THE JOURNAL OF BIOLOGICAL CHEMISTRY

Printed m U.S.A. Vol. 256, No. 2, Issue of January 25, pp. 956-963, 1981

The Small Nuclear RNAs of the Cellular Slime Mold Dictyostelium discoideum ISOLATION AND CHARACTERIZATION*

(Received for publication, May 16, 1980)

Jo Ann Wise and Alan M. Weiner From the Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06510

Three species of small nuclear RNA from the lower eucaryote Dictyostelium discoideum have been isolated and characterized with regard to size, cellular abun- dance, modified nucleotide content, and 5’-end struc- tures.

Previous studies had shown that the nuclei of mam- malian cells contain a number of discrete low molecular weight, nonribosomal, nontransfer RNA molecules known as small nuclear RNAs. The mammalian small nuclear RNAs range in size from approximately 100 to 250 nucleotides and are quite abundant, in some cases approaching ribosomal RNA in number of copies/cell. Some of these molecules have an unusual cap structure at their 5’-ends similar to that found on eucaryotic messenger RNAs, and a number contain a characteris- tic set of internal modifications as well.

Our results indicate that the small nuclear RNAs of Dictyostelium resemble their counterparts in higher eucaryotic cells structurally, but are present in signif- icantly fewer copies/cell. The implications of these findings for small nuclear RNA function are discussed.

In recent years, a substantial body of literature has begun to accumulate on a comparatively neglected class of RNA molecules found in all eucaryotic cells, the small nuclear RNAs. In mammalian cells, a t least eight species of snRNA,’ ranging in size from approximately 100 to 250 nucleotides, have been extensively characterized (for a review, see Ref. 1). U1, U2, and U3 snRNA appear to be metabolically stable, and U1 is nearly as abundant in the nuclei of mammalian cells as the ribosomal RNAs are in the cytoplasm (2). U3 snRNA has been localized primarily within the nucleolus (3), while U1 and U2 (as well as several other snRNAs) are found in distinct small ribonucleoprotein particles (4, 5). Five snRNAs from rodent cells (4.5 S , 4.5 Sr, U1, U2, and U3) have been com- pletely sequenced. U1, U2, and U3 are uridine-rich and possess 5’-cap structures resembling those found on messenger RNA, but more highly methylated (6-8); U2 contains substantial amounts of pseudouridine, as well as both 2“O-ribose and base methylations (7); 4.5 S and 4.5 SI have 5’-triphosphates and are unmodified. Both U1 and 4.5 S snRNA have been postulated to play a role in processing heterogeneous nuclear

* These studies were supported by Grants PCM 76-81524 and PCM 78-21799 awarded by the National Science Foundation and by Grant GM 26312 awarded by the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduer- tisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

’ The abbreviations used are: snRNA, small nuclear RNA; Mes, 2- (N-morpho1ino)ethanesulfonic acid.

RNA because they can form base-paired regions with the sequences flanking splice junctions in various nuclear precur- sors of messenger RNA (5,9). Rodent 4.5 S RNA also displays remarkable homology to a sequence found at the origin of replication in three different papovaviruses, simian virus 40, polyoma, and BK, suggesting that it may play an additional role in the initiation of cellular DNA replication (11).

The existence of snRNAs in lower eucaryotes has been inferred from the gel electrophoresis patterns of nuclear RNA from a number of organisms (reviewed in Ref. 12), but has been most dramatically demonstrated by the elegant nuclear transplantation studies of Goldstein and his collaborators with Amoeba proteus (13, 14). Nuclei from labeled amoebae were transplanted to unlabeled amoebae to form binucleate cells: certain snRNA species remained in the original nucleus (non- shuttling RNAs), while others equilibrated between the two nuclei (shuttling RNAs).

Here we report the initial isolation and characterization of small nuclear RNAs from the cellular slime mold Dictyoste- lium discoideurn; in a separate publication (47), we describe the genomic organization, cloning, and sequencing of the genes encoding one of these snRNA species. The lower eucaryote Dictyostelium was chosen because its DNA se- quence organization and RNA metabolism have been the subject of intensive study (15) and because vegetative amoe- bae can be induced to undergo a synchronous program of changing RNA and protein synthesis during the develop- mental life cycle (16, 31). Thus, modulation of snRNA gene expression during development might give some indication of the function of this class of molecules, which has remained largely in the realm of speculation until now. Our experiments show that the slime mold does indeed possess several species of nuclear RNA which are comparable in both size and struc- ture to the snRNAs of mammalian cells. The most striking difference between the snRNAs of Dictyostelium and their mammalian counterparts is the abundance of the molecules: amoeba1 snRNAs are present in only 1 to 2% as many mole- cules/nucleus, although the cell mass and generation times are similar for cultured mammalian cells and amoebae grown axenically in shaker culture.

EXPERIMENTAL PROCEDURES

Materials Trypticase peptone for growth of Dictyostelium was obtained from

BBL; yeast extract was from Difco. Carrier-free ‘”PO4 was purchased from either New England Nuclear or Amersham/Searle. [5”’”P]pCp was obtained from New England Nuclear. Sodium dodecyl sulfate, acrylamide, and N,N”methylenebisacrylamide were purchased from Bio-Rad. Nucleotide pyrophosphatase, diethylpyrocarbonate, and yeast RNA used as carrier were obtained from Sigma. The yeast RNA was phenol-extracted and ethanol-precipitated before use. T1 and T2 RNases were obtained from Sankyo (Tokyo) through Calbi-

956

Small Nuclear RNAs of Dictyostelium 957

ochem, RNase A was from Worthington, and PI nuclease was from Yamasa Shoyu (Tokyo). Cellogel strips were purchased from Kalex, polyethyleneimine cellulose thin layer plates from Brinkmann, and plastic-backed cellulose thin layer plates were from Eastman. Re- agents for chemical RNA sequencing were obtained from the sources reported by Peattie (17).

Methods

Culturing a n d Radioactive Labeling of Cells-D. discoideum strain AX3 was grown in shaker cultures in Mes-HL5 medium (18) maintained at room temperature (-19°C). For labeling with "Po,, cells were diluted to a density of 3 X lO'/ml in Mes-HL5 medium. Streptomycin sulfate was added to a concentration of 0.3 mg/ml followed by "'PO, at 40 to 80 pCi/ml. Cells were then grown for 2 days to a density of 5 X 10"/ml. Using this labeling protocol, 10,000 to 40,000 cpm of each snRNA species could be obtained.

Cell Fractionation and RNA Preparation-For preparation of total cellular RNA, the cells (1.4 X lo9) were collected by centrifu- gation and washed three times with ice-cold 20 mM potassium phos- phate (pH 6.5). The cell pellet was frozen in dry ice/ethanol for 10 min and then dislodged from the bottom of the tube. Ten milliliters of redistilled phenol and 20 ml of BC buffer (0.1 M Tris base, 0.1% sodium dodecyl sulfate, and 5 mM Na? EDTA) at 37°C were added to the cells and vortexed vigorously for 1 to 2 min. The lysis mixture was then centrifuged at 4,000 X g for 5 min a t 4°C followed by removal of the upper phase to a fresh tube. All subsequent operations were performed on ice. The aqueous phase was extracted again with 10 ml of phenol followed by three successive extractions with 30 ml of chloroform/isoamyl alcohol (98:2). The deproteinized RNA was then precipitated by addition of 0.1 volume of 4 M sodium acetate and 2.5 volumes of 100% ethanol. The RNA was recovered by centrifugation at 10,000 X g for 20 to 30 min.

For preparation of nuclear and cytoplasmic RNA, cells were col- lected, washed, and frozen as described above. Three different buffers were employed for isolation of nuclei. The dislodged cell pellet was taken up in 10 ml of HMK (5% sucrose, 2% NPT-12,40 mM MgC12,20 mM KC1, and 50 mM 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid, adjusted to pH 7.5 with NH4OH) (18), J B (10% sucrose, 0.5% NP-40, 40 mM KCl, 20 mM potassium phosphate, pH 7.5, 0.5 mM magnesium acetate, and 0.1 m Naz EGTA) (19), or TP (10% sucrose, 0.5% Triton X-100,5 mM magnesium acetate, and 50 mM 4-(2-hydrox- yethy1)-I-piperazineethanesulfonic acid, adjusted to pH 8.0 with NH,OH) (20) buffer and gently agitated on a vortex mixer until the pellet thawed, and then for 30 s longer. The nuclei were recovered from the lysate by centrifugation at 10.000 X g for 5 min at 4°C. The supernatant cytoplasmic extract was then transferred to a tube con- taining phenol/BC and held on ice while the nuclei were washed twice with 10-ml aliquots of lysis buffer without detergent. The final nuclear pellet was resuspended in 1 ml of buffer before deproteiniza- tion. Extraction of nuclear and cytoplasmic RNA was performed in parallel as described above for total cellular RNA. All buffers used in the isolation of RNA were treated with 0.1% diethylpyrocarbonate and autoclaved before use.

Preparation of Messenger RNA-Poly(A)-containing RNA from Dictyostelium was prepared essentially as described by Dottin et al. (21). except that vegetative, rather than starved cells were used.

Polyacrylamide Gel Electrophoresis-Analytical electrophoresis was performed in polyacrylamide slabs (300 X 175 X 1.5 mm), with a slot width of 18 mm. Preparative gels were 3-mm thick with a 120- mm slot. Gels were poured and run in different dilutions of 10 X TBE buffer (22), which contains 108 g of Tris base, 55 g of boric acid, and 0.93 g of disodium EDTA/liter, giving a final pH of 8.3. Gels were polymerized at room temperature in 1 X TBE buffer containing 9.67% acrylamide, 0.33% N,N"methylenebisacrylamide (10% gels) or 11.6% acrylamide, 0.4% bis (12% gels), 7 M urea, 0.067% amonium persulfate, and 0.023% N,N,N',N"tetramethylethylenediamine. Gels were run a t room temperature or a t 4°C in 1/2 X TBE buffer a t 13 V/cm. Before

min in loading buffer (7 M urea, 0.1% bromophenol blue, and 0.1% application to the gels, RNA samples were heated a t 60°C for 3 to 5

xylene cyano1 FF). Nuclear RNA from Dictyostelium was also electrophoresed in a

urea gradient gel as described by Gross et al. (23). A polyacrylamide slab gel was poured with a continuous horizontal gradient of urea running from 0 to 7 M, and the RNA was applied as a single broad band to the top of the slab. Cross-linker ratio, loading buffer, and running buffer were as described above.

Whenever necessary, the snRNAs were further purified by electro- phoresis through a 5% gel lacking urea. The urea gradient gel, in conjunction with a similar gel poured with a horizontal acrylamide gradient, indicated that this would optimize snRNA separation. Elec- trophoresis conditions were identical with those for the 10% acryl- amide gels containing 7 M urea.

Autoradiography and Elution of RNA-After preparative gel electrophoresis, the RNA bands of interest were located by autora- diography using Kodak XR film. The bands were cut out with a flamed disposable scalpel and placed in silanized glass scintillation vials; they were sometimes crushed by expulsion through a 5 ml syringe at this point. The radioactivity in each band was then meas- ured by Cerenkov radiation of 32P. The RNA was eluted from the gel by shaking vigorously at 37°C in 5 ml of 0.5 M potassium acetate, pH 7.5, treated with 0.1% diethylpyrocarbonate. The RNA was then precipitated with 2.5 volumes of 100% ethanol using 20 to 100 pg of yeast RNA as carrier. Typically, 80 to 90% yields were obtained using this procedure.

Fingerprint Analysis of RNA-Dictyostelium snRNAs (5,000 to 10,000 cpm) were digested to completion with 5 to 10 pl of T1 RNase at 2,500 units/ml in 10 mM Tris-HC1 (pH 7.5). The resulting oligo- nucleotides were fractionated by electrophoresis on Cellogel strips a t pH 3.5 followed by homochromatography on polyethyleneimine cel- lulose as described by Squires et al. (24). A C15 homomix was used for the fingerprints shown here.

Thin Layer Chromatography for Base Composition a n d Modified Base Analysis-RNAs labeled uniformly in viuo with "'PO, were analyzed for their base composition and modified nucleotide content by the method of Silberklang et al. (27). Briefly, this involves complete digestion of the RNA (5,000 to 10,000 cpm) with nuclease PI (5 pl of P1 at 1 mg/ml in 0.05 M ammonium acetate, pH 5.2, for 1 h a t 37°C) followed by two-dimensional chromatography on cellulose thin layer plates. The first dimension solvent is isobutyric acid/concentrated NH,OH/H,O (66/1/33, v/v/v); the second dimension is run in 0.1 M sodium phosphate (pH 6.8)/ ammonium sulfate/l-propanol (100/60/ 2, v/w/v). Plates were dried overnight between running of the first and second dimensions. When necessary, radioactive spots were iden- tified by their location relative to unlabeled standards. PI digestion of yeast carrier RNA produced PA, pG, PC and pU; pm'G and PI) were provided by Sanford Silverman of Dieter Soli's laboratory, Yale University. The cold marker nucleotides were visualized by ultraviolet light.

Thin Layer Chromatography for Analysis of 5'-End Structures- Cap structures were analyzed as follows. Labeled RNAs (at least 10,OOO cpm) were digested to completion with 5 to 10 pl of a mixture of RNases A (100 pg/ml), T1 (500 units/ml), and T2 (25 units/ml) in 0.05 M ammonium acetate (pH 5.2) a t 37°C for 3 h. The digestion products were then spotted on polyethyleneimine cellulose plates which had been chromatographed once in distilled water and then air dried. The chromatograms were developed for 16 h in 2 M pyridinium formate, pH 3.4 (28). Cap spots were located by autoradiography and eluted with 30% (v/v) triethyl ammonium carbonate. After removal of the triethyl ammonium carbonate by repeated lyophilization from distilled water, the eluted oligonucleotides were redigested with either P1 nuclease or a combination of P1 nuclease and nucleotide pyro- phosphatase (5 pl of 2.5 units/ml in 0.02 M Tris-HCI, pH 7.5, 0.02 M magnesium chloride for 1 h at 37OC) (29). The digestion products were then chromatographed on cellulose thin layer plates as described above.

3"End Labeling and RNA Sequencing-Low specific activity D2 RNA (1000 cpm/pg) was purified by the procedures described above using 10-fold more cells and 50-fold less "'PO,. The 3'-end of this RNA was labeled at high specific activity (greater than 5 X 10" cpm/ pg) with [5'-3zP]pCp by bacteriophage T4-encoded RNA ligase (25), and the end-labeled RNA was repurified by electrophoresis through a 5% polyacrylamide gel lacking urea. The RNA was then subjected to the chemical modification and cleavage procedures described by Peattie (17) and the products displayed on a 25% sequencing gel. Partial c o n f i a t i o n of this sequence was obtained by two procedures. First, the 3'-end-labeled RNA was subjected to complete alkaline hydrolysis followed by electrophoresis on Whatman No. 3" paper to identify the first labeled nucleotide (26). Second, a "wandering spot" analysis was performed in which RNA partially cleaved with alkali was subjected to two-dimensional fingerprinting as described above. The spacing of radioactive oligonucleotides on the autoradi- ogram is characteristic of the base removed; thus, the sequence can be read directly from such a fingerprint (27).

958 Small Nuclear RNAs of Dictyostelium

"5.8s 1 1 1 5.6s

e

D w 5 S

a ~ c o e r

a b

FIG. 1. Polyacrylamide gel electrophoresis patterns of Dic- tyostelium RNA labeled in uiuo with [3'P]phosphate. Cytoplas- mic (Lane a) and nuclear RNA (Lane b) isolated from cells lysed in HMK buffer was electrophoresed on a 10% polyacrylamide gel con- taining 7 M urea.

RESULTS

Isolation and Initial Characterization of snRNAs-Gel electrophoretic analysis of RNA from cells separated into nuclear and cytoplasmic fractions (Fig. 1) reveals that there are a number of bands in the nuclear lane which are not present in cytoplasmic RNA. The three bands above 5.8 S RNA labeled Dl, D2, and D3 seem to be almost exclusively nuclear, and even highly overloaded gels do not show cyto- plasmic bands at these positions (data not shown). In addition, a series of closely spaced bands can be seen immediately below 5.8 S RNA. These are referred to collectively as 5.6 S RNA and seem to be more abundant in the nucleus than in the cytoplasm; however, overloaded gels show that in contrast to Dl, D2, and D3, the 5.6 S RNAs appear in cytoplasmic fractions as well as in the nucleus.

Fig. 2 shows an experiment which confirms that the pattern of Dictyostelium snRNAs does not depend on the method of cell lysis. Lysis of cells into the nondenaturing HMK buffer normally used for cell fractionation (Lane a ) does produce RNA bands which are not present in RNA from whole cells lysed directly into sodium dodecyl sulfate/phenol either a t low (Lane c) or high (Lane d ) temperature. The increased intensity of these bands upon incubation of the HMK lysate before phenol extraction (Lane b ) indicates that they are discrete break-down products of higher molecular weight RNAs resulting from the action of endogenous RNases. RNA

- D3

" 0 2 'Dl "5 .85

5.6s

-5s

tRNA

" D 3

u2- . " D 2

-Dl

u4-

5s- /-5s u 5- U 6-

a b

FIG. 2 (left). Polyacrylamide gel patterns of RNA isolated from cells lysed by different procedures. A 12% gel containing 7 M urea was employed. Lane a shows the pattern of total RNA derived from cells lysed in HMK buffer a t 0°C followed immediately by phenol extraction. Lane b is identical with Lane a except that the lysate was held on ice for 10 min before deproteinization of the RNA. Bands which increase in intensity are marked autolysis products. Lane c displays total RNA isolated by lysing cells directly into phenol/BC at 37°C. Lane d is identical with Lane c except that phenol extraction was performed at 60OC. Lanes e and f show cyto- plasmic and nuclear RNA patterns, respectively, isolated using HMK buffer for cell lysis. For a description of buffers and RNA isolation procedures, as well as electrophoresis conditions, see "Methods."

FIG. 3 (right). Polyacrylamide gel comparing electrophoretic mobilities of Dictyostelium and Ehrlich ascites snRNAs. Lane a shows the pattern of nuclear RNA isolated from mouse Ehrlich ascites cells; Lane b displays Dictyostelium nuclear RNA. Methods for preparation and identification of mouse snRNAs have been de- scribed (4). conditions for gel electrophoresis were identical with those used in Fig. 1.

extracted from nuclei isolated using HMK buffer (Lane f , contains insignificant quantities of these autolysis products compared to cytoplasm (Lane e), and none of them co-migrate with Dictyostetium snRNAs.

The sue of each Dictyostelium snRNA was estimated by comparing its electrophoretic mobility under partially dena- turing conditions with that of mammalian snRNAs whose lengths are known precisely from complete primary sequence analysis. Since fingerprint analysis suggested that Ehrlich ascites snRNAs are identical with the sequenced Novikoff hepatoma snRNAs (4, 5), labeled nuclear RNA from Dictyo- stelium and Ehrlich ascites cells was applied to adjacent lanes of a 10% polyacrylamide gel run in 7 M urea (Fig. 3). U1 RNA is 171 nucleotides long (6); U2 is 196 nucleotides in length (7); and U3B RNA is 216 nucleotides long (8). Dl runs between U1 and U2 and appears to be -185 nucleotides long; D2 is slightly larger than U2, or about 210 nucleotides in length; and D3, which is somewhat larger than the others, appears to be 250 nucleotides long. Sue estimates based on electropho- retic mobility under partially denaturing conditions can be misleading, however, since adenovirus VA RNA is only 156

Small Nuclear RNAs of Dictyostelium 959

TABLE I Estimated numbers of molecules/cell of selected RNAs from

Dictyostelium and HeLa cells Data for HeLa cell RNAs reproduced from Ref. 2. RNA species Source No. of rnolecules/cell

Dl Dictyostelium 3 x 10' D2 Dictyostelium 2 X lo4 D3 Dictyostelium 5 x 10' 5s Dictyostelium 3 x 10" u1 HeLa cells 1 x 10" u2 HeLa cells 5 x 10" u 3 HeLa cells 2 x lo5 5s HeLa cells 5 x 10"

residues long but moves more slowly in this gel system than

In order to determine the cellular abundance of the Dicty- ostelium snRNAs, ""P-labeled snRNA bands were excised from the gel shown in Fig. 1 and counted by Cerenkov radia- tion. Since the cells were labeled for 2 days (4 to 5 generations), all the RNA should be at the same maximal specific activity. Thus, by knowing the amount of radioactivity above back- ground in a given gel band, the number of cell equivalents of RNA applied to the lane, and the specific activity of the RNA, the number of molecules of each snRNA species/cell can be calculated. Quantitation of 5 S ribosomal RNA was used as a control in this procedure since the cellular abundance of this species can be independently determined by measuring the optical density of unlabeled ribosomal RNAs resolved by sucrose gradient sedimentation (30). The results of such cal- culations are shown in Table I. Identical numbers were ob- tained whether RNA was extracted from nuclei or whole cells. For comparison, values are also given for the abundance of HeLa cell snRNAs and 5 S RNA as determined by Weinberg and Penman (2). Dictyostelium snRNAs are on the average only 1 to 2% as abundant/cell as the HeLa snRNAs, although both cells contain comparable numbers of ribosomes.

Optimizing Gel Purification of snRNAs-Urea concentra- tion has been known for almost a decade to exert dramatic effects on the relative electrophoretic mobilities of RNA spe- cies in polyacrylamide gels. Presumably, urea acts as a weak denaturant, and each RNA responds uniquely to partial de- naturation depending on the stability of its secondary (and possibly tertiary) structure. In the course of studies on sea urchin histone mRNAs, Gross et al. (23) realized that a vertical polyacrylamide gel containing a horizontal gradient of urea concentration could be used to distinguish between conformers of a single RNA species and co-migration of dis- tinct RNA species. Fig. 4 shows the pattern obtained when labeled Dictyostelium nuclear RNA is resolved by electropho- resis through a 10% polyacrylamide gel containing a horizontal gradient of urea from 0 to 7 M. Bands which consist of several co-migrating RNA species should split as the urea concentra- tion increases, while conformers of a single RNA species would be expected to coalesce. The results clearly support the notion that D2 and D3 are single RNA species since they migrate as single bands at all urea concentrations. D l RNA also produces a single band at most urea concentrations, but splits into two bands a t -2.5 M urea. This suggests the existence of two equally stable conformers at intermediate concentrations of urea.

Fingerprint Analysis of snRNAs-Ribonuclease T1 finger- prints of Dl, D2, D3, and the 5.6 S RNAs are shown in Fig. 5. Dl, D2, and D3 appear to be single species of RNA since the number of distinct oligonucleotides in each fingerprint is consistent with the snRNA's size and base composition. This

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J. Boyle, personal communication.

r -D3 ,D2 -Dl -5.8s 5.6s

-5s

tRNA

7M - FIG. 4. Horizontal urea gradient gel pattern of total nuclear

RNA from Dictyostelium Urea concentration increases from Left to right as indicated. For a description of the pouring and running of the gel see "Methods."

does not rule out the possibility of microheterogeneity within the sequence of any particular snRNA, and we present below evidence for minor heterogeneity in the D2 RNA sequence (see below, Fig. 9). The 5.6 S RNA species were fingerprinted as a group for two reasons. First, the bands were so closely spaced that it was impossible to dissect out a single species. Second, we initially thought that they might be conforma- tional isomers of a single RNA, analogous to those described for bacterial 5 S RNA (31, 32). However, the complexity of the fingerprint shown in Fig. 5 demonstrates that 5.6 S RNA consists of many species, and the electrophoretic behavior of 5.6 S RNA on the urea gradient gel (Fig. 4) supports this contention: the ladder of 5.6 S RNAs shows no tendency to coalesce at high urea concentrations, and several of the bands actually intersect as though distinct RNA species were re- sponding very differently to partial denaturation.

Comparison of the Dl , D2, and D3 fingerprints with those of Dictyostelium 5 S and 5.8 S ribosomal RNA demonstrates that the large (and hence, characteristic) oligonucleotides of these molecules are absent from the snRNA fingerprints (data not shown). Since abundant ribosomal RNAs are the major potential contaminant in an snRNA preparation, the absence

960 -

Small Nuclear RNAs of Dictyostelium

FIG. 5. Fingerprint analyses of uniformly 32P-labeled Dictyostelium snRNAs. RNA samples were digested to completion with RNase T1 and the resulting oligonucleotides fractionated by electrophoresis on Cellogel strips followed by homochromatography on polyethyleneimine plates.

FIG. 6. Modified base analysis of D3 RNA by two-dimen- sional chromatography. a, products of complete digestion with nuclease PI; b. 5-fold longer exposure of a after cutting out the major spots. Chromatography solvents and other details can be found under “Methods.”

of 5 S and 5.8 S RNA implies that the snRNAs are pure. Further examination of the Dl , D2, and D3 fingerprints indicates that they share no characteristic oligonucleotides. Thus, the Dictyostelium snRNAs are unrelated as judged by fingerprint analysis, and are also not cross-contaminated.

Base Composition a n d Modified Nucleotide Content-The snRNAs used in these experiments were purified in two di- mensions as described under “Methods” since we wanted to be certain that the results could not be attributed to contam- inating species of RNA. D3 snRNA, uniformly labeled with

TABLE I1 Base compositions of Dictyostelium small nuclear RNAs

RNA species Nucleotide

PA PC PU PC PO c.

Dl 31 21 30 18 D2 30 18 35 17 e1 D3 26 19 30 23 =2 5 s 27 23 24 26

”P in vivo, was digested with P1 nuclease and the products were separated by chromatography in the two-dimensional system introduced by Nishimura (33) and modified by Sil- berklang et al. (27). As shown in Fig. 6, D3 is quite highly modified. In addition to the mononucleotides PA, pG, PC, and pU, it contains p$ (identified by co-migration with an unla- beled marker), pCm, and pm”A (identified by their positions relative to PC and PA). D3 also contains another modified base, marked pX, whose position does not coincide with any of the nucleotides documented by Silberklang et al. (27). The spot marked bridge is due to a P1-resistant 5’-end structure which will be described below. Similar analysis of the other Dictyostelium snRNAs indicates that D2 also contains pseu- douridine, and D l contains no internal modifications (data not shown).

The base composition of Dl , D2, and D3 was determined by quantitation of the excised radioactive spots using Ceren-

Small Nuclear RNAs of Dictyostelium 961

kov radiation. Table I1 shows the results obtained for all three snRNAs and for 5 S RNA. The modified bases in D3 other than pseudouridine were present at less than 176, and, thus, have not been included in the table.

Analysis of 5'-End Structures-Dictyostelium snRNAs la- beled uniformly in vivo were digested to completion with T2 RNase, producing 3'-mononucleotides and T2-resistant struc- tures which could be separated by polyethyleneimine thin layer chromatography in the system of Silberklang et al. (27) as shown in Fig. 7. Uniformly labeled Dictyostelium poly(A)- containing mRNA, and Ehrlich ascites U1 snRNA were di- gested with T2 RNase in parallel to produce known cap structures as markers. Since T2 RNase cannot break pyro- phosphate bonds or phosphodiester bonds bearing a 2'-0- ribose methylation (21), the T2-resistant structures derived from the slime mold snRNAs are most likely to be oligonucle- otides with internal ribose methylations, phosphorylated 5'- terminal nucleotides, or caps. The T2-resistant structures from all three snRNAs were eluted from the thin layer plate and found to be partially resistant to both bacterial alkaline phosphatase (which removes all external phosphates) and P1 nuclease (which digests RNA to 5'-mononucleotides regard- less of 2"O-ribose methylations and also possesses a 3"phos- phatase activity (34)); this suggests that the TZresistant snRNA structures contain the internal pyrophosphate linkage characteristic of caps (data not shown). The reason for the low yield of the D3 5'-end structure in this experiment is

unclear; in other preparations, the yield was similar to that found for D l and D2 snRNA. The D3 RNA used in this experiment may have been contaminated with ribosomal RNA breakdown products since it was purified from whole cell RNA rather than from isolated nuclei.

The putative cap oligonucleotide from D2 RNA was shown to be a bona fide cap by redigestion in separate experiments with either nuclease P1 or nucleotide pyrophosphatase. The T2-resistant oligonucleotide derived from D2 RNA was redi- gested fvst with P1, and the products chromatographed in the same two-dimensional thin layer system previously used to identify modified nucleotides (Fig. 8). The T2-resistant oligo- nucleotides of D l and D3 gave apparently identical P1 rediges- tion patterns (data not shown). The positions of unlabeled markers are indicated, as well as the approximate location of the P1 bridge structures I (m7GpppAm), I1 (m'GpppA), and I11 (m7GpppG) produced by P1 nuclease digestion of the T2- resistant cap structures I (m'GpppAmpAp), I1 (m'GpppAp), and IV (m'GpppGp) of Dictyostelium mRNA as determined by Dottin et al. (21). (Cap structures have been renamed to correspond to their mobility in our chromatographic system.) The digestion pattern shown in Fig. 8 indicates that D2 snRNA bears a type 0 cap without 2'-O-ribose methylation adjacent to the bridge (21) since no products other than the bridge and phosphate can be detected despite overexposure of the autoradiogram.

When the P1-resistant bridge derived from D2 RNA was further digested with venom nucleotide pyrophosphatase (29) and chromatographed in the same two-dimensional system, three products were observed: pZ, PA, and phosphate. The nucleotide designated pZ migrates in a position similar to labeled pm2* 2* 7G obtained by digestion of the T2-resistant cap of mouse Ehrlich ascites U1 snRNA (presumably identical with rat Novikoff hepatoma m'. '.'GpppAmpUmpAp (6)), al- though we were unable to obtain sufficient unlabeled pm"2. 'G to prove this rigorously.

In order to determine the RNA sequence immediately ad- jacent to the type 0 cap, an RNase T1 digest of uniformly labeled D2 RNA was chromatographed on a column of dihy- droxyborylaminoethylcellulose, which retains oligonucleo- tides bearing a cis-2',3'-diol by formation of a cyclic boryl ester (35,36). The cap oligonucleotide binds to dihydroxyborylam- inoethylcellulose through the cis-diol of the inverted nucleo- tide pZ. Internal T 1 oligonucleotides, bearing a 3'-phosphate, flow through the column, while the T1 oligonucleotide derived from the 3'-end of D2 has a free cis-diol and is also retained. The 5"and 3'-ends of D2 were eluted from the column with sorbitol and separated by electrophoresis on Cellogel at. pH

a b

FIG. 7. Detection of T2-resistant 5'-end structures py thin layer chromatography on polyethyleneimine cellulose. The large spots near the top of the autoradiograph represent mononucleo- tides. Arrows mark the position of 5'-end structures. The known FIG. 8. Analysis of 5'-end structures of D2 snRNA by two- structure of the Ehrlich ascites U 1 RNA cap is m'. '. 'Gppp- dimensional chromatography. a, digestion of the T2-resistant cap AmpUmpAp (6). Dictyostelium- mRNA caps have the following se- structure with nuclease PI. Position of nonradioactive marker nucleo- quence? m'GpppAmpAp ( I ) ; m'GpppAp ( I I ) ; m'GpppAmpUp ( I I I ) ; tides, as well as PI bridges from Dictyosteliurn mRNA (determined and m'GpppCp ( I V ) (structures as determined in Ref. 21. but re- in separate experiments), are indicated. I , m'GpppAm; II , m'CpppA; named here according to mobility in thin layer chromatography III , m'GpppG; b, digestion of the T2-resistant oligonucleotide of D2 rather than electrophoresis on DEAE-cellulose paper at pH 3.5). RNA with P1 nuclease and nucleotide pyrophosphatase.

962

G A>G C>U U

Small Nuclear RNAs of Dictyostelium

- G -U

A - -L

-U

-G

-G

- C

-u

FIG. 9. Sequencing gel of 3“end-labeled D2 RNA. Products are displayed on a 25% polyacrylamide gel run in 7 M urea after base- specific chemical modification and cleavage (17). RNA preparation and labeling are described under “Methods.”

3.5 followed by homochromatography on polyethyleneimine thin layers. When the purified 5’-end T1 oligonucleotide was redigested with nuclease P1, and the products separated by two-dimensional thin layer chromatography, the bridge struc- ture ZpppA, together with pU and pG, were obtained (data not shown). Thus, the 5’-end sequence of D2 RNA is Zppp-

Partial Sequence of 0 2 RNA-The free 3’-hydroxyl group of D2 RNA was labeled enzymatically with [5’-:”P]pCp by bacteriophage T4 RNA ligase (25) and the end-labeled RNA was then subjected to rapid polyacrylamide gel sequence analysis after base-specific chemical cleavage (17). Fig. 9 shows a 25% polyacrylamide sequencing gel from which 27 nucleotides a t the 3’-end of D2 RNA can be read easily. The RNA appears to be homogeneous at the 3’ terminus and gives a unique sequence up to position 21, where both 4 and G are present. G appears to predominate over A by a factor of 2 to 3, based on a comparison of the intensities of these bands with others in their respective lanes.

APUPGP.

DISCUSSION

We have isolated and characterized several species of small nuclear RNA from the cellular slime mold D. discoideum. Three lines of evidence indicate that these RNA molecules

represent authentic cellular constituents rather than break- down products of larger RNA species such as ribosomal RNA: (a) the snRNAs can be isolated in identical yield from nuclei prepared using several different detergents and buffers (data not shown); (6) the same yield of snRNA/nucleus can be obtained by lysing whole cells directly into a mixture of sodium dodecyl sulfate and phenol (Fig. 2, Lanes c and d ) ; and ( c ) incubation of nuclei for up to 10 min before addition of sodium dodecyl sulfate and phenol does not increase the intensity of the snRNA bands, although several autolysis products of ribosomal RNA do become more prominent (Lanes Q and 6) . The existence of specialized 5’-terminal cap structures on the snRNAs also argues that Dl, D2, and D3 are mature cellular RNA species and not excised portions of the 35 S ribosomal RNA precursor (18) or the precursors of other cellular RNA species such as tRNA.

The three most abundant slime mold snRNAs appear sim- ilar in size to the three most abundant snRNAs of mammalian cells, although none of the Dictyostelium species actually co- migrate with Ehrlich ascites cell snRNAs. The electrophoretic pattern of Dictyostelium snRNAs also correlates well with that reported for two other lower eucaryotes: both A. proteus (13) and Tetrahymena pyriformis (12) posses three species of snRNA which appear larger than 5.8 S ribosomal RNA and resemble slime mold snRNAs in their abundance relative to each other as well. Dictyostelium does not appear to contain any abundant low molecular weight nuclear RNAs corre- sponding to 4.5 SI, 4.5 SI,, or 4.5 SIII studied by Ro-Choi and Busch (l), or to the somewhat smaller 4.5 S RNA recently characterized by Jelinek and Leinwand (37) and sequenced by Harada and Kat0 (9). The slime mold nucleus does contain a conspicuous ladder of distinct RNA species centered around 5.6 S which may correspond to the much fainter array of 5 S species recently identified in mammalian cells by antibody precipitation using autoimmune serum from patients with systemic lupus erythematosus:’

The abundance of the snRNAs represents another major difference between Dictyostelium and higher cells. The most abundant of the snRNAs in higher eucaryotes, termed U1 (1) or species D (2), is present in 20% as many copies/cell as the ribosomal RNAs (2), while the most abundant snRNA in Dictyostelium is less than 1% as abundant as the rRNAs, and the other slime mold snRNAs are proportionally diminished. Thus, quantitation immediately implies that the snRNAs do not function in any capacity which is directly related to cell size or generation time such as cellular architecture or protein biosynthesis. This conclusion is reinforced by knowing that cultured mammalian cells and vegetative amoebae grown axenically in shaker culture have comparable numbers of ribosomes/cell (2, 30) and equivalent generation times (- 12 h). Since the genome of Dictyostelium (30) is 50-fold smaller than that of mammalian cells (38), one possible explanation for the relative scarcity of snRNAs in lower eucaryotes is that snRNAs play a role as either primers of DNA replication (for a review, see Ref. 39) or in stabilizing the tertiary structure of chromatin (40, 41). Quantitation of the three major snRNAs in Tetrahymena (12), whose genome is much closer in size to that of Dictyostelium than to that of mammalian cells (42), supports this notion. The cellular abundance of certain sn- RNAs might also correlate with the extent of nuclear RNA processing rather than directly with genomic size since the immediate precursor of cytoplasmic messenger RNA, termed heterogeneous nuclear RNA, is 5- to 10-fold larger than mRNA in mammalian cells (431, but no more than 20% larger than mRNA in Dictyostelium (44). In mammalian cells, both

Lerner, M., Boyle, J. A., Harding, J., and Steitz, J. (1980) Science, in press.

Small Nuclear RNAs of Dictyostelium 963

U1 (5) and 4.5 S snRNA (9) have the potential to base pair with heterogeneous nuclear RNA splice junction sequences.

Although the presence of modified bases in snRNAs cannot yet be interpreted in functional terms, it is interesting to note that the three abundant snRNAs found in slime mold and rat Novikoff hepatoma cells exhibit corresponding patterns of base modification. One of the snRNAs, D3 in Dictyostelium and U2 in the rat (7), contains many residues of pseudouridine, as well as extensive 2'-O-ribose methylations; another snRNA, D2 from the slime mold and U3 from hepatoma (8), contains only a few residues of pseudouridine; and the third snRNA, Dl from Dictyostelium and U1 in the rat (6) has no internal modifications with the exception of a single 2'-0-methylation in U1.

The functional significance of the 5'-terminal snRNA cap structure is no less mysterious than that of the modified bases. Nearly aU eucaryotic mRNAs possess caps which protect the RNA from attack by phosphatases and 5'-exonucleases (45), and interact specifically with a protein initiation factor which selectively stimulates translation of capped relative to un- capped mRNA (46). By analogy, cap structures may increase the metabolic stability of the snRNAs or be required for recognition of snRNAs by various cellular proteins.

We have shown here that the amoeba1 snRNAs are similar in both size, cap structure, and modified base content to the snRNAs of higher eukaryotes; moreover, by DNA sequence analysis of a genomic clone, we have recently found that the primary sequence of the most abundant Dictyostelium snRNA, D2, displays extensive homology with the rat nucleo- lar snRNA U3 (47). We believe these structural similarities imply that the snRNAs of lower and higher eukaryotes are functionally analogous as well. In the future we plan to deter- mine whether the synthesis and modification of snRNAs is developmentally regulated in Dictyostelium, as may be the case in other organisms (481, and whether the slime mold snRNAs are found in discrete small ribonucleoprotein particles comparable to those characterized in mammalian cells (4, 5).

REFERENCES 1. Ro-Choi, T. S., and Busch, H. (1974) in The Cell Nucleus (Busch,

2. Weinberg, R. A,, and Penman, S. (1968) J. Mol. Biol. 38,289-304 3. Zieve, G., and Penman, S. (1976) Cell 8, 19-31 4. Lerner, M. R., and Steitz, J . A. (1979) Proc. Natl. Acad. Sei. U.

5. Lerner, M. R., Boyle, J . A., Mount, S. M., Wolin, S. J., and Steitz,

6. Reddy, R., Ro-Choi, T. S., Henning, D., and Busch, H. (1974) J.

7. Shibata, H., Ro-Choi, T. S., Reddy, R., Choi, Y. C., Henning, D.,

8. Reddy, R., Henning, D., and Busch, H. (1979) J. Biol. Chem. 254,

9. Harada, F., and Kato, N. (1980) Nucleic Acids Res. 8, 1273-1285 10. Ro-Choi, T. S., Reddy, R., Henning, D., Takano, T., Taylor, C.

W., and Busch, H. (1972) J. Biol. Chem. 247,3205-3222 11. Jelinek, W. R., Toomey, T. P., Leinwand, L., Duncan, C. H., Biro,

P. A., Choudary, P. B., Weissman, S. M., Rubin, C. M., Houck, C. M., Deininger, P. L., and Schmid, C. W. (1980) Proc. Natl. Acad. Sei. U. 5'. A. 77, 1398-1402

H., ed) pp. 151-208, Academic Press, New York

S. A. 76,5495-5499

J. A. (1980) Nature 283, 220-224

Biol. Chem. 249, 6486-6494

and Busch, H. (1975) J. Biol. Chem. 250,3909-3920

11097-11105

12.

13.

14. 15.

16. 17.

18.

19.

20. 21.

22.

23.

24.

25. 26.

27.

28.

29. 30.

31.

32.

33 34.

35.

36. 37. 38.

39.

40.

41. 42.

43.

4 4 . 45.

Hellung-Larsen, P., and Frederiksen, S. (1977) Comp. Biochem.

Goldstein. L., Wise. G . E., and Beeson, M. (1973) Exp. Cell Res. Physiol. B Comp. Biochem. 58,273-281

76,2811288 Goldstein. L.. and KO. C. (1974) Cell 2. 259-269 Firtel. R. A.. Timm. R.. Kimmel. A. R.: and McKeown, M. (1979)

~ .~~~~ ~~~~

, .

46. Sonenberg, L., Rupperchi, K. M., Heicht, S. M., and Shatkin, A.

47. Wise, J. A,, and Weiner, A. M. (1980) CeZZ22, 109-118 48. Frederiksen, S., and Hellung-Larsen, P. (1974) in Biochemistry of

the Cell Nucleus: Mechanism and Regulation of Gene Expres- sion (Hidvegi, E. J., Siimegi, J., and Venetianer, P., eds) pp. 175-180, North-Holland/American Elsevier, New York

J . (1979) Proc. Natl. Acad. Sei. U. S. A. 76,4345-4349

~ ~~~

Proc. Natl. Acad. Sci. U. S. A . 76,6206-6210 Alton, T. H., and Lodish, H. F. (1977) Deu. Biol. 60, 180-206 Peattie, D. A. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 1760-

Batts-Young, B., Maizels, N., and Lodish, H. F. (1977) J. Biol.

Bakke, A. C., Wu, J.-R., and Bonner, J. (1978) Proc. Natl. Acad.

Pederson, T. (1977) Biochemistry 16,2771-2777 Dottin, R. P., Weiner, A. M., and Lodish, H. F. (1976) Cell 8,233-

Peacock, A. C., and Dingman, C. W. (1967) Biochemistry 6,1818-

Gross, K., Probst, E., Schaffner, W., and Birnstiel, M. (1976) Cell

Squires, C., Lee, F., Bertrand, K., Squires, C. L., Bronson, M. J.,

England, T. E., and Uhlenbeck, 0. C. (1978) Nature 275,560-561 Barrell, B. G. (1971) in Procedures in Nucleic Acids Research

(Cantoni, G. L., and Davies, D. R., eds) Vol. 2, pp. 751-759, Harper Row, New York

Silberklang, M., Gillum, A. M., and RajBhandary, U. L. (1979) Methods Enzymol. 59, 58-109

Southern, E. M., and Mitchell, A. R. (1971) Biochern. J. 123,613- 617

Cory, S., and Adams, J . M. (1975) J. Mol. Biol. 99, 519-547 Loomis, W. F. (1975) Dictyostelium discoideum: A Developmental

Aubert, M., Scott, J. F., Reynier, M., and Monier, R. (1968) Proc.

Forget, B. G., and Weissman, S. M. (1967) Nature (Lond.) 213,

Nishimura, S. (1972) Prog. Nucleic Acid Res. Mol. Biol. 12, 49 Fugimoto, M., Kuminaka, A,, and Yoshino, H. (1974) Agric. Biol.

Weith, H. L., Wiebers, J . L., and Gilham, P. T. (1970) Biochem-

Rosenberg, M. (1974) Nucleic Acids Res. 1, 653-670 Jelinek, W., and Leinwand, L. (1978) Cell 15, 205-214 Hood, L. E., Wilson, J. H., and Wood, W. B. (1975) Molecular

Biology of Eukaryotic Cells, p. 37, W. A. Benjamin, Inc., Menlo Park, California

Kornberg, A. (1980) DNA Replication, W. H. Freeman and Co., San Francisco

Stontington, 0. G., and Pettijohn, D. E. (1971) Proc. Natl. Acad. Sci. U. S. A. 68.6-9

Benyajati, C. , and Worcel, A. (1976) Cell 9, 393-407 Woodard, J., Kaneshiro, E., and Gorovsky, M. (1972) Genetics 70,

251-260 Jelinek, W., Adesnik, M., Salditt, M., Sheiness, D., Wall, R.,

Molloy, G., Phillipson, L., and Darnell, J. R. (1973) J . Mol. Biol. 75, 515-532

1764

Chem. 252,3952-3960

Sei. U. S. A. 75, 705-709

244

1827

8,455-469

and Yanofsky, C. (1976) J. Mol. Biol. 103,351-381

System, Academic Press, New York

Natl. Acad. Sci. U. S. A. 61, 292-299

878-882

Chem. 38, 1555-1561

istry 9,4396-4401

Firtel, R. A., and Lodish, H. F. (1973) J. Mol. Biol. 79, 295-314 Shatkin, A. J. (1976) Cell 9. 645-653