Nucleic Acids Research

Screening of [lambda] gt11 cDNA expression library

A [lambda] gt11 HeLa S3 cDNA library (Clontech no. HL3021b, lot no. 37208) was screened according to established procedures (27-29) using a concatenated, radiolabeled double-stranded oligonucleotide corresponding to the human U6 SPH motif [NONOCT(long); 14]. Approximately 5.5 × 10⁵ phages were screened at a density of 1.5 × 10⁴ per filter. Two positive clones ([lambda]SBF1 and [lambda]SBF2) were selected that withstood four rounds of probe binding. Inserts were excised by restriction with EcoRI and subcloned into the pGEM3Zf(+) plasmid vector (Promega). The insert from [lambda]SBF2 was sequenced entirely on both strands using the dideoxy method with multiple internal primers. This partial cDNA sequence of human SPH-binding factor has been deposited in GenBank under accession no. AF071771.

Production of recombinant protein

Production of polyclonal antisera against hSBF and western blots

Antisera were generated from two rabbits injected with recombinant hSBF(76-626) that was gel purified and emulsified along with the slice of polyacrylamide. Sera were assayed initially by ELISA and western blotting and then by inhibition of SBF-DNA complexes in an electrophoretic mobility shift assay (EMSA).

For the western blots, ~100 µg of total protein from a HeLa S100 extract were fractionated on a 7% polyacrylamide-SDS gel and electrophoretically transferred to a nitrocellulose filter. The filter was incubated with a 1:1000 dilution of anti-hSBF antiserum and visualized by a colorimetric assay using Western Blue stabilized substrate for alkaline phosphatase (Promega).

DNA binding assays

In vitro transcription in HeLa S100 extract

S100 extracts were prepared from HeLa spinner cells as described (30). In order to remove endogenous SBF, the extract was immunodepleted using polyclonal anti-hSBF antiserum. Protein A-Sepharose resin was preincubated with immune serum, preimmune serum or no serum for 1 h at room temperature. After washing twice with phosphate-buffered saline and three times with buffer D (0.1 M KCl, 20 mM HEPES, pH 7.9, 20% glycerol, 0.1 mM EDTA, 2 mM dithiothreitol), the antibody-protein A-Sepharose resin was incubated with the S100 extract (20 µl resin/200 µl S100) for 2 h at 4°C with gentle agitation, followed by a brief centrifugation to separate the depleted extract.

Transcription reactions were carried out with 50 ng of plasmid DNA containing the U6/CFREE reporter plus 450 ng of pGEM3Zf(-) DNA. Final reactions (25 µl) contained 40 mM KCl, 8 mM HEPES (pH 7.5), 2 mM MgCl₂, 2 mM dithiothreitol and ~100 µg of protein from a HeLa cell S100 extract. After preincubation at 30°C for 90 min, transcription proceeded with the addition of nucleoside triphosphates to final concentrations of 0.5 mM ATP, 0.5 mM UTP, 20 µM unlabeled GTP and 10 µCi [[alpha]-³²P]GTP (800 Ci/mmol; DuPont/NEN), along with [alpha]-amanitin to 2 µg/ml. After further incubation at 30°C for 30 min, nucleic acids were isolated by phenol/CHCl₃ extraction and ethanol precipitation. RNAs were separated by electrophoresis on 12% polyacrylamide/8.3 M urea gels, visualized by autoradiography or quantitated using a Fujix BAS2000 PhosphorImager (Fuji).

Molecular cloning of cDNA encoding human SPH-binding factor

In order to isolate a cDNA clone that encodes human SBF, we screened a HeLa [lambda] gt11 expression library with a radiolabeled, concatenated oligonucleotide containing the human U6 snRNA gene SPH motif. Two independent plaques were obtained after screening ~550 000 phage. Limited sequencing of the first candidate indicated that we had obtained a partial cDNA encoding human Sµbp-2, a protein that binds to the immunoglobulin µ switch region (31). However, it is unlikely that hSBF and Sµbp-2 are the same since the predicted molecular weight of Sµbp-2 (~110 000) is significantly larger than that of hSBF (~85 000), a value that was determined by protein-DNA crosslinking (14). Partial sequencing of a second candidate ([lambda]SBF2) showed that it was highly homologous to human ZNF143 (26) and Xenopus laevis Staf (23). Complete sequencing of the [lambda]SBF2 insert (GenBank accession no. AF071771) demonstrated that it contained a partial cDNA, including nucleotides that encode amino acids 76-626 of the ZNF143 coding sequence, with a single nucleotide difference leading to an E->Q change at amino acid 549. The deduced hSBF sequence contains seven putative CCHH zinc fingers in the central region of the primary structure. Our partial cDNA clone encodes two of the four 15mer repeats noted previously in the sequences of Staf from Xenopus and mouse (23,24). In the 3[prime]-untranslated region (3[prime]-UTR), the HeLa-derived hSBF sequence is truncated relative to ZNF143 with divergent sequence prior to the polyadenylation site of hSBF.

In order to obtain the remainder of the coding sequence of HeLa cell SBF, we employed RT-PCR with a sense strand primer corresponding to the start codon region of ZNF143, an antisense primer including hSBF sequence just 3[prime] to the C-terminal 15mer repeat and total RNA isolated from HeLa cells. The sequence obtained was identical to that of the N-terminal region of ZNF143 (results not shown). Human SBF is highly homologous to Xenopus Staf (463 out of 626 residues are identical = 74%; 23) and the recently cloned mouse Staf (608 out of 626 residues are identical = 97%; 24).

DNA-binding specificity of recombinant hSBF

In order to investigate the DNA-binding properties of hSBF encoded by the cDNA clone, we constructed a bacterial expression plasmid containing the hSBF sequence. Recombinant hSBF was expressed in bacteria and partially purified by urea solubilization out of inclusion bodies (Fig. 1A). This protein preparation bound the SPH motif of the human U6 distal region in a DNase I footprint assay (Fig. 1B). Furthermore, we noticed two details of the DNase I protection pattern of recombinant SBF that matched what was found with a HeLa cell fraction of SBF (see fig. 6 in 14), namely an extended region of protection 5[prime] to the SPH motif and a hypersensitive band at the junction of the OCT and SPH elements.

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Figure 1. Recombinant hSBF(76-626) binds to the human U6 SPH motif. (A) SDS-PAGE showing expression of hSBF(76-626) in E.coli. Lane 1 shows total protein from cells lysed after 3 h IPTG induction at 37°C and lane 2 shows partially purified protein recovered from inclusion bodies after urea solubilization and removal of urea by dialysis. (B) Binding of human U6 SPH motif by recombinant hSBF(76-626) detected by DNase I footprinting. Lane 1 shows the pattern of digestion with naked DNA probe, lane 2 shows protection by a recombinant protein containing the Oct-1 POU domain and lanes 3 and 4 show the cleavage patterns after incubation with recombinant hSBF(76-626).

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Figure 2. Binding of recombinant hSBF(76-626) expressed by in vitro transcription/translation is sensitive to mutations within the U6 SPH motif. (A) Electrophoretic mobility shift assay with in vitro translated protein. Protein was expressed from the pET/hSBF(76-626) plasmid DNA or a pET5a control plasmid lacking any insert, incubated with ~3 fmol of radiolabeled probe containing the human U6 SPH+OCT elements and separated by electrophoresis on 4% polyacrylamide non-denaturing gels. Bands labeled `Oct' or `hSBF' represent complexes that were competed by unlabeled OCT oligonucleotide (300- and 3000-fold molar excess in lanes 10 and 11, respectively) or unlabeled SPH oligonucleotide (300- and 3000-fold molar excess in lanes 12 and 13, respectively). (B) Sequences of sense strand of the human U6 SPH motif embedded in plasmid DNAs used for competition experiments. Each plasmid contained a human U6 maxigene with a set of mutations in the octamer motif (OCTMUT) and the specified mutations in the adjacent SPH motif (described in 14). (C) EMSA using in vitro expressed hSBF(76-626) plus ~3 fmol of radiolabeled U6 distal region probe containing OCT+SPH motifs, and various amounts of plasmid DNAs containing no U6 promoter (pGEM), wild-type U6 promoter (maxiU6) or promoters containing mutations in the SPH motif. Quantification using a PhosphorImager is tabulated in the row labeled `% SBF-DNA complex'. A quantitative comparison using the same mutant SPH competitors (1 µg of each) in EMSA experiments with HeLa S100 extract is supplied in the bottom row (14).

In addition, we expressed hSBF(76-626) after transcription/translation in vitro and used the protein in an EMSA (Fig. 2). A strong hSBF-DNA complex was detected on a human U6 distal region probe containing both octamer and SPH motifs. This complex was formed only upon addition of a transcription/translation lysate containing the pET/hSBF(76-626) plasmid and not with addition of a pET5a control plasmid (compare lanes 5-7 with lanes 2-4 in Fig. 2A). Furthermore, the hSBF-DNA complex was competed effectively by addition of unlabeled hU6 SPH oligonucleotide (Fig. 2A, lanes 12 and 13), but not by addition of OCT oligonucleotide (Fig. 2A, lanes 10 and 11). A minor complex was formed on the probe with both pET5a control and pET/hSBF(76-626) samples that was competed upon addition of unlabeled OCT oligonucleotide. It is possible that this complex resulted from a small amount of Oct protein in the reticulocyte lysate. To further analyze the specificity of in vitro translated hSBF(76-626) protein in EMSA experiments, we added various plasmid DNA competitors that contained mutations in the SPH motif (Fig. 2B). These mutations have been shown to compete less effectively than the wild-type sequence for hSBF-DNA complex formation in gel mobility shift experiments using HeLa S100 extract (14; bottom row of Fig. 2C). All the mutations were relatively less effective competitors for binding of the recombinant hSBF protein than the wild-type SPH element (Fig. 2C). Significantly, the RM2-8 mutant was the most effective competitor among the mutants in experiments using both the in vitro translated hSBF (30% complex remaining with 1 µg competitor) and the endogenous hSBF present in S100 extract (25% complex remaining with 1 µg of competitor). Quantitative comparison of the other mutants is complicated because all are relatively ineffective, leaving >40% of hSBF-DNA complex even after addition of 1 µg of plasmid DNA. In combination, DNase I footprinting and EMSA results show that the recombinant protein produced from the SBF2 cDNA clone binds to the human U6 SPH motif with the same specifity as the native SBF present in extracts from HeLa cells.

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Figure 3. Polyclonal antibodies prepared against recombinant hSBF(76-626) bind specifically to native hSBF. (A) Western blot. Approximately 100 µg of total protein from a HeLa S100 extract were fractionated on a 7% polyacrylamide-SDS gel, electrophoretically transferred to nitrocellulose and probed with a 1:1000 dilution of serum. Antibody binding was visualized with a colorimetric assay. The positions of markers run on an adjacent lane are noted on the right side. (B) EMSA. Each lane contained ~2 fmol of radiolabeled SPH oligonucleotide probe and 4 µg of protein from a HeLa cell S100 extract. For samples in lanes 1-3, 0.5 µl of the designated antiserum was added at the same time as the S100 extract and incubated at 30°C for 30 min. For samples loaded in lanes 4-6, the SPH probe and S100 extract protein were preincubated at 30°C for 30 min, followed by a further 15 min incubation after the addition of 0.5 µl of the designated antiserum. Two antisera from rabbits separately injected with hSBF antigen (A and B) or preimmune serum (P.I.) were used in this experiment. The band marked `hSBF' was the only one competed by addition of excess unlabeled SPH oligonucleotide in samples that lacked rabbit antisera (results not shown).

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Figure 4. In vitro transcription reactions using human U6 promoter templates in hSBF-depleted HeLa S100 extracts. (A) HeLa S100 extract was depleted of hSBF using anti-SBF antiserum or mock-depleted without addition of any serum (ProtA beads) or with addition of preimmune serum (P.I. serum). Untreated S100 extract or each treated extract was used to compare the transcriptional efficacy with 50 ng of a proximal-only U6 promoter template (dl-84) or 50 ng of a template containing a single copy of the SPH element ligated to the U6 proximal promoter (NON10). Bands labeled `U6/CFREE' are transcripts derived from the U6 promoter. The band marked `tRNA^his' resulted from guanylyltransferase radiolabeling of endogenous RNA in the extract and served as a convenient recovery control for each sample. (B) Stimulation of U6 promoter transcription by addition of recombinant hSBF(76-626) to SBF-immunodepleted S100 extracts. All reactions contained immunodepleted extract such as characterized in lanes 6 and 7 from (A). Reactions analyzed in lanes 1-3 contained 50 ng of dl-84/CFREE plasmid DNA, whereas RNAs in lanes 4-6 resulted from reactions containing 50 ng of NON10 (+SPH) plasmid DNA. (C) Quantitation of transcription reactions. Transcripts that had been separated by electrophoresis on polyacrylamide gels were quantified with a Fujix BAS2000 PhosphorImager (Fuji). After background subtraction, the amount of each U6/CFREE group of bands was normalized according to the tRNA^his band intensity in that lane and compared with the signal from each promoter when hSBF(76-626) was omitted from the transcription reaction. Each bar represents the average of two independent experiments and the error bar shows the range from each experiment.

Polyclonal antibodies to hSBF inhibited assembly of SBF-DNA complexes

To further verify the authenticity of the hSBF cDNA clone and to provide a biochemical reagent for studies of SBF function, we used recombinant, gel-purified hSBF(76-626) as an antigen to prepare rabbit polyclonal antibodies. Binding of a western blot containing total protein from a HeLa S100 extract with the anti-SBF antiserum identified a major band of ~79 kDa (Fig. 3A). At present, we do not know whether the closely spaced doublet indicates mild degradation of hSBF or altered native forms of the protein. The estimated molecular mass of 79 kDa on the western blot is fairly close to the size of 85 kDa that we determined previously by UV light-mediated protein-DNA crosslinking (14).

In addition, anti-SBF antisera from both rabbits that we inoculated effectively inhibited detection of hSBF-DNA complexes on a human U6 SPH oligonucleotide probe incubated with HeLa S100 extract (Fig. 3B, compare lanes 2 and 3 with lane 1 or lanes 5 and 6 with lane 4). We detected no discrete supershifted band in this assay, although a significant amount of probe was retarded at the origin of the gel when the antiserawere added subsequent to complex formation (Fig. 3B, lanes 5and 6).

Recombinant hSBF-stimulated transcription from a U6 promoter in vitro

In previous work we have demonstrated that a human U6 promoter containing a single copy of an SPH motif was transcribed in vitro at an ~3.5-fold higher level than a promoter lacking any distal element (7). In order to examine further the capability of hSBF to stimulate polymerase III transcription of snRNA gene promoters, we used anti-SBF antiserum to immunodeplete the HeLa S100 transcription extract. The immune antiserum almost completely eliminated the ability of the extract to activate transcription of the +SPH template (NON10) when compared with the dl-84 template (Fig. 4A, compare lanes 6 and 7 with 2 and 3). Using a gel shift assay, the immunodepleted extract contained a barely detectable amount of hSBF (results not shown). Controls in which the HeLa S100 extract was treated without serum or with preimmune serum did not diminish the SPH-dependent transcriptional stimulation (Fig. 4A, lanes 4 and 5 and 8 and 9, respectively).

Next, we added back recombinant hSBF(76-626) to the immunodepleted extract and compared the transcription of the dl-84 and +SPH templates. Addition of the recombinant protein caused a marked increase in transcription, up to a 6-fold higher level with ~20 ng of protein (Fig. 4B, lanes 4-6, and C). We consistently found that the recombinant hSBF(76-626) also stimulated transcription of the dl-84 template that lacks an SPH motif (Fig. 4B, lanes 1-3, and C). However, the fold stimulation of the +SPH/U6 promoter was at least double that of the -SPH template (Fig. 4C).