Characterization of a Drosophila Proximal‐Sequence‐Element‐Binding Protein Involved in Transcription of Small Nuclear RNA Genes

Abstract

In a wide variety of eukaryotic organisms, transcription of small nuclear RNA (snRNA) genes is dependent upon a proximal sequence element (PSE) located upstream of position -40 relative to the transcription start site. There is little or no existent knowledge concerning the PSE-binding proteins of organisms other than human. Here, we report the purification of a fraction enriched in the Drosophila melanogaster PSE-binding protein (DmPBP). DmPBP forms a highly specific complex with the PSE. The protein stimulates transcription from the U1 gene promoter by RNA polymerase I1 and from the U6 gene promoter by RNA polymerase I11 in Drosophila nuclear extracts, and activation is dependent upon the presence of a PSE. The molecular mass of native DmPBP as measured by gel-filtration chromatography is 375 kDa. Two polypeptides (apparent molecular masses 59 kDa and 61 kDa) appear to be in close contact with the DNA in that they can be very efficiently and specifically crosslinked to the PSE sequence by ultraviolet irradiation.Keywords: small nuclear RNA gene ; proximal sequence element ; proximal-sequence-element-binding protein; transcription factor; transcriptional activation.The genes encoding the spliceosomal snRNAs can be divided into two classes depending upon whether they are transcribed by RNA polymerase I1 (RNAP 11) or by RNA polymerase I11 (RNAP 111). The U1, U2, U4 and U5 snRNA genes are transcribed by RNAP 11, whereas U6 genes are transcribed by RNAP 111. In vertebrates, transcription of both classes of snRNA genes is dependent upon the proximal sequence element (PSE), a cis-acting element that is located upstream of position -40 with respect to the transcription start site. The PSE is unique to snRNA genes and to a few other related genes that encode small stable cellular RNAs (reviewed by Dahlberg and Lund, 1988;Parry et al., 1989;Hernandez, 1992; Lob0 and Hernandez, 1994).Cis-acting elements that are functionally homologous to the vertebrate PSE have been identified at similar positions upstream of the RNAP 11-transcribed and RNAP 111-transcribed snRNA genes of sea urchins, insects and nematodes (Li et al., 1996;Zamrod et al., 1993;Thomas et al., 1990;Hannon et al., 1994). A functionally similar regulatory element termed the USE is present upstream of plant snRNA genes (Goodall et al., 1991). Based upon these observations, it is evident that the PSE of snRNA genes is an ancient regulatory element that has been conserved functionally (but not in sequence) from early in the evolutionary history of eukaryotic organisms. The human PSE-binding protein has been purified from HeLa cell extracts and partially characterized. Different research groups have termed it the PSE-binding protein (PBP), proximal transcription factor (PTF), or small nuclear RNA activator protein complex (SNAPc) (Waldschmidt et al., 1991;Murphy et al., 1992;Simmen et al., 1992;Sadowski et al., 1993). It is capable of activating U1 and U2 gene transcription by RNAP 11, as well as U6 and 7SK gene transcription by RNAP I11 (Sadowski et al., 1993;...

Identification and Topological Arrangement of Drosophila Proximal Sequence Element (PSE)-Binding Protein Subunits That Contact the PSEs of U1 and U6 Small Nuclear RNA Genes

“…Because each of these subunits is required for snRNA transcription by either RNAP II or RNAP III, it seems that the same protein complex functions at both classes of vertebrate snRNA gene promoters. The fact that the PSEA plays a major role in determining RNA polymerase specificity in Drosophila poses the question of whether a similar complex functions at both RNAP II and RNAP III snRNA promoters in insects.Our lab recently reported the partial purification and characterization of the D. melanogaster PSEA-binding protein (DmPBP) (27). Like SNAP c /PTF, DmPBP is a multisubunit…”

Differential Utilization of TATA Box-binding Protein (TBP) and TBP-related Factor 1 (TRF1) at Different Classes of RNA Polymerase III Promoters

“…Previous work had shown that D. melanogaster U1 and U6 snRNA genes can be faithfully transcribed in vitro by using unfractionated SNF prepared from 0 -12-h fruit fly embryos (22)(23)(24)(25). As a control for TRF1-dependent transcription, we also employed a tRNA Arg -maxi gene template (20).…”

“…As controls, three U1 snRNA gene loci and three tRNA gene loci were also analyzed. Furthermore, because DmSNAPc is required for transcription of both U1 and U6 snRNA genes (23) but not for the transcription of tRNA genes, we also examined DmSNAPc occupancy by using antibodies against DmSNAP43.…”

“…The preparation of the antibodies against D. melanogaster TBP and DmSNAP43 has been described previously (21 In Vitro Transcription Assays-The templates that contained the wild type U1 and U6 promoters have been described previously (22,23). The plasmid pArg-maxi contained a tRNA Arg gene with a 12-bp insertion between the internal promoter elements (19) and was obtained from Deborah Johnson (Department of Molecular Pharmacology and Biochemistry, University of Southern California).…”

“…Procedures for in vitro transcription and analysis of the RNA products by primer extension have been previously described in detail (22,23). Transcription reactions (25 l final volume) utilized 15 l of a soluble nuclear fraction (SNF) prepared from D. melanogaster embryos (22,24,25).…”