The Drosophila U1 and U6 Gene Proximal Sequence Elements Act as Important Determinants of the RNA Polymerase Specificity of Small Nuclear RNA Gene Promoters in Vitroand in Vivo

Abstract

Transcription of genes coding for metazoan spliceosomal snRNAs by RNA polymerase II (U1, U2, U4, U5) or RNA polymerase III (U6) is dependent upon a unique, positionally conserved regulatory element referred to as the proximal sequence element (PSE). Previous studies in the organism Drosophila melanogasterindicated that as few as three nucleotide differences in the sequences of the U1 and U6 PSEs can play a decisive role in recruiting the different RNA polymerases to transcribe the U1 and U6 snRNA genesin vitro. Those studies utilized constructs that contained only the minimal promoter elements of the U1 and U6 genes in an artificial context. To overcome the limitations of those earlier studies, we have now performed experiments that demonstrate that theDrosophila U1 and U6 PSEs have functionally distinct properties even in the environment of the natural U1 and U6 gene 5′-flanking DNAs. Moreover, assays in cells and in transgenic flies indicate that expression of genes from promoters that contain the “incorrect” PSE is suppressed in vivo. TheDrosophila U6 PSE is incapable of recruiting RNA polymerase II to initiate transcription from the U1 promoter region, and the U1 PSE is unable to recruit RNA polymerase III to transcribe the U6 gene. Transcription of genes coding for metazoan spliceosomal snRNAs by RNA polymerase II (U1, U2, U4, U5) or RNA polymerase III (U6) is dependent upon a unique, positionally conserved regulatory element referred to as the proximal sequence element (PSE). Previous studies in the organism Drosophila melanogasterindicated that as few as three nucleotide differences in the sequences of the U1 and U6 PSEs can play a decisive role in recruiting the different RNA polymerases to transcribe the U1 and U6 snRNA genesin vitro. Those studies utilized constructs that contained only the minimal promoter elements of the U1 and U6 genes in an artificial context. To overcome the limitations of those earlier studies, we have now performed experiments that demonstrate that theDrosophila U1 and U6 PSEs have functionally distinct properties even in the environment of the natural U1 and U6 gene 5′-flanking DNAs. Moreover, assays in cells and in transgenic flies indicate that expression of genes from promoters that contain the “incorrect” PSE is suppressed in vivo. TheDrosophila U6 PSE is incapable of recruiting RNA polymerase II to initiate transcription from the U1 promoter region, and the U1 PSE is unable to recruit RNA polymerase III to transcribe the U6 gene. small nuclear RNA proximal sequence element proximal sequence element A proximal sequence element B Drosophila melanogaster PSEA-binding protein Genes coding for most of the small nuclear RNAs (snRNAs)1 are transcribed by RNA polymerase II, but U6 genes are transcribed by RNA polymerase III. In all cases, however, the promoters of these genes have features that are very similar to each other yet distinct from “classical” RNA polymerase II and RNA polymerase III promoters. In most organisms studied, transcription of U1, U2, U4, and U5 genes by RNA polymerase II or U6 genes by RNA polymerase III requires a unique proximal sequence element (PSE) that is located upstream of position −40 relative to the transcription start site (Fig.1A) (1Hernandez N. McKnight S.L. Yamamoto K.R. Transcriptional Regulation. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1992: 281-313Google Scholar, 2Lobo S.M. Hernandez N.T. Conaway R.C. Conaway J.W. Transcription: Mechanisms and Regulation. Raven Press, New York1994: 127-159Google Scholar, 3Stefanovic B. Marzluff W.F. Mol. Cell. Biol. 1992; 12: 650-660Crossref PubMed Scopus (14) Google Scholar, 4Wendelburg B.J. Marzluff W.F. Nucleic Acids Res. 1992; 20: 3743-3751Crossref PubMed Scopus (13) Google Scholar, 5Li J.-M. Parsons R.A. Marzluff W.F. Mol. Cell. Biol. 1994; 14: 2191-2200Crossref PubMed Google Scholar, 6Das G. Henning D. Reddy R. J. Biol. Chem. 1987; 262: 1187-1193Abstract Full Text PDF PubMed Google Scholar, 7Zamrod Z. Tyree C.M. Song Y. Stumph W.E. Mol. Cell. Biol. 1993; 13: 5918-5927Crossref PubMed Scopus (23) Google Scholar, 8Vankan P. Filipowicz W. EMBO J. 1989; 8: 3875-3882Crossref PubMed Scopus (48) Google Scholar, 9Waibel F. Filipowicz W. Nucleic Acids Res. 1990; 18: 3451-3458Crossref PubMed Scopus (74) Google Scholar, 10Dahlberg J.E. Lund E. Birnstiel M.L. Structure and Function of Major and Minor Small Nuclear Ribonucleoprotein Particles. Springer Verlag KG, Heidelberg, Federal Republic of Germany1988: 38-70Crossref Google Scholar, 11Parry H.D. Scherly D. Mattaj I.W. Trends Biochem. Sci. 1989; 14: 15-19Abstract Full Text PDF Scopus (98) Google Scholar, 12Dahlberg J.E. Lund E. Science. 1991; 254: 1462-1463Crossref PubMed Scopus (23) Google Scholar). In vertebrates, the PSEs of U1 and U2 genes are functionally interchangeable with the PSEs of U6 genes (13Lobo S.M. Hernandez N. Cell. 1989; 58: 55-67Abstract Full Text PDF PubMed Scopus (176) Google Scholar, 14Parry H.D. Tebb G. Mattaj I.W. Nucleic Acids Res. 1989; 17: 3633-3644Crossref PubMed Scopus (37) Google Scholar). That is, if the PSE of the U6 promoter is replaced with the U1 or U2 PSE, there is no effect on the RNA polymerase III specificity of the U6 promoter. Likewise, the U6 PSE can functionally substitute for the U1 or U2 PSE in the vertebrate U1 and U2 promoters for transcription by RNA polymerase II. In echinoderms and plants, the U1, U2, and U6 PSEs (called USEs in plants) are similarly functionally interchangeable with each other (15Li J.-M. Haberman R.P. Marzluff W.F. Mol. Cell. Biol. 1996; 16: 1275-1281Crossref PubMed Google Scholar, 16Waibel F. Filipowicz W. Nature. 1990; 346: 199-202Crossref PubMed Scopus (74) Google Scholar, 17Kiss T. Marshallsay C. Filipowicz W. Cell. 1991; 65: 517-526Abstract Full Text PDF PubMed Scopus (87) Google Scholar). The RNA polymerase III specificity of vertebrate U6 snRNA genes is determined by the presence of a TATA box at a fixed distance downstream of the PSE (2Lobo S.M. Hernandez N.T. Conaway R.C. Conaway J.W. Transcription: Mechanisms and Regulation. Raven Press, New York1994: 127-159Google Scholar, 13Lobo S.M. Hernandez N. Cell. 1989; 58: 55-67Abstract Full Text PDF PubMed Scopus (176) Google Scholar, 18Mattaj I.W. Dathan N.A. Parry H.D. Carbon P. Krol A. Cell. 1988; 55: 435-442Abstract Full Text PDF PubMed Scopus (118) Google Scholar). Paradoxically, the promoters of the vertebrate snRNA genes transcribed by RNA polymerase II lack TATA boxes (Fig.1 A). In plants, on the other hand, snRNA genes transcribed by both RNA polymerases have essential TATA boxes in their promoters. In this case, the choice of RNA polymerase is determined by a 10-base pair difference in the spacing between the USE and the respective TATA box (Fig. 1 A) (16Waibel F. Filipowicz W. Nature. 1990; 346: 199-202Crossref PubMed Scopus (74) Google Scholar, 17Kiss T. Marshallsay C. Filipowicz W. Cell. 1991; 65: 517-526Abstract Full Text PDF PubMed Scopus (87) Google Scholar, 19Goodall G.J. Kiss T. Filipowicz W. Oxf. Surv. Plant Mol. Cell Biol. 1991; 7: 255-296Google Scholar). In contrast to the organisms described above, in vitroexperiments carried out in the Drosophila melanogastersystem indicated that the sequence of the PSE itself plays a major role in determining the RNA polymerase specificity of snRNA gene promoters in insects (20Jensen R.C. Wang Y. Hardin S.B. Stumph W.E. Nucleic Acids Res. 1998; 26: 616-622Crossref PubMed Scopus (39) Google Scholar). The basal promoter elements of DrosophilasnRNA genes are shown in the last two lines of Fig. 1 A. TheDrosophila proximal sequence element A (PSEA) is a 21-base pair conserved sequence that is analogous to the vertebrate PSE in that it is required for a basal level of transcription and is involved in specifying the transcription start site (7Zamrod Z. Tyree C.M. Song Y. Stumph W.E. Mol. Cell. Biol. 1993; 13: 5918-5927Crossref PubMed Scopus (23) Google Scholar). The U6 genes ofDrosophila, like those of other organisms, contain canonical TATA boxes. The proximal sequence element B (PSEB), which is found inDrosophila RNA polymerase II-transcribed snRNA genes, has no obvious counterpart in other organisms. Finally, the spacing of the PSEA from the PSEB (8 base pairs) or from the TATA box (12 base pairs) differs between the two classes of Drosophila genes. Fig.1 B shows typical sequences of the variousDrosophila U1 and U6 promoter elements. In a previous study, the cis-acting determinants of RNA polymerase specificity in the Drosophila system were studied by transcribing artificial templates that contained all possible combinations of U1 or U6 PSEA, PSEB, or TATA box and 8 or 12 base pair inter-element spacing (20Jensen R.C. Wang Y. Hardin S.B. Stumph W.E. Nucleic Acids Res. 1998; 26: 616-622Crossref PubMed Scopus (39) Google Scholar). The results of those in vitrotranscription experiments indicated that the PSEB and TATA sequences were essentially interchangeable in terms of their effects on RNA polymerase specificity (20Jensen R.C. Wang Y. Hardin S.B. Stumph W.E. Nucleic Acids Res. 1998; 26: 616-622Crossref PubMed Scopus (39) Google Scholar). Similarly, the difference in the spacing between the elements affected primarily the efficiency of transcription rather than RNA polymerase specificity. The major determinant of RNA polymerase specificity lay within the sequence of the PSEA itself (20Jensen R.C. Wang Y. Hardin S.B. Stumph W.E. Nucleic Acids Res. 1998; 26: 616-622Crossref PubMed Scopus (39) Google Scholar), even though the U1 and U6 PSEAs differed by only a few nucleotides (Fig. 1 B). To thoroughly investigate the role of the PSEA as a determinant of RNA polymerase specificity, we felt that it was important to interchange the U1 and U6 PSEAs within their natural promoter contexts and to examine the effects of such an exchange on promoter activity in living cells. These experiments validate the notion that the U1 and U6 PSEAs are functionally distinct in terms of their to transcription by RNA polymerase II or RNA polymerase the in however, the of from promoters that contain the PSEA is suppressed in vivo. The templates that contained the U1 and U6 promoter sequences have described (7Zamrod Z. Tyree C.M. Song Y. Stumph W.E. Mol. Cell. Biol. 1993; 13: 5918-5927Crossref PubMed Scopus (23) Google Scholar, Y. R.C. Stumph W.E. Nucleic Acids Res. 1996; PubMed Scopus Google Scholar). The U1 contained sequences from the gene from position to position The U6 in transcription contained sequences of theDrosophila gene from position to position To each PSEA sequence to that of the other each was at natural that the respective PSEA, and the sequence was replaced with that contained the PSEA sequence of the other gene base in the 21-base pair transcription and by were performed as described (7Zamrod Z. Tyree C.M. Song Y. Stumph W.E. Mol. Cell. Biol. 1993; 13: 5918-5927Crossref PubMed Scopus (23) Google Scholar, Y. Song Y. Wang Y. Stumph W.E. J. Biochem. PubMed Scopus Google Scholar). The was a from of and of contained an of base at position in the U6 RNA coding To the U6 PSEA in this with the U1 or PSEA, the was with which on of the the was replaced with that contained the U1 PSEA sequence or a sequence that was at nucleotide relative to the U6 These were by base of the this in a between nucleotides and of the U6 coding The were to cells in with were to in and was with of of the U6 of an that contained the gene by a Drosophila and 12 of were A of each of cells was out and for assays to for relative RNA was from the of the cells RNA and were for expression The nucleotides of the were to the U6 snRNA and the last were to the of the were on and were by gene sequences between and were the to those found in the U6 PSEA were the U1 PSEA at and as or in 1 and by the U1 promoter with and that on of the U1 that contained the sequences were to different U1 or U6 or PSEAs in the U1 as indicated in Fig. assays to cells were in with of of the of from D. as an for and of to the of to cells were were determined cells were in with of of the of a as an from R. of and Cell of and of were carried out as described a to sequences the of the gene. nucleotides in was for at position of the U1 gene. expression from the constructs were determined by of the The gene was from the Drosophila as EMBO J. PubMed Google by polymerase with that the and an upstream site and a was site of such that the site of the was upstream of the the A U1 promoter sequences from to was from the described in the by with and was in both the and site of the to the constructs and The in which the U6 PSEA replaced the U1 PSEA in the U1 promoter region, was in a similar of cells was carried out in of or The was to the of to cells were to and in The and of the cells were by a of at which the was constructs for of flies with gene by the U1 promoter were as The constructs and described were each with that the downstream of the U1 which contained the U1 or U6 These two were between and of the that a of the gene Drosophila Scholar). of flies Science. PubMed Scopus Google was carried out by at the with of the two in the flies was to lines for the To the of the lines to and of the were in and to lay on that contained flies were and the of to were and Previous transcription studies that the role of cis-acting elements in determining RNA polymerase specificity utilized templates that contained only the essential Drosophila U1 or U6 promoter elements and most of the sequences in the U1 or U6 gene 5′-flanking (20Jensen R.C. Wang Y. Hardin S.B. Stumph W.E. Nucleic Acids Res. 1998; 26: 616-622Crossref PubMed Scopus (39) Google Scholar). Those studies indicated that the U1 and U6 PSEAs as the major determinants of RNA polymerase specificity in vitro. we to this in the of the natural U1 and U6 gene 5′-flanking DNAs. To examine the effect of the U6 PSEA the of the U1 or the U1 PSEA the of the U6 the constructs at the of These were as described a nuclear protein for both RNA polymerase II and RNA polymerase III transcription Y. Song Y. Wang Y. Stumph W.E. J. Biochem. PubMed Scopus Google Scholar). The were by (7Zamrod Z. Tyree C.M. Song Y. Stumph W.E. Mol. Cell. Biol. 1993; 13: 5918-5927Crossref PubMed Scopus (23) Google Scholar, Y. Song Y. Wang Y. Stumph W.E. J. Biochem. PubMed Scopus Google Scholar), the U1 gene was by RNA polymerase II as indicated by the of the transcription to the RNA polymerase II but to the RNA polymerase III (Fig. the U1 PSEA was to a U6 PSEA a pair the transcription the promoter specificity was to RNA polymerase III (Fig. was indicated by the of the transcription to but to a pair that the U1 PSEA to a U6 PSEA was to the promoter specificity from RNA polymerase II to RNA polymerase even though the of the promoter and the transcription start site was from the U1 gene. The results of the are shown in Fig. Y. Song Y. Wang Y. Stumph W.E. J. Biochem. PubMed Scopus Google Scholar), the U6 gene was transcribed by RNA polymerase III the U6 PSEA was to a U1 PSEA, transcription by RNA polymerase II was transcription of this by RNA polymerase III was is the was from an analogous artificial promoter (20Jensen R.C. Wang Y. Hardin S.B. Stumph W.E. Nucleic Acids Res. 1998; 26: 616-622Crossref PubMed Scopus (39) Google Scholar). The for the RNA polymerase III activity of this is the TATA sequence by itself transcription by RNA polymerase III as by RNA polymerase that is of the PSEA Y. R.C. Stumph W.E. Nucleic Acids Res. 1996; PubMed Scopus Google Scholar). the U1 PSEA very at a pair spacing from the downstream PSEB or TATA box (20Jensen R.C. Wang Y. Hardin S.B. Stumph W.E. Nucleic Acids Res. 1998; 26: 616-622Crossref PubMed Scopus (39) Google Scholar). a is in theDrosophila in transcription there a of transcription by both RNA polymerases II and III that is of the effects of the U1 PSEA the U6 promoter. To overcome this of the in transcription we to an in a TATA box can for transcription by RNA polymerases II or III in nuclear the of a transcription in requires the presence of promoter or elements the TATA in the experiments described we the of the U1 and U6 PSEAs the of transcription in vivo. the Drosophila U1 PSEA functionally substitute for the U6 PSEA to transcription of the U6 gene in Drosophila cells. this the three constructs at the of The contained the U6 promoter and U6 gene with a 10-base pair between nucleotides and of the U6 RNA coding The the of the gene from the U6 The was to the for nucleotide that the U6 PSEA to a U1 PSEA, and the contained a PSEA with of base These three constructs were to cells. RNA was from the cells and by for the presence of the U6 RNA from cells with the that contained the U6 promoter a of the for of transcription at position of the U6 gene (Fig. In of the U1 PSEA the U6 promoter transcription activity to (Fig. a very was in but indicated that transcription was than relative to the U6 promoter. level was the with a or with a that contained the PSEA (Fig. 1 and these results it is that the U1 PSEA substitute for the U6 PSEA as a of the U6 promoter. The promoter activity of the U1 PSEA was suppressed in the of the U6 performed a of experiments to examine the U6 PSEA functionally substitute for the U1 PSEA in the U1 promoter. the U1 promoter RNA polymerase II, we as a gene for these The and of the to examine a of constructs that contained of the the U1 PSEA with the U6 PSEA, constructs were PSEA sequences that contained a of U1 and U6 A of this of constructs is shown at the of Fig. the we a that a U1 or U6 nucleotide is at or within the PSEA (Fig. 1 B). that U1 nucleotides are at and but U6 nucleotides at and of cells with the indicated were and The U1 promoter expression of the (Fig. B). base to those of the U6 PSEA in the of gene expression essentially to the U6 PSEA was unable to functionally substitute for the U1 PSEA to recruit RNA polymerase II to the U1 promoter. the constructs PSEAs the RNA polymerase II activity of the promoter as the U6 of the PSEA The in Fig. B results from previous that determined the in RNA polymerase specificity of constructs that contained the of PSEAs (20Jensen R.C. Wang Y. Hardin S.B. Stumph W.E. Nucleic Acids Res. 1998; 26: 616-622Crossref PubMed Scopus (39) Google Scholar). the in transcription in the assays the from RNA polymerase II to RNA polymerase III specificity in the in of of the constructs that the of were carried out to the results at the level of the RNA (Fig. with the that contained the U1 promoter a of the for of transcription at position of the U1 gene (Fig. In of the U6 PSEA the U1 promoter the of to (Fig. and that contained the and PSEAs of transcription the was essentially no by than the each the relative level of expression was by (Fig. than by activity (Fig. B). the that the is than the at the transcription these results indicate that the U6 PSEA was unable to functionally substitute for the U1 PSEA in the U1 promoter to recruit RNA polymerase II to transcribe the gene. there was a in in cells as the U6 of the PSEA was the no for of RNA polymerase III transcription in the U6 PSEA is the U1 transcription to an of the properties of the U1 and U6 PSEAs in living the which to the was downstream of the U1 promoter. A similar was in which the U1 PSEA was replaced with the U6 PSEA (Fig. constructs were as the contained the U1 promoter in relative to the the other utilized the promoter to gene expression (Fig. Drosophila cells were with these and was to the cells. The of the cells was by a of all the of the cells and to a few of cells were no in the that were with the constructs that contained the U6 PSEA or the U1 promoter in (Fig. In of cells to and on with constructs that contained the gene the of the U1 or promoters. on these cells to and and the was the these results we that the U1 promoter can expression of the gene. the U6 PSEA was unable to substitute for the U1 PSEA for this it was unable to recruit the RNA polymerase II required for of a To these results in a in similar gene constructs were the of flies by transgenic lines were that contained gene the of the U1 promoter or the of a U1 promoter in which the PSEA to a U6 of these transgenic lines were for in expression of the by flies to lay on that contained of were from flies that contained of gene downstream of the U1 promoter In the flies that contained the of the U6 PSEA in the U1 promoter no to the These results indicate that the U1 promoter can expression of the but of the U6 PSEA the RNA polymerase II activity of the U1 promoter in of transgenic lines that contain the gene by the U1 gene on on on 1 and and lines were for their to to the were on were for each each was with 8 and contained with the U1 lines contained with a of the U6 PSEA for the U1 PSEA in the U1 promoter. in a lines were for their to to the were on were for each each was with 8 and contained with the U1 lines contained with a of the U6 PSEA for the U1 PSEA in the U1 promoter. Previous in studies that the RNA polymerase specificity of Drosophila snRNA genes was determined primarily by a few nucleotide differences within the 21-base pair PSEA That was upon transcription of promoter constructs that contained features of the U1 and U6 gene promoters. it was important to validate those both in cells and in the of the natural promoter environment of the U1 and U6 genes. The that the Drosophila U1 and U6 PSEAs are functionally distinct in as as in the U1 PSEA is with transcription only by RNA polymerase II, and the U6 PSEA transcription only by RNA polymerase III. there are differences as (20Jensen R.C. Wang Y. Hardin S.B. Stumph W.E. Nucleic Acids Res. 1998; 26: 616-622Crossref PubMed Scopus (39) Google Scholar), as as those (Fig. indicated that the PSEA can as a to the RNA polymerase specificity snRNA gene promoters. That is, the U1 and U6 PSEAs in the RNA polymerase specificity. 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PubMed Scopus Google Scholar). that different upon to a U1 or U6 PSEA and that these distinct are each with the downstream of only RNA polymerase II or RNA polymerase results the the of Drosophila transgenic and to the that to RNA polymerase specificity. it to in that the RNA polymerase specificity with the U1 or U6 Reddy U6 gene for U6 and and for the are very to and for and with and and we for on the