Identification of a SPH element in the distal region of a human U6 small nuclear RNA gene promoter and characterization of the SPH binding factor in HeLa cell extracts

Abstract

Vertebrate small nuclear RNA (snRNA) gene promoters contain a distal, enhancer-like region that is composed of an octamer motif adjacent to at least one other element. Here we show that a human U6 snRNA distal region contains a SPH motif previously found in several chicken snRNA gene enhancers and the 5'-flanking region of vertebrate selenocysteine tRNA genes. SPH binding factor (SBF) was detected in either chicken or HeLa cell extracts that could bind SPH elements in a species-independent manner. Both human and chicken SBF required divalent cation to bind effectively to DNA. DNase I footprinting experiments indicated that human SBF specifically protected the human U6 SPH element. Furthermore, a SBF polypeptide of approximately 85 kDa was detected in both HeLa and chicken extracts following ultraviolet light-mediated cross-linking to human U6 or chicken U4 SPH elements. A part of the human U6 SPH element was quite sensitive to mutation, as demonstrated by both specific protein binding and transcription assays. From these data it is apparent that the distal regions of some RNA polymerase III- and RNA polymerase II-transcribed small RNA promoters are virtually identical in composition, and their mechanisms of transcriptional activation are possibly quite similar.

FIG. 1

Identification of a DNA binding activity in HeLa cell extracts similar to chicken SBF. (A) Sequence of the enhancer region in the chicken U1ψ(O,S) construction. The octamer and SPH motifs are indicated by underlining. Lower-case letters indicate changes from the wild-type sequence, but these do not significantly affect enhancer activity (19). (B) Identification of a Mg²⁺-dependent DNA binding activity in HeLa cell extracts that interacts with the chicken U1 gene enhancer. A 118-bp 32P-labeled DNA fragment containing sequences of the U1 enhancer was incubated with increasing amounts of HeLa cell nuclear extract, and protein/DNA complexes were resolved by electrophoretic mobility shift assays. Complexes observed in the absence of MgCl₂ (lanes 1-5) are indicated to the left of the autoradiograph, whereas complexes formed in the presence of 5 mM MgCl₂ (lanes 6-10) are indicated to the right. (C) Similarity of electrophoretic mobilities and Mg²⁺-dependent DNA binding properties of chicken and human SBF/DNA complexes. Assays were performed as in (B), except that in lanes 2, 3, 5, and 6 the protein fraction used contained SBF purified from chicken embryo nuclear extracts by heparin agarose and DNA affinity chromatography. The SBF used in this experiment had been dialyzed against buffer containing 12.5 mM MgCl₂. Thus, there is a minor contribution of MgCl₂ from the added SBF (e.g., ∼0.4 mM MgCl₂ final concentration in lane 6).

FIG. 2

Competition of specific complexes by synthetic oligo-nucleotides containing octamer and SPH motifs. Electropho-retic mobility shift assays were performed as in Fig. 1 using a constant amount of HeLa nuclear extract (0.5 μl) in the presence of 5 mM MgCl₂ and increasing amounts of octamer competitor (cUlOCT; 0.2, 1, 5, 25, and 125 ng, lanes 1-5), increasing amounts of SPH motif competitor (cU l SPH; 0.2, 1, 5, and 25 ng, lanes 6-9), or no competitor (lane 10). Cold competitor oligonucleotides were mixed with the labeled DNA fragment prior to adding the extract. Note that the band labeled Oct-1 was specifically competed by the octamer oligonucleotide, and the band labeled SBF was specifically competed by the SPH motif oligonucleotide. Although the molecular compositions of the X and Y complexes were not directly investigated in this study, the data are consistent with the possibility that the X complex contains two molecules of Oct-1, and that the Y complex contains both Oct-1 and SBF.

FIG. 3

Competition of the human U6 NONOCT(SPH) complex by oligonucleotides containing vertebrate snRNA gene distal region elements. (A) Radiolabeled DNA probe containing wild-type human U6 NONOCT(SPH) and OCTCON sequences was used for electrophoretic mobility shift assays as described in the Materials and Methods section. Each sample contained approximately 3 fmol of probe DNA and 2 μg of protein from a HeLa cell SI00 extract. Competitor double-stranded oligonucleotides (sequences given in Table 1) were mixed into binding reactions prior to addition of the HeLa extract in amounts denoted above each lane. In (B), a longer version of the NONOCT-(SPH) region sequence was used [NONOCT(long) in Table 1]. Comparison of the efficiency of competition by the NONOCT(long) and NONOCT [“short”; (A) and (6)] oligonucleotides indicated a greater than 10-fold higher affinity for the longer oligonucleotide (results not shown).

FIG. 4

Competition of the chicken SBF/DNA complex by the human U6 NONOCT (SPH) element. Electrophoretic mobility shift reactions in lanes 2–21 contained chicken SBF [approximately 5 μg of the heparin agarose fraction (19)] and 5 mM MgCl2. Double-stranded oligonucleotides containing the chicken U1 SPH motif (lanes 1-11) or human NONOCT(SPH) element (lanes 12–22) were used as radiolabeled probes. Approximately 300 pg of radiolabeled probe was used in each binding reaction. Unlabeled competitor oligonucleotides corresponding to the chicken U1 or human U6 SPH motif (or a nonspecific oligonucleotide, NS) were added as indicated above the lanes in the following amounts: 5 ng in lanes 2, 5, 8, 13, 16, 19; 25 ng in lanes 3, 6, 9, 14, 17, 20; and 100 ng in lanes 4, 7, 10, 15, 18, 21. No competitor was added in lanes 11 and 12. The sequences of the cUlSPH and NONOCT(long) oligonucleotides used as probes and competitors are shown in Table 1. The nonspecific oligonucleotide competitor (NS) consisted of the following annealed sequences: 5′-GATCGGTTCAGGGAGCGCGCCGGCGCGCTGTGACGTAG-3′ and 5′-GA TCCTACGTCACAGCGCGCCGGCGCGCTCCCTGAACC-3′.

FIG. 5

Comparison of sequences of snRNA distal region SPH motifs and spacing to octamer motifs. The sequences of several SPH motifs and of a consensus derived from them are shown. Also noted are the distance to and the orientation of the octamer motif that is nearby the SPH element for all but the Xenopus tRNA^(Ser)Sec promoter. The consensus sequence of OCTforw is ATGCAAAT and OCTrev is ATTTGCAT. The sequences listed are from the following references: chicken U1 (20), chicken U4B and U4X (9), X. laevis tRNA^(Ser)Sec (16), bovine tRNA^(Ser)Sec (7), and human U6 (13).

FIG. 6

DNase I footprint over the NONOCT(SPH) region of the human U6 distal region. A singly end-labeled DNA fragment containing the human U6 NONOCT(SPH) and octamer motifs was prepared, incubated with fractionated HeLa cell extract, treated with DNase I, and electrophoresed as described in the Materials and Methods section. The human SBF fraction (approximately 15 μg) that was incubated with the probe to generate the sample in lane 2 was HeLa SI00 extract fractionated in series over phosphocellulose and DEAE-cellulose as described previously (6). Lane 1 displays the result of Maxam-Gilbert G-reaction cleavage of the probe that was used as a marker, and lane 3 shows DNase I cleavage of the probe carried out in the absence of protein. The lane marked “X” was a misloaded sample irrelevant to the results presented here.

FIG. 7

Ultraviolet light-mediated cross-linking to identify a DNA binding polypeptide of human and chicken SBF. An internally radiolabeled double-stranded oligonucleotide probe containing the human U6 NONOCT(SPH) and octamer motifs (A) or chicken U4B SPH motif (B) was prepared, incubated with protein, irradiated with ultraviolet light, treated with nucleases, and electrophoresed as described in the Materials and Methods section. (A) The human U6 SPH motif probe was incubated with HeLa extract protein fractionated over phosphocellulose as described (6). During the binding reactions, samples contained unlabeled double-stranded oligonucleotide competitors: lane 1: 3 pmol nonspecific DNA (N.S.); lane 2: 3 pmol OCTCON; lane 3: 3 pmol NONOCT(long). The sequences of OCTCON and NONOCT(long) are shown in Table 1. The N.S. oligonucleotide contained the following sequences that had been previously annealed: 5′-GATCCAGTCTGATCAGACTG-3′ and 5′-GATCCAGTCTGATCAGACTG-3′. Samples were electrophoresed on a 10% polyacrylamide gel. The arrow marked “hSBF” delineates the polypeptide that is specifically cross-linked to the NONOCT(SPH) element. Although this particular experiment did not include a lane of coelectrophoresed protein markers, the average apparent molecular weight of the hSBF polypeptide from four other experiments was approximately 85,000. (B) The chicken U4B SPH probe was incubated with the chicken SBF heparin agarose fraction (lanes 1-5) or with the HeLa P.35 fraction (lanes 6-10). Binding reactions for lanes 1, 5, 6, and 10 contained no added competitor oligonucleotides. Samples run in the other lanes contained competitor oligonucleotides as follows: lanes 2, 7: 3 pmol NONOCT(long); lanes 3, 8: 3 pmol cUlSPH; lanes 4, 9: 3 pmol of a nonspecific oligonucleotide with the sequence described in the legend to Fig. 4. Reactions loaded in lanes 5 and 10 were not irradiated with UV light, nor treated with nucleases. Samples were electrophoresed in an 8% polyacrylamide gel alongside protein markers whose mobilities are delineated to the right.

FIG. 8

Mutations located in the human U6 NONOCT(SPH) region decrease U6 maxigene expression in transfected cells and are inefficient competitors in electrophoretic mobility shift assays. (A) Locations of mutations in templates used in these experiments. Sequences are aligned according to the SPH consensus from Fig. 5. The NONOCTMUT clustered point mutant was described previously (6). Random mutations were introduced using a degenerate primer by PCR as described in the Materials and Methods section. Two degenerate primers, RM1 and RM2, were used in separate PCR preparations, and the region of degeneracy for each primer is delineated by the lines. All random mutation (RM) plasmids also contained a clustered point mutation to disrupt the consensus octamer motif [OCTCONMUT in (6)]. (B) Transient expression of U6 maxigene templates containing random mutations in the region of the NONOCT(SPH) element after transfection of human 293 cells. U6 maxigene expression in transfected cells was detected by primer extension analysis and electrophoresis on 10% polyacrylamide/8.3 M urea gels. “MaxiU6” represents the primer extension product from U6 maxigene RNA, and “cβ3” represents the major primer extension product from transcripts initiated from a chicken 0-tubulin gene contained in a cotransfected plasmid used as a control to normalize for variable transfection efficiency and RNA recovery. The lane marked “M” contained radiolabeled DNA fragments from Mspl digestion of pGEMl plasmid DNA. The “dl-148” plasmid used to generate the sample electrophoresed in lane 2 was a U6 maxigene template lacking 5′-flanking sequence upstream of –148, and, hence, missing the distal region. (C) Quantitation of transfection experiments. Primer extension products that had been electrophoresed on polyacrylamide gels were quantitated with a Fujix BAS2000 Phosphorimager (Fuji). After background subtraction, the level of each maxiU6 band was normalized according to the cβ3 band intensity in that lane and compared with that from a wild-type (w.t.) U6 maxigene template included in each experiment. The height of each bar represents the average value from at least four separate experiments (actual number of samples tested given in parentheses above each bar), and the height from the midpoint of the error bar shows one standard deviation from the mean. All mutant templates contained a disrupted consensus octamer motif. The “NONOCT-/OCTCONMUT” template contained both disrupted octamer and NONOCT(SPH) motifs [NONOCTMUT in (A)]. (D) Electrophoretic mobility shift assays using NONOCT(SPH) region random mutant templates as competitors. A radiolabeled probe containing both the NONOCT(SPH) and octamer motifs from the human U6 distal region was prepared and used for gel shift assays as described in the Materials and Methods section except all binding reactions contained 20 μg/ml poly(dl-dC)·poly(dl-dC) and 20 μg/ml plasmid DNA. Binding reactions contained 2 μg of protein from a HeLa SI00 extract. To effect specific competition of the hSBF/DNA complex, 50, 300, or 1000 ng of plasmid DNAs containing a normal NONOCT(SPH) motif (RM2-13) or mutant templates were substituted for the same amount of pGEM3Zf(–) DNA in binding reactions. Radioactivity in hSBF/DNA complexes separated on native polyacrylamide gels was determined using a Fujix BAS2000 Phosphorimager (Fuji). After background subtraction, the amount of radioactivity at each titration point was compared as a percentage to the signal from the sample where only 1 μg, pGEM3Zf(–) vector, and no specific competitor, was used as the plasmid DNA. Each data point is the average of two experiments.