The tandemly repeated gene set encoding the sea urchin U6 gene has been cloned from the sea urchin Strongylocentrotus purpuratus. The U6 gene is transcribed by RNA polymerase III in a sea urchin nuclear extract. Like that of the vertebrate U6 genes, transcription of the sea urchin U6 gene does not require any internal sequences or 3' sequences but requires onl 5' flanking sequences. Only 88 nucleotides of 5' flanking sequence are required for maximal expression in vitro. Mutagenesis experiments demonstrated the requirement for three elements, a CACGTG element at -80, a proximal sequence element at about -55, and the TATA-like box at -25. The major protein in sea urchin extracts that interacts with the CACGTG element is sea urchin USF, and immunodepletion of sea urchin USF greatly reduces transcription. The USF binding site in the U6 gene is highly homologous (11 of 13 nucleotides) with the USF binding sites found in the promoter of the S. purpuratus spec genes.The genes encoding the spliceosomal small nuclear RNAs (snRNAs) and some other snRNAs in metazoans have unusual promoter structures (27) Here we report the sequence requirements for expression of the sea urchin U6 snRNA gene. As with the vertebrate U6 snRNA genes, there is no internal promoter element, and there is a required TATA-like box. There are also two other elements absolutely required for transcription in vitro, a sequence at the expected position for a PSE between -45 and -65 and an E-box sequence at -80. The E-box sequence binds to the sea urchin homolog of USF (suUSF), and immunodepletion experiments demonstrate that suUSF is required for U6 transcription by RNA polymerase III. MATERUILS AND METHODSPreparation of sea urchin nuclear extract. Sea urchins (Strongylocentrotus purpuratus or Lytechinus variegatus) were grown to the hatching blastula stage, and nuclei were prepared from embryos 1 to 2 h after hatching. Nuclei were prepared exactly as previously described (23). The nuclei were stored in liquid N2. Nuclear extract was prepared as previously described (23) except for the final dialysis step, which was done for 4 h against 80 mM KCl-25 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) (pH 7.5)-0.1 mM EDTA-20% glycerol-1 mM dithiothreitol (DTT)-0.1 mM phenylmethylsulfonyl fluoride. For some of the mobility shift experiments, nuclei were prepared from frozen embryos processed by the method of Calzone et al. (4). These nuclei were also purified by centrifugation through 2 M sucrose as described previously (23)

The tandemly repeated gene set encoding the sea urchin U6 gene has been cloned from the sea urchin Strongylocentrotus purpuratus. The U6 gene is transcribed by RNA polymerase III in a sea urchin nuclear extract. Like that of the vertebrate U6 genes, transcription of the sea urchin U6 gene does not require any internal sequences or 3' sequences but requires only 5' flanking sequences. Only 88 nucleotides of 5' flanking sequence are required for maximal expression in vitro. Mutagenesis experiments demonstrated the requirement for three elements, a CACGTG element at -80, a proximal sequence element at about -55, and the TATA-like box at -25. The major protein in sea urchin extracts that interacts with the CACGTG element is sea urchin USF, and immunodepletion of sea urchin USF greatly reduces transcription. The USF binding site in the U6 gene is highly homologous (11 of 13 nucleotides) with the USF binding sites found in the promoter of the S. purpuratus spec genes.