A nuclear extract from HeLa cells has been separated by DEAE-cellulose chromatography into two frac- tions, both of which are required for mRNA splicing in ...
Proc. Nati. Acad. Sci. USA Vol. 82, pp. 4351-4355, July 1985
Biochemistry
Isolation and characterization of two fractions from HeLa cells required for mRNA splicing in vitro (RNA processing/adenovirus/I-globin)
HENRY M. FURNEAUX*, KAREN K. PERKINS*, GREG A. FREYERt, JAIME ARENAS*, AND JERARD HURWITZ* *Graduate Program in Molecular Biology and Virology, Sloan-Kettering Institute for Cancer Research, New York, NY 10021; and tCold Spring Harbor Laboratory, P.O. Box 100, Cold Spring Harbor, NY 11724
Contributed by Jerard Hurwitz, March 15, 1985 A nuclear extract from HeLa cells has been ABSTRACT separated by DEAE-cellulose chromatography into two fractions, both of which are required for mRNA splicing in vitro. Both fractions are heat labile and sensitive to N-ethylmaleimide. The activity of one of the fractions was abolished by preincubation with micrococcal nuclease, while the other fraction was unaffected by this treatment. This abolition indicates an essential nucleic acid component. Fractions I and II are required for the in vitro splicing of human ,B-globin and adenovirus transcripts.
Maturation of many mRNA molecules in eukaryotic cells is accompanied by the elimination of intervening sequences present in the primary transcript (1). This process, called RNA splicing, is a critical control of gene expression, in that proteins of different amino acid composition can be made from the same primary transcript. Recently, a more detailed insight into the mechanism of this process has been gained by the development of cell-free extracts that splice precursor mRNA in vitro (2-5). In particular, the ability of nuclear extracts to efficiently utilize a precursor mRNA synthesized by a bacterial RNA polymerase (6) represents a considerable advance in the development of assays for the various activities likely to be involved in splicing. It is clear that a precise understanding of the mechanism of mRNA splicing in vitro will result from the identification and purification of the components involved. This paper reports the beginning ofthis process: the chromatographic resolution of a nuclear extract from HeLa cells into two discrete mutually dependent fractions.
MATERIALS AND METHODS Materials. Bacteriophage SP6 RNA polymerase fraction V (blue dextran-Sepharose pool) was purified by the method of Butler and Chamberlin (7). Restriction enzymes were obtained from New England Biolabs, RNase T1 from Calbiochem, micrococcal nuclease from P-L Biochemicals, and [a-32P]GTP from New England Nuclear. Phage and Plasmid DNA. The SP6-p-globin plasmids pSp64-H,3 6 (containing an intron of 130 nucleotides) and pSp64-HP 6-IVS 1,2 (cDNA) were generously provided by M. Green (Harvard University). SP6 denotes a plasmid containing an efficient SP6 bacteriophage promoter. The M13 phage pJAW was a gift of P. Sharp (Massachusetts Institute of Technology). The SP6-adenovirus major late plasmid pKT1 was constructed by one of us (G.A.F.) and will be described elsewhere. RNA Splicing Reaction in Vitro. SP6 transcripts were prepared essentially as described by Ruskin et al. (6) except that the transcript was labeled with [a-32P]GTP and primed
with m"GpppG'. SP6 transcript was purified by polyacrylamide/urea gel electrophoresis, eluted from the gel slice with 0.5 M ammonium acetate/0.1% sodium dodecyl sulfate/1 mM EDTA, and precipitated with 2.5 vol of ethanol. The transcript was resuspended in 20 mM Hepes buffer, pH 7.6/1 mM EDTA and stored at -200C. In all experiments the RNA concentration is expressed as fmol of guanine nucleotide. SP6 RNA transcripts were shown to have capped ends by labeling with [a-32P]ATP and digestion with RNase T2. Two-dimensional thin-layer chromatography on polyethyleneimine cellulose yielded labeled m GpppGnpAp, indicating that virtually all of the transcript had been primed with m7GpppGm. RNA splicing reaction mixtures (0.05 ml) containing 20 mM Hepes buffer at pH 7.6, 3 mM MgCl2, 2 mM dithiothreitol, 0.4 mM ATP, 20 mM creatine phosphate, 2.6% (vol/vol) polyvinyl alcohol, labeled RNA transcripts (as indicated in figure legends), and various enzyme fractions (as indicated in legends) were incubated at 30°C for 2 hr. The reaction was terminated by the addition of 0.2 ml of sodium dodecyl sulfate buffer (200 mM Tris HCl, pH 7.5/25 mM EDTA/300 mM NaCl/2% sodium dodecyl sulfate) and water to 0.4 ml. After extraction with phenol/chloroform and precipitation with ethanol the RNA products were dissolved in formamide and then analyzed by polyacrylamide/urea gel electrophoresis and visualized by autoradiography. Single-Stranded cDNA Hybridization Analysis. Various 32plabeled RNA products were analyzed by hybridization to single-stranded cDNA according to the method of Keohavong et al. (8). A single-stranded ,B-globin cDNA was constructed by the insertion of the HindIII/BamHI fragment from pSp64-H,3 6-IVS 1,2 into the HindIII and BamHI sites of M13 mp8. Nuclear Extract Preparation and DEAE-Cellulose Chromatography. Nuclear extracts were prepared from 101 of HeLa cells as described by Dignam et al. (9). The ionic strength of the nuclear extracts (30 ml, 16 mg of protein per ml) was adjusted to 0.2 M KCl and then applied to a DEAE-cellulose column (9.6 x 2.7 cm), previously equilibrated with 0.2 M KCl in buffer A [20 mM Hepes, pH 7.6/0.1 mM EDTA/1 mM dithiothreitol/10% (vol/vol) glycerol]. After collection of the material that passed through the column (fraction I, 50 ml, 6 mg of protein per ml), the column was washed with 3 column volumes of 0.2 M KCI in buffer A. The column was then eluted with 1 M KCl in buffer A. Fractions (10 ml) were then collected and the peak protein fractions were pooled to yield fraction 11 (20 ml, 2.3 mg/ml). Fractions I and II were dialyzed against 0.1 M KCl in buffer A and stored in aliquots at -70°C. Fractions retained activity for at least 2 months at -70°C and for at least 3 days at 4°C.
RESULTS In these studies we have used the RNA splicing assay devised by Ruskin et al. (6). In this procedure, a short SP6-f8-globin transcript of 497 nucleotides containing an intron of 130
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Proc. Natl. Acad Sci. USA 82
nucleotides was incubated with nuclear extracts from HeLa cells and the RNA products were analyzed by polyacrylamide/urea gel electrophoresis. RNA splicing activity in the extract was measured by the appearance of discrete RNA species. Fig. lA shows the result of such an assay with a nuclear extract from HeLa cells. Creatine phosphate plus ATPdependent formation of five RNA species, 450, 370, 252, 155, and 140 nucleotides (as determined by comparison with DNA size markers), was observed. With the exception of the 450-nucleotide species, these reaction products agree well with those described by Ruskin et al. (6). The 450-nucleotide species does in fact correspond to the 380-nucleotide "lariat" species described previously (6). Our RNA products were routinely analyzed on 6% polyacrylamide/urea gels to increase the separation between the lariat species and the spliced RNA product, which is 370 nucleotides in length. The identity of the 450-nucleotide lariat species was confirmed by its anomalous gel migration and by the presence of a nuclease P1-resistant digestion product expected from a branch site containing 2',5'-phosphodiester linkage (data not shown). The identity of the 370-nucleotide species as the spliced RNA product was suggested by its comigration with an SP6 transcript synthesized from the cDNA SP6-p-globin plasmid pSp64-H,8 6-IVS 1,2 (Fig. 1A). The nature of the 370nucleotide product was confirmed by hybridization analysis using the single-stranded cDNA hybridization, RNase T1protection technique described by Keohavong et al. (8). A
When this procedure is used, hybridization of cDNA to radioactive precursor transcript should result in the protection of the exon segments. Since the 8-globin cDNA used here corresponded exactly to the 5' and 3' termini of the transcript, such an analysis resulted in the formation of two labeled RNA species, 155 and 209 nucleotides long (Fig. 1B, lane 1). On the other hand, spliced RNA should be entirely protected, yielding a 370-nucleotide band. Fig. 1B (lanes 2 and 3) shows that both bona fide spliced RNA (synthesized from the cDNA SP6 plasmid, pSp64-H8 6-IVS 1,2) and the 370-nucleotide reaction product yielded 370-nucleotide bands. Separation of the Nuclear Extract into Two Fractions. The two fractions (I and II) obtained by DEAE-cellulose chromatography were assayed for RNA splicing activity. Fig. 2 (lanes 1-4) shows that each fraction alone exhibited no ATP-dependent formation of specific RNA processing products. The combination offractions I and II, however, resulted in the ATP-dependent formation of all the products expected in the RNA splicing reaction described above (Fig. 2, lanes 5 and 6). A more detailed analysis of the mutual dependence of the two fractions is presented in Fig. 3. The concentration of each fraction was varied independently, and the splicing activity was expressed as the amount of 370-nucleotide spliced product formed. The difference in response to increasing amounts of each fraction indicated that these are, in fact, separate activities and not merely the chromatographic distribution of a single activity. Under optimal conditions, the combination of fractions I and 11 (166 ,ug of protein) yielded 63 fmol of final RNA spliced product in 2 hr at 30°C,
B
12
1
M
2
3
(1985)
1 23456
M
M
LET-rn 497497-
cm
450370-
370-
I
450-
_
370-
209- 155 -
m
252-
252-
LI-LI -
155140-
ATP - +
155-
FIG. 1. Splicing of an SP6-/3-globin transcript by a nuclear extract from HeLa cells. (A) SP6-,8-globin transcript (325 fmol, 17,000 cpm/pmol) was incubated with nuclear extract (320 ,g of protein) and samples were analyzed by 6% polyacrylamide/urea gel electrophoresis. Lanes: 1 and 2, incubation in the absence and presence of ATP and creatine phosphate, respectively; M, 370nucleotide-spliced SP6 transcript synthesized from the pSp64-Ht3 6IVS 1,2 DNA template. Transcripts are indicated schematically on the left. Bars represent exons, and the line represents the intron in its linear and "lariat" forms. (B) Single-stranded cDNA hybridization analysis of the 370-nucleotide RNA species. RNA samples were hybridized to single-stranded ,-globin cDNA and analyzed. Lanes:
1,
-3
precursor
8-globin transcript; 2, the 370-nucleotide species
iso-
lated from the reaction described for A; 3, spliced RNA synthesized from pSp64-H,8 6IVS 1,2; M, DNA markers, Taq I digest of
replicative form I DNA from phage
4X174.
-
140-
w
Fraction I + + - - + + Fraction E - - + + + + ATPandCrP - + - + - + FIG. 2. Fraction I and fraction II are required for in vitro splicing of SP6-43-globin RNA. Reaction mixtures contained P3-globin transcript (325 fmol, 17,000 cpm/pmol), and fraction 1 (120 ,g) and/or fraction II (46 ug) as indicated above. Reaction products were analyzed by 6% polyacrylamide/urea gel electrophoresis. Lanes: 1-6, combinations of fraction I and II in the presence or absence of ATP and creatine phosphate (CrP); M, DNA markers, Alu I digest of 4X174 replicative form I DNA.
Proc. Natl. Acad. Sci. USA 82 (1985)
Biochemistry: Furneaux et al. A 100
Table 1. Effect of heat and N-ethylmaleimide and II
B
Fraction I
-
Fraction II
on
4353
fractions I
RNA splicing Treatment
-5E z
w
activity,
Exp. A
Fraction I Fraction II fmol/2 hr 602 None None None 30 min at 650C 111 30 min at 650C
