Small Nuclear RNA Genes: a Model System to Study Fundamental Mechanisms of Transcription

small nuclear RNA proximal sequence element distal sequence element polymerase TATA box binding protein upstream sequence element base pair(s) amino acid(s) human B“ human BRF human BRFU The human small nuclear RNA (snRNA)1 genes, which encode snRNAs that are involved in RNA processing reactions such as mRNA splicing, serve as prototypes for a family of genes whose promoters are characterized by the presence of a proximal sequence element (PSE) and a distal sequence element (DSE). From a transcription point of view, this family of genes is highly interesting because all of its members have very similar promoters, even though some of them are transcribed by RNA polymerase (pol) II and others by pol III. As a result, the snRNA genes have served as a model system to explore how RNA polymerase specificity is determined and, in general, to compare the pol II and III transcription machineries. This has led to the concept that the pol II and III transcription machineries use common factors, the best known of which is the TATA box binding protein (TBP). In addition, the relative simplicity of these promoters has also made them an attractive system to study how transcriptional activators perform their function. Fig. 1 shows the structures of snRNA promoters from Homo sapiens (Hs),Arabidopsis thaliana (At), and Drosophila melanogaster (Dm) and serves to illustrate the remarkable fact that although snRNA promoters have diverged during evolution, the close similarity between those recognized by pol II and those recognized by pol III has been conserved. In fact, in each of the examples in Fig. 1, RNA polymerase specificity can be changed by altering a single parameter, indicated in red on the figure. In the human genes, the U1 and U2 snRNA promoters serve as the prototypic pol II snRNA promoters, and the U6 snRNA promoter serves as the prototypic pol III snRNA promoter (see Ref. 1Lobo S.M. Hernandez N. Conaway R.C. Conaway J.W. Transcription, Mechanisms and Regulation. Raven Press, Ltd., New York1994: 127-159Google Scholar for a review). The human pol II snRNA core promoters contain only one essential element, the PSE, whereas the pol III snRNA core promoters consist of two elements, the PSE and a TATA box located at a fixed distance downstream. The DSE serves to enhance transcription from the core promoter. Both the DSE and the PSE can be interchanged between pol II and III snRNA promoters with no effect on RNA polymerase specificity, which is determined by the presence or absence of the TATA box. TheA. thaliana pol II and III snRNA promoters contain an upstream sequence element (USE) and a TATA box, which are both interchangeable between the pol II and III snRNA promoters. RNA polymerase specificity is determined in this case by the exact spacing between the USE and the TATA box, which is 33–34 base pairs (bp) and 23–24 bp in the pol II and III snRNA promoters, respectively (2Waibel F. Filipowicz W. Nature. 1990; 346: 199-202Crossref PubMed Scopus (74) Google Scholar). The D. melanogaster pol II snRNA promoters contain two elements referred to as the PSEA and the PSEB spaced by 8 bp, and the pol III snRNA promoters contain a PSEA and a TATA box spaced by 12 bp. The PSEA is quite conserved in various pol II and III snRNA promoters, but positions 19 and 20 of the 21-bp elements are always g/aG in the pol II and TC in the pol III snRNA promoters. RNA polymerase specificity is determined by the precise sequence of the PSEA element, with the base pairs at positions 19 and 20 playing a major role (Ref.3Jensen R.C. Wang Y. Hardin S.B. Stumph W.E. Nucleic Acids Res. 1998; 26: 616-622Crossref PubMed Scopus (39) Google Scholar, and references therein). A number of snRNA promoters have been characterized in various sea urchins. As in other species, the pol II and III snRNA promoters are closely related in structure. They all have a PSE and some have a TATA box, but the presence of the TATA box does not correlate with RNA polymerase specificity. The PSEs in different snRNA promoters show little sequence identity and yet can be exchanged with no effect on polymerase specificity. The determinants of RNA polymerase specificity are not known (Ref. 4Li J.M. Haberman R.P. Marzluff W.F. Mol. Cell. Biol. 1996; 16: 1275-1281Crossref PubMed Google Scholar, and references therein). In Saccharomyces cerevisiae, only the pol III U6 snRNA promoter has been studied. It consists of a TATA box located upstream of the transcription start site and A and B boxes typical of gene-internal tRNA promoters. The A box is located, as in tRNA genes, within the RNA coding region, but the B box is located at an anomalous position 3′ of the gene (5Brow D.A. Guthrie C. Genes Dev. 1990; 4: 1345-1356Crossref PubMed Scopus (110) Google Scholar, 6Burnol A.F. Margottin F. Schultz P. Marsolier M.C. Oudet P. Sentenac A. J. Mol. Biol. 1993; 233: 644-658Crossref PubMed Scopus (58) Google Scholar, 7Eschenlauer J.B. Kaiser M.W. Gerlach V.L. Brow D.A. Mol. Cell. Biol. 1993; 13: 3015-3026Crossref PubMed Scopus (80) Google Scholar). The snRNA promoters in vertebrates, A. thaliana,D. melanogaster, and sea urchins all contain an element, variously called the PSE, PSEA, or USE, centered 50–70 bp upstream of the transcription start site. The factor binding to this element has been best characterized in the human system and is variously known as PBP, PTF, or SNAPc. It is a complex containing five types of subunits, SNAP190, SNAP50 (PTFβ), SNAP45 (PTFδ), SNAP43 (PTFγ), and SNAP19 (see Ref. 8Henry R.W. Ford E. Mital R. Mittal V. Hernandez N. Cold Spring Harbor Symp. Quant. Biol. 1998; 63: 111-120Crossref PubMed Scopus (32) Google Scholar, and references therein). SNAP190 forms the backbone of the complex, with SNAP19 and SNAP45 associating toward the N and C terminus, respectively, of the molecule. SNAP43 can associate with the same region of SNAP190 as SNAP19, and SNAP50 joins the complex by associating with SNAP43 (see Fig. 3 for an illustration of SNAPc). The ability to assemble recombinant SNAPc and thus mutant forms of SNAPc has allowed a study of the role of SNAPc subunits for binding to DNA and for basal and activated transcription by RNA polymerases II and III. The smallest subassembly of SNAPc subunits tested that binds to the PSE with the same specificity as the complete complex consists of SNAP190 aa 84–505, SNAP43 aa 1–268, and SNAP50 (9Ma B. Hernandez N. J. Biol. Chem. 2001; 276: 5027-5035Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). This observation is consistent with UV cross-linking experiments that suggest that within SNAPc, both SNAP190 and SNAP50 are in close contact with the DNA (see Ref. 8Henry R.W. Ford E. Mital R. Mittal V. Hernandez N. Cold Spring Harbor Symp. Quant. Biol. 1998; 63: 111-120Crossref PubMed Scopus (32) Google Scholar, and references therein). The specific binding of SNAPc to the PSE is mediated in part by an unusual Myb domain extending from aa 263 to 503 within SNAP190 and containing a half-repeat followed by four repeats (8Henry R.W. Ford E. Mital R. Mittal V. Hernandez N. Cold Spring Harbor Symp. Quant. Biol. 1998; 63: 111-120Crossref PubMed Scopus (32) Google Scholar, 10Mittal V. Ma B. Hernandez N. Genes Dev. 1999; 13: 1807-1821Crossref PubMed Scopus (58) Google Scholar). In the human system, the very same SNAPc is involved in transcription by pol II and pol III (11Henry R.W. Mittal V. Ma B. Kobayashi R. Hernandez N. Genes Dev. 1998; 12: 2664-2672Crossref PubMed Scopus (65) Google Scholar). The polypeptide composition of PSE binding factors from other species is unknown, but there are indications that at least in some cases, the same PSE binding factor is also recruited to both pol II and III snRNA promoters. Thus, both in sea urchins and D. melanogaster, similar complexes bind to the PSEs of pol II and III snRNA promoters as judged from electrophoretic mobility shift assays (3Jensen R.C. Wang Y. Hardin S.B. Stumph W.E. Nucleic Acids Res. 1998; 26: 616-622Crossref PubMed Scopus (39) Google Scholar, 4Li J.M. Haberman R.P. Marzluff W.F. Mol. Cell. Biol. 1996; 16: 1275-1281Crossref PubMed Google Scholar), and in the latter case, site-specific protein-DNA photo-cross-linking experiments reveal the same set of polypeptides in close proximity to the DNA in both cases. Interestingly, however, the precise cross-linking patterns of these polypeptides to the U1 and U6 PSEAs are significantly different. Thus, in D. melanogaster, RNA polymerase specificity may ultimately be determined by different conformations of the same factor, which are dictated by the exact PSEA sequence (12Wang Y. Stumph W.E. Mol. Cell. Biol. 1998; 18: 1570-1579Crossref PubMed Google Scholar). Transcription from TATA box-containing mRNA promoters can be reconstituted with a combination of recombinant and well defined factors, as shown in Fig. 2 A. In vitro, these factors can be added sequentially to the promoter to form a functional transcription initiation complex, and each step can be monitored by electrophoretic mobility shift assay. TBP or the TBP-containing complex TFIID binds first to the TATA box, followed by TFIIB, a TFIIF-pol II complex, TFIIE, and TFIIH. TFIIA can join the initiation complex at any stage of assembly, and its main role for mRNA core promoter function appears to be counteracting repressors that associate with TBP and prevent its binding to DNA (13Orphanides G. Lagrange T. Reinberg D. Genes Dev. 1996; 10: 2657-2683Crossref PubMed Scopus (852) Google Scholar). In the case of the pol II snRNA promoters, the transcription initiation complex has not yet been assembled in a stepwise fashion in vitro, but many of its components have been identified functionally by depletion of transcription extracts with specific antibodies and reconstitution of transcription with recombinant factors. Thus, many of the players are known, but their mode of assembly on snRNA promoters is not, and thus their location in Fig.2 A is arbitrary. Depletion and reconstitution experiments indicate that recombinant TBP (but not the TBP-containing complexes TFIID or TFIIIB), TFIIB, TFIIA, TFIIF, and TFIIE are required (Ref. 14Kuhlman T.C. Cho H. Reinberg D. Hernandez N. Mol. Cell. Biol. 1999; 19: 2130-2141Crossref PubMed Scopus (53) Google Scholar, and references therein). TFIIA appears to perform a more direct function in snRNA transcription complex assembly than just counteracting TBP-associated repressors. The role, if any, of TFIIH in pol II transcription of snRNA genes is not clear. Depletion and reconstitution experiments suggest that U1 transcription either does not require TFIIH or requires much lower levels than transcription from a mRNA promoter. If TFIIH is indeed not required, this raises the interesting question of how open complex formation is achieved at snRNA promoters. A combination of all the general transcription factors and SNAPc does not initiate transcription from the U1 promoter, suggesting that additional as yet unidentified factors are required (14Kuhlman T.C. Cho H. Reinberg D. Hernandez N. Mol. Cell. Biol. 1999; 19: 2130-2141Crossref PubMed Scopus (53) Google Scholar). The key player for recruitment of pol II to a promoter is the factor TFIIB because it contacts the RNA polymerase directly. In the case of pol III, this role is played mainly by the multisubunit factor TFIIIB. TFIIIB was completely defined first in S. cerevisiaeand consists of three subunits, TBP, a tightly associated subunit referred to as the TFIIB-related factor BRF1 (PCF4/TDS4) (see Ref. 15Hernandez N. Genes Dev. 1993; 7: 1291-1308Crossref PubMed Scopus (564) Google Scholar, and references therein), and a more loosely associated polypeptide called B“ (TFIIIB90/TFC5/TFC7) (16Kassavetis G.A. Nguyen S.T. Kobayashi R. Kumar A. Geiduschek E.P. Pisano M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9786-9790Crossref PubMed Scopus (91) Google Scholar, 17Rüth J. Conesa C. Dieci G. Lefebvre O. Dusterhoft A. Ottonello S. Sentenac A. EMBO J. 1996; 15: 1941-1949Crossref PubMed Scopus (77) Google Scholar). This TFIIIB complex is involved in transcription from all types of yeast pol III promoters tested including the gene-internal tRNA-type promoters and the U6 promoter, which, as described above, contains a TATA box and A and B boxes (18Joazeiro C.A. Kassavetis G.A. Geiduschek E.P. Mol. Cell. Biol. 1994; 14: 2798-2808Crossref PubMed Scopus (72) Google Scholar). TBP was shown to be required for pol III transcription of vertebrate snRNA genes before TBP was known to be a subunit of TFIIIB (see Ref. 15Hernandez N. Genes Dev. 1993; 7: 1291-1308Crossref PubMed Scopus (564) Google Scholarfor a review). Ironically, however, the composition of mammalian TFIIIB and the role of TFIIIB polypeptides other than TBP in snRNA gene transcription have been determined only recently. A human homologue of yeast B“ was recently cloned (19Schramm L. Pendergrast P.S. Sun Y. Hernandez N. Genes Dev. 2000; 14: 2650-2663Crossref PubMed Scopus (111) Google Scholar). The protein shows strong similarity to the yeast protein within and around a 59-aa domain called the SANT domain, which is essential for transcription in yeast. Depletion of human B” (hB“) from transcription extracts debilitates transcription from the U6 promoter and a tRNA-type promoter, and transcription can be restored in both cases by addition of recombinant hB” (19Schramm L. Pendergrast P.S. Sun Y. Hernandez N. Genes Dev. 2000; 14: 2650-2663Crossref PubMed Scopus (111) Google Scholar). hB“ is, therefore, shown as part of the initiation complex assembled on both U6 and tRNA-type promoters in Fig. 2 B. The first human homologue of yeast BRF cloned was called TFIIIB90 (20Wang Z. Roeder R.G. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7026-7030Crossref PubMed Scopus (109) Google Scholar) or human BRF (hBRF) (21Mital R. Kobayashi R. Hernandez N. Mol. Cell. Biol. 1996; 16: 7031-7042Crossref PubMed Scopus (67) Google Scholar). Like its yeast counterpart and like its cousin TFIIB, the protein has a zinc binding domain at its N terminus followed by a core domain consisting of two degenerate repeats. The C-terminal half of the protein is poorly conserved with the yeast protein and has no counterpart in TFIIB. Depletion and reconstitution experiments have shown that human BRF is required for transcription from tRNA-type promoters but not for transcription from the U6 snRNA promoter (21Mital R. Kobayashi R. Hernandez N. Mol. Cell. Biol. 1996; 16: 7031-7042Crossref PubMed Scopus (67) Google Scholar). Remarkably, as shown in Fig. 2 B, the U6 snRNA promoter uses another homologue of yeast human BRFU (19Schramm L. Pendergrast P.S. Sun Y. Hernandez N. Genes Dev. 2000; 14: 2650-2663Crossref PubMed Scopus (111) Google Scholar), also called M. Wang Z. Roeder R.G. Proc. Natl. Acad. Sci. U. S. A. 2000; PubMed Scopus Google Scholar). another of the TFIIB-related family of and has conserved zinc and core and a C-terminal BRFU was cloned both base of similar to (19Schramm L. Pendergrast P.S. Sun Y. Hernandez N. Genes Dev. 2000; 14: 2650-2663Crossref PubMed Scopus (111) Google Scholar) and of a complex consisting of BRFU and four tightly associated polypeptides M. Wang Z. Roeder R.G. Proc. Natl. Acad. Sci. U. S. A. 2000; PubMed Scopus Google Scholar). The role of the polypeptides is not clear. In one of U6 transcription be restored in a by addition of recombinant BRFU in (19Schramm L. Pendergrast P.S. Sun Y. Hernandez N. Genes Dev. 2000; 14: 2650-2663Crossref PubMed Scopus (111) Google Scholar), and a combination of pol III and recombinant SNAPc, TBP, and direct U6 transcription S. P. Hernandez N. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). In another of depletion of BRFU transcription from a promoter, but transcription not be reconstituted by addition of recombinant transcription only be reconstituted by addition of a complex from BRFU M. Wang Z. Roeder R.G. Proc. Natl. Acad. Sci. U. S. A. 2000; PubMed Scopus Google Scholar). Thus, it is not the polypeptides in the complex are essential for U6 another factor by an BRF may also be required for U6 transcription V. Hardin P. W. J.M. S.M. EMBO J. 2000; 19: PubMed Scopus Google Scholar). the zinc domain and the first that are in BRF and conserved in the other of the TFIIB as well as the C-terminal domain in Depletion of extracts with antibodies all BRF U6 and transcription be reconstituted by addition of from V. Hardin P. W. J.M. S.M. EMBO J. 2000; 19: PubMed Scopus Google Scholar). the that the U6 transcription complex contains two related to BRFU and The of BRFU is an step toward a complete of how RNA polymerase specificity is determined at the human snRNA promoters. in mRNA promoters, TFIIB with TBP to the TATA box and, in a on the zinc domain, with the pol complex (13Orphanides G. Lagrange T. Reinberg D. Genes Dev. 1996; 10: 2657-2683Crossref PubMed Scopus (852) Google Scholar). Thus, at least for mRNA promoters, TFIIB can be as the key factor that TBP or TFIID with the and it that TFIIB the same role in the pol II snRNA promoters. Like TFIIB, BRF also contacts the RNA which is pol III in this The precise BRF domain required for this function is not known, but it is not the zinc domain because or of the zinc does not RNA polymerase recruitment but open complex formation (see Ref. L. Pendergrast P.S. Sun Y. Hernandez N. Genes Dev. 2000; 14: 2650-2663Crossref PubMed Scopus (111) Google Scholar, and references therein). It that in the human U6 promoter, the recruitment of pol III is by either the zinc domain as for TFIIB and pol II or another part of the protein as for BRF and pol III at tRNA-type promoters. Thus, the of RNA polymerase specificity may ultimately on TFIIB or BRFU is recruited to the promoter. The human snRNA promoters are activated by a The DSE is of various protein binding but one of them is the sequence In addition, it has that the of many snRNA genes contain an element referred to as the The element was first identified in the snRNA gene Stumph W.E. Mol. Cell. Biol. 1990; 10: PubMed Scopus Google Scholar and Z. Stumph W.E. Nucleic Acids Res. 1990; 18: PubMed Scopus Google Scholar, and references and in the of the tRNA whose promoter contains a PSE and TATA box E. A. P. Nucleic Acids Res. PubMed Scopus Google Scholar, E. C. A. P. J. Mol. Biol. 1993; PubMed Scopus Google Scholar). It that a functionally element located upstream of the sequence in the human U6 snRNA promoter called the element D.A. O. Mol. Cell. Biol. 1993; 13: PubMed Scopus Google Scholar) in fact, to an element T.C. O. Stumph W.E. 1996; Google Scholar) and that elements are in the of many snRNA promoters M. E. C. A. P. EMBO J. 16: PubMed Scopus Google Scholar). In the human U6 snRNA promoter, both the and elements the formation of complexes Nucleic Acids Res. 1998; 26: PubMed Scopus Google Scholar). The in a transcription factor called or binding factor which was cloned first M. E. C. A. P. EMBO J. 16: PubMed Scopus Google Scholar, C. E. A. P. EMBO J. 1995; 14: PubMed Scopus Google Scholar) and from H. T. T. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar) and E. A. P. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Nucleic Acids Res. 1998; 26: PubMed Scopus Google Scholar). is a zinc protein containing zinc of the different of which can be to bind to different DNA In zinc 1 is required for binding to the tRNA but not to the U6 of a zinc 1 binding site with binding of the protein recruited to the sequence (Ref. M. E. A. P. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, and references contains two of transcription from and promoters C. A. P. Mol. Cell. Biol. 1998; 18: PubMed Scopus Google Scholar). The human and, to a are similar to similar DNA binding and can pol II and III snRNA gene transcription E. A. P. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, Nucleic Acids Res. 1998; 26: PubMed Scopus Google Scholar). The sequence the transcription as by the of which the of snRNA the presence of transcription within and the of to snRNA promoter in by experiments (Ref. G. W. Nature. 1995; PubMed Scopus Google Scholar, and references Pendergrast P.S. Hernandez N. Mol. Cell. 2001; 7: Full Text Full Text PDF PubMed Scopus Google Scholar). snRNA gene transcription not only its but also its domain, a DNA binding domain consisting of two DNA binding an domain and a C-terminal domain, by a W. Genes Dev. 1995; PubMed Scopus Google Scholar). As described this from the ability of the domain to bind with SNAPc and thus SNAPc to the The of many of the factors that bind to snRNA promoters has allowed a study of how these factors with each other to form a transcription initiation of this is in Both TBP and SNAPc have that prevent their binding to DNA on their In the case of TBP, this of DNA binding in the N terminus of the because of this the ability of the protein to bind to TATA boxes V. Hernandez N. PubMed Scopus (77) Google Scholar). the N terminus of TBP the DNA binding domain of the as in Fig. 3 although other are In the case of SNAPc, the of DNA binding within the C-terminal of SNAP190 because a these binds much more to DNA than complete SNAPc V. Ma B. Hernandez N. Genes Dev. 1999; 13: 1807-1821Crossref PubMed Scopus (58) Google Scholar). Both TBP and from the DNA and bind with sequence specificity, these may serve to that these factors not bind to in the Fig. 3 B SNAPc, TBP, and the domain to The domain and to their DNA binding and SNAPc and TBP (see Ref. 8Henry R.W. Ford E. Mital R. Mittal V. Hernandez N. Cold Spring Harbor Symp. Quant. Biol. 1998; 63: 111-120Crossref PubMed Scopus (32) Google Scholar, and references therein). the same of SNAPc and TBP that serve as of DNA binding are required for Thus, the domain of TBP is required for binding with SNAPc, because as in B, it is in a with SNAPc V. Hernandez N. PubMed Scopus (77) Google Scholar). the C-terminal domain of SNAP190 is required for binding with and in this case it is that binding is on a direct contact between the two which a at position within the domain in Fig. 3 and a at position within SNAP190 region in E. M. Hernandez N. Genes Dev. 1998; 12: PubMed Scopus Google Scholar). Thus, binding of these factors that the of DNA binding that contact and the factor binding to a site. This mode of DNA binding in that factors bind to located in promoter than to The binding of the domain and characterized on containing closely spaced and In the snRNA promoters, however, the sequence and the PSE are by bp, and this distance binding of and SNAPc on DNA of and in the presence of a between the sequence and the PSE in both the U1 and U6 snRNA promoters Pendergrast P.S. Hernandez N. Mol. Cell. 2001; 7: Full Text Full Text PDF PubMed Scopus Google Scholar, S. 2000; PubMed Scopus Google Scholar). in assembly in the of a at the same location Pendergrast P.S. Hernandez N. Mol. Cell. 2001; 7: Full Text Full Text PDF PubMed Scopus Google Scholar, W. Mol. Cell. Biol. PubMed Scopus Google Scholar). assembly the domain to transcription from the U6 promoter. It also binding of the domain and SNAPc, and this binding is on the same direct contact as binding to closely spaced on DNA Pendergrast P.S. Hernandez N. Mol. Cell. 2001; 7: Full Text Full Text PDF PubMed Scopus Google Scholar). suggest that the role of the is to close proximity the sequence and the PSE such that SNAPc and the domain can contact and each other to the as in Fig. 3 B. Thus, this is a case a does not transcription but is a functional of the transcription for on the