Transcription of snRNA genes by either RNA polymerase II (U1 to U5) or RNA polymerase III (U6) is dependent upon a proximal sequence element (PSE) located approximately 40 to 60 bp upstream of the transcription start site. In Drosophila melanogaster, RNA polymerase specificity is determined by as few as three nucleotide differences within the otherwise well-conserved 21-bp PSE. Previous photo-cross-linking studies revealed that the D. melanogaster PSE-binding protein, DmPBP, contains three subunits (DmPBP45, DmPBP49, and DmPBP95) that associate with the DNA to form complexes that are conformationally distinct depending upon whether the protein is bound to a U1 or a U6 PSE. We have identified and cloned the genes that code for these subunits of DmPBP by virtue of their similarity to three of the five subunits of SNAP c , the human PBP. When expressed in S2 cells, each of the three cloned gene products is incorporated into a protein complex that functionally binds to a PSE. We also find that the conformational difference referred to above is particularly pronounced for DmPBP45, herein identified as the ortholog of human SNAP43. DmPBP45 cross-linked strongly to DNA for two turns of the DNA helix downstream of the U1 PSE, but it cross-linked strongly for only a half turn of the helix downstream of a U6 PSE. These substantial differences in the cross-linking pattern are consistent with those of a model in which conformational differences in DmPBP-DNA complexes lead to selective RNA polymerase recruitment to U1 and U6 promoters.In eukaryotes, small nuclear RNAs (snRNAs) are required for pre-mRNA splicing. Most snRNAs, such as U1, U2, U4, and U5, are synthesized by RNA polymerase II, but U6 snRNA is synthesized by RNA polymerase III (2,3,6,10,11,18,26). Transcription of snRNA genes by either RNA polymerase is dependent upon a proximal sequence element (PSE) located upstream of position Ϫ40 relative to the transcription start site. In the insect Drosophila melanogaster, the PSE is referred to more specifically as the PSEA to distinguish it from a second conserved element termed the PSEB (38).Although the PSEAs of all D. melanogaster snRNA genes share extensive sequence similarity, the PSEAs of the three U6 genes present in the fly genome consistently vary at a few nucleotide positions from the PSEAs of the RNA polymerase II-transcribed snRNA genes (13). Indeed, RNA polymerase specificity can be determined by as few as three base pair differences within the otherwise well-conserved U1 and U6 PSEAs (13). As a result, the fly U1 and U6 PSEAs are not interchangeable either in vitro or in vivo (13,22).Nonetheless, both PSEAs are recognized by the same D. melanogaster PSE-binding protein, DmPBP (29,33). DmPBP contains three distinct subunits (DmPBP45, DmPBP49, and DmPBP95) that can be specifically photo-cross-linked to DNA containing U1 or U6 PSEA sequences. Interestingly, the pattern of photo-cross-linking was different depending upon whether DmPBP was bound to a U1 or a U6 PSEA (33). Those results, together with the functio...

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Most small nuclear RNAs (snRNAs) are synthesized by RNA polymerase II, but U6 and a few others are synthesized by RNA polymerase III. Transcription of snRNA genes by either polymerase is dependent on a proximal sequence element (PSE) located upstream of position ؊40 relative to the transcription start site. In contrast to findings in vertebrates, sea urchins, and plants, the RNA polymerase specificity of Drosophila snRNA genes is intrinsically encoded in the PSE sequence itself. We have investigated the differential interaction of the Drosophila melanogaster PSE-binding protein (DmPBP) with U1 and U6 gene PSEs. By using a site specific protein-DNA photo-cross-linking assay, we identified three polypeptide subunits of DmPBP with apparent molecular masses of 95, 49, and 45 kDa that are in close proximity to the DNA and two additional putative polypeptides of 230 and 52 kDa that may be integral to the complex. The 95-kDa subunit cross-linked at positions spanning the entire length of the PSE, but the 49-and 45-kDa subunits cross-linked only to the 3 half of the PSE. The same polypeptides cross-linked to both the U1 and U6 PSE sequences. However, there were significant differences in the cross-linking patterns of these subunits at a subset of the phosphate positions, depending on whether binding was to a U1 or U6 gene PSE. These data suggest that RNA polymerase specificity is associated with distinct modes of interaction of DmPBP with the DNA at U1 and U6 promoters.In higher eukaryotes, four of the major small nuclear RNAs (snRNAs) found in spliceosomes (U1, U2, U4, and U5) are synthesized by RNA polymerase II (RNAP II), but U6 snRNA is synthesized by RNA polymerase III (RNAP III) (2,3,9,18,22). The U6 gene promoter is representative of an unusual class of RNAP III promoters that contain a TATA box but lack internal promoter elements (5,15,19,30). Promoters of snRNA genes transcribed by either RNAP II or RNAP III contain an essential proximal sequence element (PSE) at a conserved location approximately 40 to 65 bp upstream of the transcription start site that is required for the initiation of snRNA transcription (4,9,18,26,29,30,32,36).In Drosophila melanogaster, the PSE is more specifically named the PSEA to distinguish it from a second, even more proximal conserved element, PSEB, present in the promoters of Drosophila snRNA genes transcribed by RNAP II (36). The PSEB is located at the TATA box position but is a poor TATA box sequence (consensus CATGGAg/aA) (16). PSEB is separated from the upstream PSEA by 8 bp of nonconserved sequence (16,36). Drosophila U6 genes, on the other hand, contain a canonical TATA box rather than a PSEB, and the TATA box is separated by 12 bp from the upstream PSEA (4).In vertebrates and sea urchins, the PSEs of U1 and U2 genes are interchangeable with the PSEs of U6 genes (14,17,23). In these organisms, the PSEs are therefore not responsible for the determination of RNAP specificity. Surprisingly, recent results from our lab revealed that the U1 and U6 PSEAs are not interchangeable in t...

Most small nuclear RNA (snRNA) genes are transcribed by RNA polymerase II, but some (e.g., U6) are transcribed by RNA polymerase III. In vertebrates a TATA box at a fixed distance downstream of the proximal sequence element (PSE) acts as a dominant determinant for recruiting RNA polymerase III to U6 gene promoters. In contrast, vertebrate snRNA genes that contain a PSE but lack a TATA box are transcribed by RNA polymerase II. In plants, transcription of both classes of snRNA genes requires a TATA box in addition to an upstream sequence element (USE), and polymerase specificity is determined by the spacing between these two core promoter elements. In these examples, the PSE (or USE) is interchangeable between the two classes of snRNA genes. Here we report the surprising finding that the Drosophila U1 and U6 PSEs cannot functionally substitute for each other; rather, determination of RNA polymerase specificity is an intrinsic property of the PSE sequence itself. The alteration of two or three base pairs near the 3'-end of the U1 and U6 PSEs was sufficient to switch the RNA polymerase specificity of Drosophila snRNA promoters in vitro. These findings reveal a novel mechanism for achieving RNA polymerase specificity at insect snRNA promoters.

In animals, most small nuclear RNAs (snRNAs) are synthesized by RNA polymerase II (Pol II), but U6 snRNA is synthesized by RNA polymerase III (Pol III). In Drosophila melanogaster, the promoters for the Pol II-transcribed snRNA genes consist of ∼21 bp PSEA and ∼8 bp PSEB. U6 genes utilize a PSEA but have a TATA box instead of the PSEB. The PSEAs of the two classes of genes bind the same protein complex, DmSNAPc. However, the PSEAs that recruit Pol II and Pol III differ in sequence at a few nucleotide positions that play an important role in determining RNA polymerase specificity. We have now performed a bioinformatic analysis to examine the conservation and divergence of the snRNA gene promoter elements in other species of insects. The 5′ half of the PSEA is well-conserved, but the 3′ half is divergent. Moreover, within each species positions exist where the PSEAs of the Pol III-transcribed genes differ from those of the Pol II-transcribed genes. Interestingly, the specific positions vary among species. Nevertheless, we speculate that these nucleotide differences within the 3′ half of the PSEA act similarly to induce conformational alterations in DNA-bound SNAPc that result in RNA polymerase specificity.

We have identified and sequenced two members of a chicken middle repetitive DNA sequence family. By reassociation kinetics, members of this family (termed CRl) are estimated to be present in 1500-7000 copies per chicken haploid genome. The first family member sequenced (CRlUla) is located approximately 2 kb upstream from the previously cloned chicken Ul RNA gene. The second CRl sequence (CRl)Va) is located approximately 12 kb downstream from the 3' end of the chicken ovalbumin gene. The region of homology between these two sequences extends over a region of approximately 160 base pairs. In each case, the 160 base pair region is flanked by imperfect, but homologous, short direct repeats 10-15 base pairs in length. When the CRl sequences are compared with mammalian ubiquitous interspersed repetitive DNA sequences (human Alu and Mouse Bl families), several regions of extensive homology are evident. In addition, the short nucleotide sequence CAGCCTGG which is completely conserved in ubiquitous repetitive sequence families from several mammalian species is also conserved at a homologous position in the chicken sequences. These data imply that at least certain aspects of the sequence and structure of these interspersed repeats must predate the avian-mammalian divergence. It seems that the CRl family may possibly represent an avian counterpart of the mammalian ubiquitous repeats.

Transcription of a Drosophila U1 small nuclear RNA gene was functionally analyzed in cell extracts derived from 0- to 12-h embryos. Two promoter elements essential for efficient initiation of transcription in vitro by RNA polymerase II were identified. The first, termed PSEA, is located between positions -41 and -61 relative to the transcription start site, is crucial for promoter activity, and is the dominant element for specifying the transcription initiation site. PSEA thus appears to be functionally homologous to the proximal sequence element of vertebrate small nuclear RNA genes. The second element, termed PSEB, is located at positions -25 to -32 and is required for an efficient level of transcription initiation because mutation of PSEB, or alteration of the spacing between PSEA and PSEB, severely reduced transcriptional activity relative to that of the wild-type promoter. Although the PSEB sequence does not have any obvious sequence similarity to a TATA box, conversion of PSEB to the canonical TATA sequence dramatically increased the efficiency of the U1 promoter and simultaneously relieved the requirement for the upstream PSEA. Despite these effects, introduction of the TATA sequence into the U1 promoter had no effect on the choice of start site or on the RNA polymerase II specificity of the promoter. Finally, evidence is presented that the TATA box-binding protein is required for transcription from the wild-type U1 promoter as well as from the TATA-containing U1 promoter.

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...