(Received for publication, July 31, 1995)
From the
The two U4 genes in the chicken genome code for distinct sequence variants of U4 small nuclear RNA that are differentially expressed during development. Whereas U4B RNA is constitutively expressed, U4X RNA is specifically down-regulated relative to U4B in a tissue-specific manner during development. To investigate mechanisms controlling the differential expression of the U4B and U4X genes, chimeric U4 genes were constructed and their transcriptional activities assayed by injection into Xenopus oocytes or by transfection of CV-1 cells. The proximal regulatory region of the U4B gene and the enhancers of both the U4B and U4X genes functioned efficiently in each expression system. However, the proximal region of the U4X gene was inactive. To localize and identify the responsible nucleotides, reciprocal point mutations were introduced into the U4X and U4B proximal regulatory regions. The results indicate that the U4X gene contains a suboptimal proximal sequence element, and that this results primarily from the identities of the nucleotides at positions -61 and -57 relative to the transcription start site.
The small nuclear RNAs (snRNAs) ()of the U family
(U1, U2, U3, etc.) are evolutionarily conserved and metabolically
stable RNAs that function in pre-mRNA and pre-rRNA processing in the
nuclei of eukaryotic cells. The genes for vertebrate snRNAs possess
similar promoter structures and, with the exception of U6, are
transcribed by RNA polymerase II. Two distinct evolutionarily conserved
regions in the 5`-flanking DNA contribute to the expression of
vertebrate snRNA genes(1, 2, 3, 4) .
The more distal region, generally positioned between nucleotides
-250 and -180 relative to the transcription start site,
functions as a transcriptional enhancer. It normally contains at least
one copy of the octamer motif (recognized by the Oct-1 transcription
factor), and it usually contains functional binding sites for one or
more additional proteins, such as a GC box, SPH motif, CCAAT box,
cyclic AMP response element,
etc.(5, 6, 7, 8, 9, 10, 11, 12) .
The proximal sequence element (PSE) is an essential promoter element centered near position -55 and is required for accurate initiation of snRNA transcription. The PSE is recognized by a transcription factor variously named PSE binding protein(13, 14) , PSE transcription factor (15) , or snRNA activating protein complex(16) . The PSE binding factor is believed to play a role in recruiting the TATA box-binding protein to snRNA promoters(16, 17, 18, 19) .
In the chicken, there are two and only two genes that code for U4 snRNA (20, 21) . These genes, designated U4B and U4X, are closely linked within 500 base pairs (bp) of each other in the chicken genome. The two genes encode distinct sequence variants of U4 RNA that differ at seven nucleotide positions. Moreover, the relative amount of U4X and U4B RNA is differentially regulated during chicken development. The U4X and U4B genes are expressed at nearly equal levels during early embryogenesis, but U4X gene expression is specifically down-regulated in a tissue-specific manner relative to U4B as development proceeds (22) .
Although there is no direct evidence linking the differential expression of the chicken U4X and U4B RNA genes to transcriptional control, their promoter structures are consistent with such a mechanism. First there is no obvious similarity between the U4B and U4X gene 5`-flanking DNA other than the PSE and the enhancer region. Second, both the enhancer and PSE contain a number of base differences in crucial regulatory regions that could affect the relative expression of the two U4 genes(20, 23) . Third, just 5` of, and partially overlapping, the U4X PSE there exists a unique dyad symmetry element that is not found in the U4B proximal region. This palindromic sequence is recognized by a sequence-specific DNA binding factor, proximal palindrome binding factor, whose DNA binding activity is inversely related to the accumulation of U4X RNA(23) .
To gain further insight into the molecular basis for the differential expression of the U4X and U4B genes, we have generated constructs that possess chimeric U4B/U4X promoters, including families of constructs with reciprocal point mutations in the PSE and dyad symmetry elements. We find that the proximal regulatory region, not the enhancer, is responsible for the differential activity of the U4B and U4X genes in transient expression assays. Moreover, we identify two specific nucleotide positions that differ between the U4X and U4B PSEs that are crucial for U4 snRNA gene expression in these assays.
To construct template BXX, U4B sequences from -227 to -142 were joined to position -132 of the U4X gene. Template -BX was constructed by deleting U4B sequences from positions -280 to -118 in the 5`-flanking region of the BBX construct. Template XBX contained, in order, U4X sequences from -323 to -97, two nucleotides (AT) of linker sequence, and U4B 5`-flanking DNA from position -117 to the beginning of the gene, followed by the U4X RNA coding region and 3`-flanking DNA.
To construct the U4B
maxigene, 17 nucleotides from the pUC19 polylinker were inserted
between positions 8 and 9 of the U4B gene as follows. The construct
U4B-227 (9) was cut with EcoRI and HindIII, and the fragment containing the U4B gene sequence was
isolated. This fragment was then cut with restriction endonuclease FspI, giving blunt ends between nucleotides 8 and 9 of the U4B
DNA coding region. These fragments were then cloned between the EcoRI and SmaI sites and between the HindIII
and HincII sites of the pUC19 vector. Thus the plasmid
polylinker sequence between the HincII and SmaI sites
became a part of the U4B maxigene transcript. The U4mB/AS template was
constructed by exchanging the 5`-flanking DNA of U4mB with that of
U4B/AS(24) . The remaining U4X and U4B constructs that
contained point mutations were generated by site-directed mutagenesis
using polymerase chain reaction or by cloning synthetic
oligonucleotides between pre-existing restriction sites.
The primer extension
reactions were performed as follows: 5 ng of the specific primer
(500,000 cpm) was mixed with 10 µg of total RNA in 10 µl
of 1
annealing buffer solution (250 mM KCl, 10 mM Tris-Cl, pH 8.3). The mixture was heated at 80 °C for 3 min
and transferred to a 45 °C water bath for 45 min. After 45 min of
annealing, 10 µl of ice-cold nuclease-free water was added to each
sample. To 10 µl of the annealed primer/RNA mixture, 12 µl of
reverse transcription mixture (24 mM Tris-Cl, pH 8.3, 16
mM MgCl
, 0.8 mM each dNTP, 8 mM
dithiothreitol, 125 ng actinomycin D/µl, and 1.5 units of avian
myeloblastosis virus reverse transcriptase/µl) were added. This
mixture was incubated at 45 °C for 45 min to allow extension of the
primer. The reaction was stopped by adding 8 µl of stop solution
(100% formamide with 0.3% bromphenol blue and 0.3% xylene cyanol FF).
The samples were boiled for 3 min and then loaded on either a 20 or 10%
denaturing polyacrylamide gel for analysis of U4maxiB or U4X RNA,
respectively. The labeled bands were detected by autoradiography and
quantitated by PhosphorImager analysis.
Figure 1:
Transcriptional activities of chimeric
U4 gene constructs. A, chimeric constructs (diagrammed in C) were expressed by injection into the nuclei of Xenopus oocytes together with [-
P]GTP to label
the synthesized RNA. A 5 S RNA gene plasmid was coinjected to serve as
an internal control for injection efficiency and sample recovery. RNA
was isolated on the day following injection and aliquots were
electrophoresed through a denaturing polyacrylamide gel. Bands
corresponding to U4B or U4X RNA and to 5 S RNA are indicated. B, four of the chimeric constructs were transfected into CV-1
cells, total RNA was isolated, and the accumulation of U4X RNA was
assayed by primer extension using a primer specific for chicken U4X
RNA. The upper U4X band represents the full-length U4X
extension product, and the lower band corresponds to a shorter
product resulting from a strong stop for reverse
transcriptase(21) . C, a diagrammatic representation
of the chimeric U4 constructs is pictured to the left.
Relative expression levels in oocytes or in CV-1 cells is shown in the
corresponding columns to the right (nt, not tested). All
values are normalized to a U4XBX expression efficiency of
100%.
More definitive conclusions were obtained from experiments employing the chimeric constructs that combined the U4B enhancer with the U4X proximal region, and vice versa. The U4B enhancer region was unable to activate U4X gene expression from a template containing the U4X proximal region (BXX, Fig. 1A, lane 3). In contrast, the U4X enhancer fully supported a high level of transcriptional activity from a template containing the U4B proximal sequence (XBX, cf. lanes 8 and 9 versus lane 7). Taken together, these results indicate that the U4X proximal region, not the enhancer, is the regulatory region primarily responsible for the inactivity of the U4X gene in frog oocytes.
We next examined the relative activities of the enhancer and proximal regions of the U4X and U4B genes in a different expression system. Four of the chimeric constructs that contain the U4X gene coding region were transfected into monkey CV-1 cells, total RNA was prepared, and the accumulation of U4X RNA was assayed by primer extension using a 17-mer primer complementary to the 3` end of U4X RNA. This primer was chosen because it does not cross-hybridize with any form of U4 RNA found in CV-1 cells(21, 22) . As shown in Fig. 1B, the two constructs that contained the U4X proximal region, XXX and BXX, were not detectably expressed (lanes 1 and 3). In contrast, the two constructs that contained the U4B proximal region, BBX and XBX, were efficiently expressed (lanes 2 and 4). Overall, the results in Fig. 1indicate that the U4X proximal region is unable to support transcription in either frog oocytes or monkey CV-1 cells, whereas the U4B gene proximal region is an efficient promoter in both expression systems.
Figure 2: Transcriptional activity of U4X gene constructs with base substitutions in the proximal region. Following transfection of CV-1 cells and isolation of total RNA, primer extension analysis of U4X RNA was carried out as in Fig. 1B, using the U4XBX construct as a positive control for normalization to 100% efficiency. The sequence alignment shows the point mutations that were introduced into the PSE region of the U4X gene, gradually converting it into identity with the U4B PSE. Dashes indicate nucleotides identical to those in the wild type U4X sequence, whereas differences are explicitly shown as lower case letters in the mutant U4X constructs or as capital letters in the wild type U4B proximal region sequence (bottom line). A 16-bp dyad symmetry element (palindromic sequence) that overlaps the U4X PSE is underlined. Note that when the U4X and U4B PSE sequences are aligned to maximize homology, the U4B PSE is two nucleotides closer to the transcription start site. (The numbering above the sequence alignment indicates U4X nucleotide positions, and the numbering below is for U4B).
The U4mB/AS construct was efficiently expressed in CV-1 cells (Fig. 3, lanes 1 and 7). However, a single C to T point mutation at position -55 resulted in a 10-fold reduction in promoter activity (lane 2) and a single T to G change at position -59 resulted in a 6-fold reduction (lane 3). When the two point mutations were combined (lane 4), the residual level of expression was very low and was detectable only on long exposures or by PhosphorImager analysis.
Figure 3: Transcriptional activity of U4maxiB gene constructs having mutations within or near the PSE. Total RNA was prepared from transfected CV-1 cells and primer extension analysis was carried out using a U4maxiB specific primer. The sequence alignment at the bottom shows the alterations from the U4mB/AS sequence shown in the top line. The underlined regions in the U4mB/pal and U4mB/pal59 sequences indicate a 16-bp palindromic sequence identical to that found upstream of the U4X PSE. Expression levels relative to U4mB/AS (taken as 100%) are shown in the column to the right.
In an attempt to divorce the effect of the nucleotide substitutions at positions -55 and -59 from potential regulatory effects of the palindromic sequence, a construct was made in which the palindrome was displaced 4 bp in the 5` direction. In this construct (U4mB/pal59), the wild type U4B PSE sequence was maintained to position -63, and the complete palindromic sequence extended 16 bp upstream beginning at position -59. Fig. 3, lane 10, shows that this construct was expressed at a wild type level in CV-1 cells. Moreover, a construct that was mutated at six positions within the palindromic sequence was expressed at a similar level (lane 11). Thus, expression of the U4B gene in CV-1 cells was not significantly affected by either the presence or the absence of the palindromic sequence near, but not extensively overlapping, the U4B PSE.
We have investigated the cis-acting DNA sequences important for the differential expression of the U4X and U4B genes in frog oocytes and monkey CV-1 cells. The chicken U4B gene was efficiently expressed in both oocytes and CV-1 cells, whereas the U4X gene was not detectably expressed in either system. To localize the DNA sequences responsible for this differential activity, enhancer and promoter swap experiments were carried out using chimeric constructs. The results indicated that the proximal region of the U4X gene was inactive in both transient expression systems.
To define the specific nucleotides responsible for the difference in activity, point mutations were introduced into the U4X proximal region to determine whether specific changes (designed to match the sequence of the U4B PSE) could rescue U4X expression. Indeed, the alteration of two nucleotides at positions -57 and -61 was sufficient to raise the activity of the U4X PSE to a level comparable to that of the U4B PSE (Fig. 2). Moreover, some increased level of activity was alternatively obtained by replacing the U4X sequences between -52 and -37 with the corresponding nucleotides of the U4B PSE. These findings strongly suggest that the U4X gene contains a suboptimal PSE sequence, and that the nucleotides responsible for this effect are to some extent delocalized throughout the PSE. However, it is clear that the greatest down-effects on expression result from the presence of a G at position -61 and a T at position -57.
The importance of the bases of these two positions was confirmed by making the reciprocal changes at the homologous positions in the U4B PSE (positions -59 and -55). These two nucleotide changes resulted in 10- and 6- fold reductions in the U4B PSE activity, respectively, when the mutations were assayed individually, and, when present together in the same construct, resulted in essentially complete loss of activity (Fig. 3). It is perhaps significant that none of the other cloned chicken U1, U2, or U4B genes contain either a G or a T at these respective positions(20) . Our data localizing the critical nucleotides to the proximal region are reminiscent of studies of developmentally-regulated sea urchin U2 RNA genes performed by Stefanovic and Marzluff(25) . In those studies, the sequences responsible for the differential expression of the early and late U2 genes was localized to a 14-bp region within the U2 PSE.
The two critical nucleotide positions identified above are not only located within the U4X PSE, but they also are members of a 16-bp dyad symmetry element that overlaps the U4X PSE. Thus, in addition to disrupting DNA recognition by the PSE-binding factor, mutations at these positions could also affect DNA recognition by another factor, proximal palindrome binding factor, that binds specifically to the palindromic element(23) . In an attempt to separate and distinguish between these two potential effects, a construct was prepared that contained an intact palindromic sequence upstream of the U4B PSE (U4mB/pal59, Fig. 3). In this construct, the wild type U4B PSE was maintained, but the palindromic sequence was displaced 4 bp upstream relative to its homologous position in the wild type U4X proximal region. As shown in Fig. 3, the palindromic sequence at this position had no significant effect on expression of the U4mB transcript. Thus, in this transient expression system, U4 gene transcription was more closely correlated with the identities of the nucleotides at U4B positions -59 and -55 (U4X positions -61 and -57) rather than with the presence or the absence of the intact palindromic sequence. Nonetheless, it remains a possibility that the palindromic sequence in its natural chromosomal context in chicken cells may play a role in regulating the developmental expression of the U4X gene in vivo. Such effects may not be observable in the heterologous transient expression systems investigated here. Another possibility, not addressed by our experiments, is that U4X gene activity conceivably could be regulated in response to the methylation state of the multiple CpG dinucleotides in the palindromic sequence (CGCGCCGGCGCG)(26) .
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