(dA-dT) per ml. The TM250 fraction was applied to the affinity column (equilibrated in TM1000.1% NP-40), and the SBF activity was recovered from the column by elution with TM1000 buffer (same as TM100 buffer, except for 1,000 mM KCl)-0.1% NP-40. This affinitypurified fraction was dialyzed into TM100-0.1% NP-40; when desired, it was concentrated to a volume of 200 to 500 ,ul by spinning in a Centricon-10 microconcentrator (Amicon Corp., Danvers, Mass.). DNA-binding activity was stable for several weeks when the fraction was stored in a liquidnitrogen freezer. DNase I footprint analysis. A DNA fragment containing the wild-type enhancer sequences was end labeled with 32p on either the template or the nontemplate strand at the naturally occurring Asp718 site located at position -246 relative to the start of Ul transcription. The fragment extended from the labeled Asp718 site to a Sau3AI site at position -154. Footprinting reactions were initiated by mixing 25 jxl of DNA probe mixture (containing 20,000 cpm of labeled fragment, 0.5 ,ug of poly(dI-dC) poly(dI-dC), and 4% polyvinyl alcohol) with 25 ,l of protein mixture (variable amounts of affinity-purified SBF made up to 25 ,ul with 25 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid [HEPES] [pH 7.51-100 mM KCl-1 mM dithiothreitol-10 p.M ZnSO4-20%o glycerol-0.1% NP-40). After 30 min of incubation at 200C, 50 ,u1 of 10 mM MgCl2-5 mM CaCl2 was added, followed by the addition of 2 plI (0.1 U) of DNase I (Promega Biotec, Madison Wis.). After 90 s at room temperature, 90 p.l of DNase stop solution (20 mM EDTA [pH 8.0], 1% sodium dodecyl sulfate, 250 mM NaCl, 250 p.g of yeast RNA per ml) was added. The samples were extracted once with phenol and once with chloroform-isoamyl alcohol and ethanol precipitated. After centrifugation, the dried pellets were each suspended in 2 ,ul of formamide loading buffer and electrophoresed in a 10% polyacrylamide gel containing 8 M urea. -
Gel mobility shift assays. DNA fragments were isolated from the various Ul mutant templates by digestion with EcoRI and HindIII. The resulting 420-bp fragments were each labeled by a fill-in reaction with E. coli DNA polymerase (Klenow fragment), [(a-32P]dATP, and nonradioactive dTTP, dCTP, and dGTP. After digestion with Sau3AI, the
70-bp enhancer-containing fragments, spanning positions -219 to -154, were isolated by polyacrylamide gel electrophoresis and used for mobility shift assays as previously described (35), with the following minor modifications. For the assays shown in Fig. 7B and C, poly(dA-dT) poly(dAdT) was used as a nonspecific competitor. To increase the intensity of the signals in Fig. 7C, we ran samples in a thinner gel (1.5- versus 3-mm thickness) and exposed them at -70°C rather than 4°C. -
RESULTS Definition of the borders of the SPH motif. We previously reported that the chicken Ul distal element, located near position -200, acts as a transcriptional activator and is composed of multiple functional motifs (35). The top four rows of Fig. 1 summarize those earlier findings. Note that the octamer and GC box, although required for full enhancer activity, were unable to measurably stimulate Ul gene expression in a construction lacking the SPH motif (pU1A-246dl). By contrast, the SPH motif, which consists of approximately 24 to 28 nucleotides immediately 3' of the octamer, was able to enhance gene expression more than 10-fold even in the absence of the GC box and octamer motif (template pUlA-206 versus template pUlA-188). We therefore set out to define more precisely the functional DNA sequences of the SPH motif. To facilitate our mutational analysis, we first introduced into the Ul enhancer several specific substitution mutations to create unique restriction endonuclease recognition sites at the borders of the SPH motif. The initial mutant constructions are depicted in the lower portion of Fig. 1. The transcriptional activity of the mutant Ul templates was determined by measuring the accumulation of chicken Ul RNA after microinjection of the various plasmid constructions into X. laevis oocytes together with [c-32P]GTP. Figure 2A shows typical results. The relative transcriptional efficiencies of the various templates are summarized in Fig. 1. In agreement with our earlier results (35), a plasmid construction that contained the wild-type Ul enhancer, pU1A-246, was efficiently expressed as compared with a control template, pU1A-188, which lacked most of the enhancer sequences (Fig. 2A, lanes 1 and 2 and lanes 9 and 10). Deletion of the GC box (pU1A-219) or deletion of the GC box plus the octamer motif (pU1A-205) caused less than a twofold reduction in enhancer activity (compare lanes 1, 3, 4, 9, and 15 in Fig. 2A). Most of the substitution derivatives of pU1A-219 (pU1A-219sl, pU1A-219s2, and pUlT) were expressed at a comparable level, which was about half that of the wild-type template (Fig. 2A, lanes 5, 6, 7, 11, and 13). Together, these results indicate that DNA sequences upstream of position -206 and the A residue at nucleotide position -184 are not essential for a relatively high level of enhancer activity under noncompetitive conditions. Alterations of the C residues at positions -205 and -206 had quite disparate effects on Ul gene expression. The C-to-G substitution at position -205 (pUlTsl) caused a reduction of Ul gene expression, whereas a C-to-G substitution at position -206 (pU1A-219s4) had a positive effect on Ul gene expression. Interestingly, an insertion of
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NAME U1A-246 UlA-246dl U1A-206 U1A-188 UlA-219 U1A-205 U1A-219s1 U1-219s2
ZXPRESSION IN RELATIVE
BoxP R Q SMOTV ... GGGGGCGGGGACATGCAAATTAAACCGCGCGCTGCATGCCGGGAGCACqCAC I
COMPETITION WITH U4B
... GGGGGCGGGGACATGCAAAT--------------------------- -CAC
---------gaattCATGCAAATTAAACCGCGCGCTGCATGCCGGGAGCACCAC ---------gaatt--------------CGCGCGCTGCATGCCGGGAGCACCAC ---------gaattCATGCAAATTAAACCGCGCGCTGCATGCCGGGAGCtCCAC ---------gaattCATGCAAATgtACCGCGCGCTGCATGCCGGGAGCACCAC
U1l U1"ls1 U1A-219s4
Yen No _
FIG. 1. Templates used to define the boundaries of the SPH motif in the chicken Ul RNA gene enhancer. The wild-type sequence of the chicken U1-52a gene enhancer region from nucleotide positions -180 to -230 is shown at the top. Sequence motifs involved in enhancer function are delineated by overlining. Wild-type sequences are shown in uppercase letters; sequences that differ from wild-type sequences are shown in lowercase letters and are underlined. Broken lines represent upstream vector DNA or internal deletions within the Ul enhancer sequence. Plasmid nomenclature is as follows: A followed by a negative number, 5'-flanking deletion of Ul sequences to the corresponding endpoint relative to the transcription initiation site; s, substitution mutation; d, internal deletion; i, insertion mutation. P in the pseudo-wild-type construction (pUlP) indicates that the plasmid contains an engineered KpnI-Asp7l8 restriction endonuclease site between the octamer and SPH motifs and a T residue at position -184 to create an SstI site at the 3' border of the SPH motif. Plasmid templates derived from the pseudo-wild-type construction also contain T. Bars to the right of each sequence represent the approximate relative expression levels under noncompetitive conditions (see Fig. 2A). The ability of the various Ul constructions to be expressed when in competition with a chicken U4B RNA gene template is summarized in the far right-hand column (from experiments presented in Fig. 2B). Data for the pU1A-246dl and pU1A-206 templates are taken from Roebuck et al. (35).
four nucleotides between positions -208 and -207 (pUlTiACGT) also increased the level of Ul RNA synthesis (Fig. 2A, lane 16). Finally, the Ul deletion template pUlTsldl, which lacked DNA sequences in the SPH motif from positions -197 to -183, was not detectably expressed in the oocyte assay, even though the octamer motif remained intact. These results extend our earlier findings (35) demonstrating that the SPH motif, which spans approximately positions -206 to -183, is essential for Ul enhancer function in oocytes. Sequence requirements for the formation of a stable transcription complex. We and others have shown that the snRNA gene enhancer is required for the formation of a transcription complex that is stable to challenge by a second snRNA transcription unit (24, 25, 27, 35, 41). Although the SPH motif is essential for chicken Ul enhancer activity, it is insufficient for the assembly of a stable transcription complex on a construction lacking both the GC box and octamer motif (35). To further investigate the sequence requirements for stable complex formation, we coinjected a constant amount of the chicken U4B RNA gene (16, 25) into oocytes with an eightfold molar excess of either the wild-type Ul template or one of the various mutant Ul templates (Fig. 2B). A summary of these results is shown in Fig. 1. Both the wild-type Ul template, pU1A-246 (which contained an intact enhancer), and the pU1A-219 template (which lacked a GC box) were efficiently expressed in the competition assay as compared with control templates. Similarly, the various Ul templates that contained point mutations at the borders of the SPH motif were each efficiently expressed in the competition assay. In general, the relative levels of Ul expression paralleled those observed under noncompetitive conditions (compare Fig. 2A and B). In contrast, the Ul
template pU1A-205, which lacked the octamer motif, was not detectably expressed in the competition assay even though it was highly expressed under noncompetitive conditions (compare Fig. 2B, lane 3, with Fig. 2A, lanes 4 and 15). These results indicate that the octamer and SPH motifs are both required for the formation of an active stable snRNA transcription complex. Effect of internal deletions within the SPH motif on Ul enhancer activity. The U1T plasmid construction, which we will refer to as the pseudo-wild-type template, was used to construct the series of deletion, substitution, and insertion templates shown in Fig. 3. Figure 4A shows typical results obtained when the different mutant pUlT templates were expressed by injection into X. laevis oocytes. The relative transcriptional efficiencies of the various templates are summarized in Fig. 3. A Ul deletion template, pUlPdl, which lacked four nucleotides at the SstI restriction site, was expressed in oocytes at approximately the same level as the pseudowild-type template (compare lanes 1 and 8 in Fig. 4A). This result indicates that the DNA sequences 3' of position -188 are not essential for function when upstream sequences of the enhancer remain intact. Likewise, an internal deletion of five nucleotides at the BssHII site (pUlTd2; Fig. 4A, lane 6) had a relatively minor effect on Ul gene expression. On the other hand, an internal deletion of four nucleotides at the SphI site (pUlTd3) had a more severe effect on enhancer activity (Fig. 4A, lane 7). Larger deletions in the SPH motif or deletions extending into the octamer motif resulted in templates with very little or nondetectable transcriptional activity (pUlTd4 through pUlPdll and pUlTA-193). Together, these results indicate first that sequences important for Ul enhancer activity are spread throughout the SPH
A Expression (no competition) c
Ir- c N CM C%
B Expression (competition with U4B)
a) In) It .- Co
C'J -r-C%J CM
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qw 40 -*_ ,D 40 *
7 8 9 10 11 1213 1 2 34 5 6 9 10 1112131415 1617 1 2 3 4 5 6 7 8 FIG. 2. Effect of mutations in the Ul enhancer region on Ul RNA gene expression in X. laevis oocytes. Oocytes were injected with [a-32P]GTP and the Ul mutant templates shown in Fig. 1 (except for pUlPs7, which is shown in Fig. 3). Total RNA was isolated 18 to 24 h later from pooled oocytes, run in 8 or 10% denaturing polyacrylamide gels, and subjected to autoradiography. In all injections, an X. laevis 5S RNA gene plasmid (0.4 ±ug/ml) was coinjected as a positive internal control to normalize (based upon short autoradiographic exposures) for the efficiency of injection into the oocyte nuclei. (A) Ul RNA gene expression under noncompetitive conditions. The plasmids injected are indicated above each lane. Lanes 1 through 8 and lanes 9 through 17 represent different sets of injection experiments. The bands corresponding to Ul RNA and 5S RNA are indicated. (B) Ul RNA gene expression in competition with a chicken U4B RNA gene. The injected Ul plasmids which gave rise to the corresponding RNA samples are indicated above each lane. In each case, the Ul gene plasmid was in eightfold molar excess over the coinjected wild-type U4B gene plasmid. Lanes 1 through 6 and lanes 7 through 13 represent different sets of injection experiments. The bands corresponding to Ul RNA, U4B RNA, and 5S RNA are indicated.
motif and second that the octamer and SPH motifs work together to enhance Ul gene expression. Finally, a template that contained the enhancer sequence in the inverse orientation (pUlTinv) was expressed in oocytes (Fig. 4A, lane 9). Thus, the chicken Ul enhancer, like mRNA enhancers, is capable of stimulating gene expression in either orientation. We also measured the transcriptional activity of selected pUlT deletion mutations under conditions of competition with the wild-type chicken U4B RNA gene (Fig. 4B). Under these competition conditions, the pseudo-wild-type template and the deletion template which lacked four nucleotides at the SstI site (pUlTdl) were both efficiently expressed (compare lanes 1, 2, and 3 in Fig. 4B). In contrast, Ul deletion templates lacking sequences at either the BssHII or the SphI site were expressed very poorly in competition with U4B (Fig. 4B, lanes 4, 5, and 6). The template containing an inverted enhancer was also expressed at a reduced level as compared with the wild type (Fig. 4B, lane 7). Taken together, the data shown in Fig. 4 indicate that DNA sequences comprising the SphI site and, to a lesser degree, the BssHII site not only are important for a high level of Ul RNA gene expression but also contribute to the formation of a stable transcription complex. Effect of substitutions and insertions within the SPH motif. In the internal deletion templates discussed above, the spacing between potential functional motifs in the enhancer is generally not preserved. Moreover, sequences that are normally not adjacent in the wild-type enhancer become juxtaposed. To exclude any potential complications that
might arise from these effects, we also examined a series of templates containing substitutions at the BssHII and/or SphI sites. Substitution mutations at either the BssHII site (pUlTs2) or the SphI site (pUlTs4) significantly reduced Ul gene expression, with the substitution at the SphI site having the greater effect (Fig. 5). Similarly, a mutation (pUlTs7) which altered the sequences spanning both restriction sites reduced Ul gene expression to an essentially nondetectable level. The Ul template (pUlTs5) that contained substitutions at the BssHII site plus a four-nucleotide deletion at the SstI site was very weakly expressed, even though an identical four-nucleotide deletion at the SstI site in the pseudowild-type enhancer failed to reduce Ul gene expression (pUlPdl; Fig. 4A, lane 8). Thus, the functional contribution of the nucleotides at the SstI site became apparent only when the activity of the SPH motif was already partially impaired by upstream alterations at either the BssHII (pUls5) or the SphI (pUlMd5) site. The above-mentioned results indicate that the DNA sequences from positions -206 to -192 are crucial for enhancer activity. This region of the SPH motif consists of two related alternating purine-pyrimidine sequences (the BssHII and SphI sites). To examine whether enhancer activity could be directly correlated with the length of perfectly alternating purine-pyrimidine tracts, we constructed three templates that contained a single-nucleotide insertion (G, C, or T) between the BssHII and SphI sites. Whereas the insertion of a C or T does not increase the alternating character of the region, the insertion of a G results in a 15-bp stretch of
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