Identification of SNAPc Subunit Domains That Interact with Specific Nucleotide Positions in the U1 and U6 Gene Promoters

Abstract

The small nuclear RNA (snRNA)-activating protein complex (SNAPc) is essential for transcription of genes coding for the snRNAs (U1, U2, etc.). In Drosophila melanogaster, the heterotrimeric DmSNAPc recognizes a 21-bp DNA sequence, the proximal sequence element A (PSEA), located approximately 40 to 60 bp upstream of the transcription start site. Upon binding the PSEA, DmSNAPc establishes RNA polymerase II preinitiation complexes on U1 to U5 promoters but RNA polymerase III preinitiation complexes on U6 promoters. Minor differences in nucleotide sequence of the U1 and U6 PSEAs determine RNA polymerase specificity; moreover, DmSNAPc adopts different conformations on these different PSEAs. We have proposed that such conformational differences in DmSNAPc play a key role in determining the different polymerase specificities of the U1 and U6 promoters. To better understand the structure of DmSNAPc-PSEA complexes, we have developed a novel protocol that combines site-specific protein-DNA photo-cross-linking with site-specific chemical cleavage of the protein. This protocol has allowed us to map regions within each of the three DmSNAPc subunits that contact specific nucleotide positions within the U1 and U6 PSEAs. These data help to establish the orientation of each DmSNAPc subunit on the DNA and have revealed cases in which different domains of the subunits differentially contact the U1 versus U6 PSEAs.The Drosophila melanogaster small nuclear RNA (snRNA)-activating protein complex (DmSNAPc) is a heterotrimeric transcription factor (21) that is required for the synthesis of the U1, U2, U4, U5, and U6 spliceosomal snRNAs (2, 22, 31). Homologous protein complexes are required for snRNA gene expression in humans (1,11,12,18,(33)(34)(35) and for spliced leader RNA synthesis in trypanosomes (6,7,30). This indicates that SNAPc appeared early in eukaryotic evolution and continues in contemporary times to be utilized for the transcription of important noncoding RNA molecules in diverse organisms. DmSNAPc binds sequence-specifically to an essential, conserved ϳ21-bp promoter element termed the proximal sequence element A (PSEA) that is located approximately 40 to 60 bp upstream of the transcription start site of fly snRNA genes (8,13,19,23).In animals, the U1 to U5 snRNA genes are transcribed by RNA polymerase II (Pol II), but U6 snRNA genes are transcribed by RNA Pol III (4,5,10,14,19,24,29,31). Surprisingly, the primary determinant of the RNA polymerase specificity of the D. melanogaster snRNA genes is the precise sequence of the PSEA. A few conserved nucleotide differences in the 3Ј half of the 21-bp PSEA are sufficient to determine the polymerase specificity of the fly snRNA genes in vitro and to restrict the polymerase specificity in vivo (2,19,20,26).The three subunits of DmSNAPc are termed DmSNAP190, DmSNAP50, and DmSNAP43 so that the names correspond to the most widely used nomenclature for the three orthologous human SNAPc subunits, SNAP190, SNAP50, and SNAP43. These three human subunits are also known as PTF␣, PTF␤, an...

MED26 regulates the transcription of snRNA genes through the recruitment of little elongation complex

“…A recent study showed that depletion of ICE1 also decreased the occupancy of other LEC components at snRNA genes, indicating that ICE1 functions as a scaffold protein for LEC and contributes to the recruitment of LEC to the promoter 13 . It has also been reported that the minor difference in U1 and U6 promoters results in a slight conformational change of SNAPc and could determine the Pol II and Pol III specificities of the U1 and U6 promoters 39 . The results of those studies and our study raise the possibility that a combination of MED26, ICE1, SNAPc and other factors contributes to the LEC recruitment to snRNA genes.…”

The Little Elongation Complex Functions at Initiation and Elongation Phases of snRNA Gene Transcription

“…snRNA genes can be transcribed by either Pol II or Pol III. Slight differences in snRNA promoter elements may put SNAPc into different conformations that can be differentially recognized by Pol II or Pol III (Kim et al, 2010). …”

Mutation of zebrafish Snapc4 is associated with loss of the intrahepatic biliary network

“…The mutant form of Snapc4 is similar to that of invertebrate Snapc4 (Hernandez Jr et al, 2006; Kim et al, 2010) or the mini-Snapc4 used for in vitro studies (Ma and Hernandez, 2001). As expected from these in vitro studies, the truncated mutant form of Snapc4 still can regulate the expression of most of its target snRNAs, and thus can be considered to be a hypomorphic allele.…”