No abstract

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 proximal sequence element (PSE) for the sea urchin U6 small nuclear RNA gene has been defined. The most critical nucleotides for expression, located 61 to 64 nucleotides (nt) from the transcription start site, are 4 nt, AACT, at the 5 end of the PSE. Two nucleotide mutations in this region abolish transcription of the sea urchin U6 gene in vitro. The same two nucleotide mutations greatly reduce the binding of specific factors detected by an electrophoretic mobility shift assay. There is also a conserved AC dinucleotide 57 nt from the start site of the sea urchin U1 and U2 PSEs. The sea urchin U1 and U2 PSEs were substituted for the sea urchin U6 PSE, with the conserved AC sequences aligned with those of the U6 PSE. Both of these genes were expressed at levels higher than those observed with the wild-type U6 gene. Similar complexes are formed on the U1 and U2 PSEs, and formation of the complexes is inhibited efficiently by the U6 PSE. In addition, the E-box sequence present upstream of the PSE enhances U6 transcription from both the U1 and U2 PSEs. Finally, depletion of a nuclear extract with a DNA affinity column containing the U6 PSE sequence reduces expression of the U6 genes driven by the U6, U1, or U2 PSE but does not affect expression of the 5S rRNA gene. These data support the possibility that the same factor(s) interacts with the PSE sequences of the U1, U2, and U6 small nuclear RNA genes expressed in early sea urchin embryogenesis.The spliceosomal small nuclear RNAs (snRNAs) are among the most abundant transcripts synthesized in metazoans. Most of the spliceosomal snRNAs are synthesized by RNA polymerase II (Pol II), with the exception of the U6 snRNA, which is synthesized by Pol III (7,20). snRNA promoters differ from promoters of most genes transcribed by Pol II and Pol III (18). Vertebrate snRNA promoters, including the U6 snRNA, contain two major elements: a proximal sequence element (PSE) located at about position Ϫ55 which has been loosely conserved and a well-conserved distal sequence element (DSE) located around position Ϫ200. The vertebrate snRNA genes transcribed by Pol II lack a TATA box, while the U6 genes contain an essential TATA box (8, 18). There are common factors, including Oct-1, which bind to the DSE and enhance transcription from both the Pol II and Pol III snRNA genes (14,15,28). Recently, two active PSE-binding protein complexes, SNAPc (for snRNA-activating protein complex [5]) and PTF (for PSE-binding transcription factor [31]), have been purified from HeLa cells. Both complexes bind to the PSE sequence and direct transcription from U1 and U6 genes, providing direct evidence that the same PSE binding complex is involved in the transcription of both Pol II and Pol III snRNA genes (5, 31, 32). Two subunits of the SNAPc complex have been cloned and shown to be novel proteins (5, 32), although the other factors in this complex have not yet been completely characterized.In early sea urchin embryogenesis there is a rapid synthesis of snRNAs starting at about the 32-cell stage and co...

The BGR-like gene may play an important role in spermatogenesis/testicular development and may be correlated with male infertility.