The Small Nuclear RNAs of the Cellular Slime Mold Dictyostelium

7/30/2019 The Small Nuclear RNAs of the Cellular Slime Mold Dictyostelium

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THE OURNAL F BIOLOGICALH EMISTRYPrinted m U . S . A .Vol. 256, No. 2 , Issue of January 25, pp. 956-963, 981

The Small Nuclear RNAs f the Cellular SlimeMold DictyosteliumdiscoideumISOLATION AND CHARACTERIZATION*

(Received for publication, May 16, 1980)

Jo Ann Wise and AlanM. WeinerFrom the Department ofMolecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06510

Three species of small nuclear RNA from the lowereucaryote Dictyostelium discoideum have been isolatedand characterized with regard to size, cellular abun-dance, modified nucleotide content, and 5-end struc-tures.Previous studies had shown that the nucleif mam-malian cells contain a numberf discrete low molecularweight,nonribosomal,nontransfer RNA moleculesknown as small nuclear RNAs. The mammalian smallnuclear RNAs range in size from approximately100 to250 nucleotides and are quite abundant, in some asesapproaching ribosomalRNA in number of copies/cell.Some of hese molecules have an unusual cap structureattheir5-endssimilartothatfoundoneucaryoticmessenger RNAs, and a number contain a characteris-tic set of internal modifications s well.Our results indicate that the small nuclear RNAs ofDictyostelium resemble heircounterparts nhighereucaryotic cells structurally, but are present in signif-icantly ewer copies/cell. The mplications of thesefindings for small nuclear NA function are discussed.

In recent years, a substantial body of literature has begunto accumulate on a comparatively neglected class of RNAmolecules found in all eucaryotic cells, the small nuclearRNAs. In mammalian cells, a t least eight species of snRNA,ranging n size from approximately 100 to 250 nucleotides,have been extensively characterized (fora review, see Ref. 1).U1, U2, and U3 snRNA appear toe metabolically stable, andU1 is nearly as abundantn th e nuclei of mammalian cells asthe ribosomal RNAs are in the cytopl asm2 ) . U3 snRNA hasbeen localized primarily within the nucleolus (3), while U1and U2 (as well as several other snRNAs) areound in distinctsmall ribonucleoprotein particles (4, 5). Five snRNAs fromrodent cells (4.5 S , 4.5 Sr,U1, U2, and U3) have been com-pletely sequenced.U1, U2, and U3 are uridine-rich and possess5-cap struc tures resembling those found on messenger RNA,but more highly methylated (6-8); U2 contains substantialamounts of pseudouridine, as well as both 2O-ribose andbase methylations (7 ) ; 4.5 S and 4.5 SI have 5-triphosphatesand are unmodified. Both U1 and 4.5 S snRNA have beenpostulated to play a role in processing heterogeneous nuclear

* These studieswere supported by Grants PCM76-81524 and PCM78-21799 awarded by the Nationa l Science Foundation and by Gran tGM 26312 awarded by the National Institutes f Health. The costs fpublication of this articl e were defrayed in par t by the payment ofpage charges. This article must therefore be hereby markedaduer-tisement in accordance with 18U.S.C. Section 1734 solely to indi catethis fact. The abbreviations used are: snRNA, smallnuclear RNA; Mes, 2-

(N-morpho1ino)ethanesulfonic cid.

RNA because they can form base-paired regionswith thesequences flanking splice unctions in various nuclear precur-sors of messenger RNA (5,9) . Rodent.5 S RNA also displaysremarkable homology to a sequence found at the origin ofreplication in three different papovaviruses, simian virus 40,polyoma, and BK, suggesting that it may play an additionalrole in the initiationof cellular DNA replication (11).Th e existence of snRNAs in lower eucaryotes has beeninferred from thegel electrophoresis patterns of nuclear RNAfrom a number of organisms (reviewed in Ref. 12), but hasbeen most dramatically demonstrated by the elegant nucleartransp lantat ion studie s f Goldstein and his ollaborators withAmoeba proteus (13, 14). Nuclei from labeled amoebae weretransplanted to unlabeled amoebae to form binucleate cells:certain snRNA species remained in the original nucleus (non-shuttling RNAs), while others equilibrated between the twonuclei (shuttling RNAs).

Here we report the initial solation and characterizat ion ofsmall nuclear RNAs from thecellular slime mold Dictyoste-lium discoideurn; in a separate publication (47), we describethe genomicorganization, cloning, and sequencing of th egenesencoding one of these nRNA species. The lowereucaryote Dictyosteliumwaschosenbecause its DNAse-quence organization andRNA metabolism have been thesubject of intensive study (15) and because vegetative amoe-bae can be nduced to undergo a synchronous program ofchanging RNAand protein synthesis during the develop-menta l life cycle (16, 31). Thus, modulation of snRNA geneexpression during development might ive some indication ofthe function of this class of molecules, which has remainedlargely in the realm of speculation until ow. Our experimentsshow tha t th e lime mold does indeed possess several speciesof nuclear RNA which are comparable n both size and struc-ture to the snRNAs of mammalian cells. The most strikingdifference between the snRNAs of Dictyostelium and theirmammalian counterpart s 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 timesare similar for cultured mammalian ells and amoebae grownaxenically in shaker culture.

EXPERIMENTAL PROCEDURESMaterials

Trypticase peptone for growthf Dictyostelium was obtained fromBBL; yeast extrac t was from Difco. Carrier-free PO4was purchasedfrom either New England Nuc lear or Amersham/Searle. [5P]pCpwas obtained from New England Nuclear. Sodium dodecyl sulfate,acrylamide, and N,Nmethylenebisacrylamide were purchased fromBio-Rad. Nucleotidepyrophosphatase,diethylpyrocarbonate, ndyeast RNA used as carrier were obtained from Sigma. Th e yeastRNA was phenol-extracted and ethanol-precipitated before use. T1and T 2 RN ases were obtained from Sankyo (Tokyo) through Calbi-

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Smalluclear RNAs of Dictyostelium 957ochem, RNase A was from Worthington, and PI nuclease was fromYamasa Shoyu (Tokyo). Cellogel strips were purchased from Kalex,polyethyleneimine cellulose th in layer p lates from Brinkmann, andplastic-backed cellulose thin layer pla tes were from Eastman. Re-agents for chemical RNA sequencing were obtained from the sourcesreported by Peattie (17).

MethodsCulturing and RadioactiveLabe ling of Cells-D. discoideumstrain AX3 was grown in shaker cultures in Mes-HL5 medium (18)maintained a t room temperatu re (-19C). For labeling with "Po,,cells were diluted to a density of 3 X lO'/ml in Mes-HL5 medium.Streptomyc in sulfate was added o a concen tration of 0.3 mg/mlfollowed by "'PO, at 40 to 80 pCi/ml. Cells were then grown for 2days toa density of 5 X 10"/ml. Using this labeling protocol, 10,000 to40,000 cpm of eac h snRNAspecies could be obtained.Cell Fractionation a nd RNA Preparation-For preparation of

total cellular RN A, the cells (1.4 X lo9)were collected by centrifu-gation and washed thre e times with ice-cold 20 mM potassium phos-pha te (pH 6.5). Th e cell pellet was frozen in dry ice/ethanol for 10min and then dislodged from the bottom of the tube. Tenmillilitersof redistilled phenol and 20 ml of BC buffer (0.1 M Tris base, 0.1%sodium dodecyl sulfate, and5 mM Na? EDTA) at7C were added tothe cells and vortexed vigorously for 1 to 2 min. T he lysis mixturewas then centrifuged at 4,000 X g for 5 min a t 4C followed byremoval of the upper phase to fresh ube. All subsequent operationswere performed on ice. The aq ueous phase was extracted again with10 ml of phenol followed by three successive ex tractions with 30 ml ofchloroform/isoamyl alcohol (98:2). Th e deproteinized RNA was thenprecipitated by addition of 0.1 volume of 4 M sodium acetate and 2.5volumes of 100%ethanol. Th e RNA was recovered by centrifugationat 10,000X g for 20 to 30 min.

For prepara tion of nuclear and cytoplasmic RNA, cells were col-lected, washed, and frozen as described above. Thr ee different bufferswere employed for isolation of nuclei. Th e dislodged cell pellet wastaken up n 10ml of HMK (5% sucrose, 2% NPT-12,40 mM MgC12,20mM KC1, and 50 mM 4-(2-hydroxyethyl)-l-piperazineethanesulfonicacid, adjust ed to pH 7.5 with NH4OH) (18), B (10% sucrose, 0.5%NP-40, 40 mM KCl, 20 mM potassium phosphate, p H 7.5, 0.5 mMmagnesium acetate, and0.1m az EGTA ) (19),or TP (10% ucrose,0.5% Triton X-100,5 mM magnesium ac etate, and50 mM 4-(2-hydrox-yethy1)-I-piperazineethanesulfonic cid, adjusted topH 8.0 withNH,OH) (20) buffer an d gently agitated on a vortex mixer unti l th epellet thawed, and then for 30 s longer. Th e nuclei were recoveredfrom the lysate by centrifugation at 10.000 X g for 5 min a t 4C. Thesupernatant cytoplasmic extract was then transferred to a tube con-tainingphenol/BC and held on ice while the nuclei were washedtwice with 10-ml aliquots of lysis buffer without detergent. Th e finalnuclear pellet was resuspended in 1ml of buffer before deproteiniza-tion. Extraction of nuclear and cytoplasmic RNA was performed inparallel as described above for otal cellular RNA. All buffers used inthe isolation of RNA were treated with 0.1% diethylpyrocarbonateand autoclaved before use.Prepa rat ion of Messenger RNA-Poly(A)-containing RNA fromDictyostelium was prepared essentially as described by Dottin et al.(21). except tha t vegetative, rather than starv edcells were used.

Polyacrylam ide Gel Electrophoresis-Analytical electrophoresiswas performed in polyacrylamide slabs (300 X 175 X 1.5 mm), with aslot width of 18 mm. Prepara tive gels were 3-mm thick with a 120-mm slot. Gels were poured an d run n different dilutions of 10X TBEbuffer (22),which contains 108g of Tris base, 55g of boric acid, and0.93 g of disodium EDTA /liter , giving a final pH of 8.3. Gels werepolymerized a t room tem perat ure in 1X TBE buffer containing 9.67%acrylamide, 0.33%N,N"methylenebisacrylamide (10%gels) or 11.6%acrylamide, 0.4% bis (12% gels), 7M urea, 0.067% amonium persulfate,and 0.023% N,N,N',N"tetramethylethylenediamine. Gels were run a troom tempera ture or at 4C in 1/2 X TB E buffer at 13 V/cm. Beforemin in loading buffe r (7 M urea, 0.1% bromophenol blue, and 0.1%application to the gels, RNA samples were heated a t 60C for 3 to 5xylene cyano1 FF).

Nuclear RNA from Dictyostelium was also electrophoresed in aurea gradient gel as described by Gross et al. (23). A polyacrylamideslab gel was poured with a continuous horizontal gradient of urearunning from 0 to 7 M , and the RNA was applied as a single broadband to the top of the slab. Cross-linker ratio, loading buffer, andrunning buffer were a s described above.

Whenever necessary, the snRNA swere furthe r purified by electro-phoresis through a 5% gel lacking urea. Th e urea gradient gel, inconjunction with a similar gel poured with a horizontal acrylamidegradient, indicated that this would optimize snRNA separation.Elec-trophoresis conditions were identical with those for the 10%acryl-amide gels containing 7 M urea.Autora diography an d Elutio n of RNA-After preparative gelelectrophoresis, the RNA bands of interest were located by autora-diography using Kodak X R film. Th e bands were cut out with aflamed disposable scalpel and placed in silanized glass scintillationvials; they were sometimescrushed by expulsion throu gh a5 mlsyringe at this point. Th e radioactivity in each band was then meas-ured by Cerenkov radiation of 32P. Th e RNA was eluted from th e gelby shaking vigorously at 37C in 5 ml of 0.5 M potassium acetate, pH7.5, treated with 0.1% diethylpyrocarbonate. TheRNA was thenprec ipita ted with 2.5 volumes of 100% ethanol using 20 to 100 pg ofyeast RNAas carrier. Typically,80 to 90 % yields were obtained usingthis procedure.Finge rprin t Analy sis of RNA-Dictyostelium snRNAs (5,000 to10,000 cpm) were digested t o completion with 5 to 10pl of T1 RNaseat 2,500 units/ml in 10 mM Tris-HC1 (pH 7.5). The resulting oligo-nucleotides were fractionated by electrophoresis on Cellogel strips atpH 3.5 followed by homochromatography on polyethyleneimine cel-lulose as described by Squires et al. (24). A C15 homomix was usedfor the fingerprints shown here.Thin Layer Chromatography for Baseomposition a n d ModifiedBa se Analysis-RNAs abeled uniformly in viuo with "'PO, wereanalyzed for their base composition and modified nucleotide contentby the meth od f Silberklang et al. 27). Briefly,his involves completedigestion of th e RNA (5,000 to 10,000 cpm) with nuclease PI (5 pl ofP1 at 1mg/ml in 0.05 M ammonium acetate, pH 5.2, for 1 h at 37C)followed by two-dimensional chromatography on cellulose thin layerplates. Th e first dimension solvent s sobutyric acid/concen tratedNH,OH/H,O (66/1/33, v/v/v ); the second dimension is run in 0.1 Msodium phosphate (pH6.8)/ ammonium sulfate/l-propanol (100/60/2, v/w/v ). Plates were dried overnight between running of the firstand second dimensions. When necessary, radioactivepots were iden-tified by their location relative to unlabeled standards. PI digestionof yeast carr ier RNA produced PA, pG, PC and pU; pm'G and PI)were provided by Sanford Silverman of Diete r Soli's laboratory, YaleUniversity. T he cold marker nucleotides were visualized by ultravioletlight.

Thin Layer Chromatography for Analysisf 5'- End Structures-Cap struct ures were analyzed a s follows. LabeledRNAs (at least10OOO cpm) were digested to completion with 5 to 10pl of a mixtureof RNases A (100 pg/ml), T1 (500 units/ml), and T2 (25 units/ml) in0.05 M ammo nium aceta te (pH 5.2) at 37C for 3 h. The digestionproducts were then spotted on polyethyleneimine cellulose plateswhich had been chromatographed once in distilled water and then airdried. Th e chromatograms were developed for 16 h in 2M pyridiniumformate, pH3.4 (28). Cap spotswere located by autoradiography andeluted with 30% (v/v) triethyl ammonium carbonate. After removalof the trieth yl ammonium carb onate y repeated lyophilization fromdistilled water, he elu ted ligonucleotides were redigested witheitherP1 nuclease or a combination of P1 nuclease and nucleotide pyro-phosphatase (5 pl of 2.5 uni ts/ml in 0.02 M Tris-HCI, pH 7.5, 0.02 Mmagnesiumchloridefor 1 h a t 37OC) (29). Th e digestion prod uctswere then chromatog raphed n cellulose thin layer plates as escribedabove.

3"End Labe ling an d RNA Sequencing-Low specific activity D2RNA (1000 cpm/pg) was purified by th e procedures described aboveusing 10-fold more cells and 50-fold less "'PO,. The 3'-end of thisRNA was labeled a t 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 througha 5% polyacrylamide gel lacking urea . T he RN A was then subjectedto t he chemical modification and cleavage procedures described byPeat tie (17) and the produ cts displayed on a 25% sequencing gel.Partial co nf ia ti on of this sequence wasobta ined by two procedures.First , he 3'-end-labeled RNA was subjected o complete alkalinehydrolysis followed by electrophoresis on Whatman No. 3 paperto identify the first labeled nucleotide (26). Second, a "wanderingspot" analysis was performed in which RNA partially cleaved withalkali was subjec ted to two-dimensional fingerprinting as describedabove. The spacing of radioactive oligonucleotides on the autoradi-ogram is characteristic of the base removed; thus, the sequence canb e read directly from such a fingerprint (27).


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958 Small NuclearRNAs of Dictyostelium

" 5 . 8 s1 1 1 5.6seD w 5 S

a ~ c o e r

a bFIG. 1. Polyacrylamide gel electrophoresis patterns ofDic-tyostelium RNA labeled in uiuo with [3'P]phosphate. Cytoplas-

mic ( L a n e a )nd nuclear RNA ( L a n e b )solated from cells lysed inHMK buffer was electrophoresed on a 10% polyacrylamide gel con-taining 7 M urea.RESULTS

Isolation and Initial Characterization of snRNAs-Gelelectropho retic analysis of RNA from cells separ ated ntonuc lear an d cytoplasmic fractions (Fig. 1) reveals that ther ea re a numbe r of bands in the nuclear lane which are notpresent in cytoplasmic RNA. T h e three bands above 5.8 SRNA labeled Dl, D2, and D3 see m to be almost exclusivelynuclear, a nd even highly overloaded gels do not show cyto-plasmic band s at these posit ions (data not hown). In addition,a series of closely spaced b ands can be seen immediatelybelow 5.8 S RNA. These are referred to collectively as 5.6 SR NA a nd se e m t o be more a bunda n tn th e nucleus than inth e cytoplasm; however, overloaded gels show th at in con trastt o Dl, D2, and D3, th e 5.6 S RN As appear in cytoplasmicfractions aswell as n th e nucleus.Fig. 2 shows an experimen twhich confirms hat the pat te rnof Dictyostelium snRNAs does not depend on the methodfcell lysis. Lysis of cells into th e non dena turin g H MK buffernormally used for cell fractionation (Lane a ) does produceRNA bands which are not present in RNA from whole cellslysed directly into sodium dodecyl sulfate/phen ol either a tlow (Lane c) or high (Lane d ) emperature . T he increasedintensi ty of these ban ds upon ncubation of the HM K lysatebefore pheno l extraction (Lane b ) indica tes tha t they a rediscrete break-down products of highermolecular weightRNAs resul t ing f rom the ac t ionf endogenous RNases. RNA

- 3" 0 2'Dl" 5 . 8 55.6s

-5s

tRNA

" D 3u2- . " D 2-Dl

u4-

5s- /-5su5-U6-

a bFIG. 2 (left). Polyacrylamide gel patterns of RNA isolatedfrom cells lysed by different procedures. A 12% gel containing 7

M urea was employed. Lane a shows the pattern of total RNA derivedfrom cells lysed in HMK buffer a t 0C ollowed immediately byphenol extraction. Lane b is identical with Lane a except that thelysate w a s held on ice for 10min before deproteinization of the RNA.Bands which increase in intensity are marked autolysis products.Lane c displays total RNA isolated by lysingcells directly intophenol/BC at 37C. Lane d is identical with Lane c except thatphenol extraction was performed a t 60OC. Lanes e and f show cyto-plasmic and nuclear RNA patterns, respectively, isolated using HMKbuffer for cell lysis. For a description of buffers and RNA isolationprocedures, as well as electrophoresis conditions, see "Methods."FIG. 3 (right). Polyacrylamide gel com paring electrophoreticmobilities ofDictyostelium and Ehrlich ascites snRNAs. Lanea shows the pattern of nuclear RNA isolated from mouse Ehrlichascites cells; Lane b displays Dictyostelium nuclear RNA. Methodsfor preparation and identification of mouse snRNAs have been de-scribed (4). conditions for gel electrophoresis were identical withthose used in Fig. 1.

extracted from nuclei isolated using HMK buffer (Lane f ,con tains insignificant q uan tities of these autolysis productscomp ared to ytoplasm (Lane e),nd none of them co-migratewith Dictyostetium snRNAs.Th e s ue of each Dictyostelium snRN A was est imated bycomparing its electropho retic mobility u nder partially dena-turing conditions with th at of mammalian snRNAs whoselengths areknown precisely from complete primary sequenceanalysis. Since fingerprintanalysis suggested that Eh rlichasci tes snRNA s are identical with th e sequenced Novikoffhepa toma snRNA s (4, 5), labeled nuclear RNA from Dictyo-stelium and Ehrlich ascitesells was applied to adjac ent lanesof a 10%polyacrylamide gel run in 7 M urea (Fig. 3). U1 RN Ais 171nucleotides long (6);U2 is 196nucleotides in leng th (7);and U3B RNA is 216 nucleotides long (8). D l runs betweenU1 and U2 and appears to be -185 nucleotides long; D2 isslightly larg er than U2, or about 210 nucleotides in length;and D3, which issomewhat larger than theothers, appe ars tobe 250 nucleotides long. Sue estimates based on electropho-retic mobility under partially denaturin g conditions can bemisleading, however, since aden ovir us VA RNA is only 156


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Smal luclearN A s of Dictyostelium 959TABLE

Estimated numbers of molecules/cell of selected RNAs fromDictyostelium and HeLa cellsData for HeLa cell RNAs reproduced from Ref.2.RNA pecies Source No. of rnolecules/cell

Dl Dictyostelium 3 x 10'D2 Dictyostelium 2 X lo4D3 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 lo55s HeLa cells 5 x 10"

residues long but moves more slowly in this gel system thanIn order to determine theellular abundance of the Dicty-osteliumsnRNAs, ""P-labeled snRNAbands were excisedfrom the gel shown in Fig. 1 and counted by Cerenkov radia-tion. Since theells were labeled or2days (4 to 5generations),all the RNA should be t the samemaximal specific activity.Thus, by knowing the amount of radioactivity above back-

ground in a given gel band, the number f cell equivalents ofRNA applied o th e lane, and the pecific activity of the RNA,the number of molecules of each snRNA species/cell can becalculated. Quantitation of 5 S ribosomal RNA was used as acontrol in this procedure since the ellular abundance of thisspecies can be independently determined by measuring theopticaldensity of unlabeled ibosomal RNAs resolved bysucrose gradient sedimentation (30).Th e results of such cal-culations are shown in Table I. Identical numbers were ob-tained whether RNA was extracted fromuclei or whole cells.For comparison, values are also given for the abundance ofHeLa cell snRNAs and5 S RNA as determined by Weinbergand Penman (2). Dictyostelium snRNAs are on the averageonly 1 to 2%as abundant/cell s the HeLa snRNAs, althoughboth cells contain comparable numbersof ribosomes.Optimizing Gel Purification f snRNAs-Urea concentra-tion has been known for almost a decade to exert dramaticeffects on the relative electrophoretic obilities of RNA spe-cies in polyacrylamide gels. Presumably, urea acts as a weakdenaturant, and each RNA responds uniquely to partial de-naturation depending on the stabil ity of its secondary (andpossibly tert iary) structure . In the course of studies on seaurchinhistone mRNAs,Gross et al. (23) realized that avertical polyacrylamide gel containing a horizontal gradientof urea concentration could be used to distinguish betweenconformers of a single RNA species and co-migration of dis-tinct RNA species. Fig. 4 shows the pattern obtained whenlabeled Dictyostelium nuclear RNA isesolved by electropho-resis through a 10%polyacrylamide gel containinga horizontalgradient of urea from0 to 7 M.Bands which consist of severalco-migrating RNA species should splitas the urea concentra-tion increases, while onformers of a singleRNA species wouldbe expected t o coalesce. The results clearly support the otionthat D2 an d D3 are single RNA species since they migrate ssingle bands at all urea concentrations. l RNA also producesa single band at most urea concentrations, but splits intowobands at -2.5 M urea. This suggests he existence of twoequally stable conformers at intermediate concentrations ofurea.Fingerprint Analysisf snRNAs-Ribonuclease T1 finger-prints of Dl , D2, D3, and the5.6 S RNAs are shownn Fig. 5.D l, D2, and D3 appear to be single species of RNA since thenumber of distinct oligonucleotides n each ingerprint sconsistent with the snRNA'size and base composition. Th is

u2 R N A . ~

J. Boyle, personal communication.

r-D3,D2-Dl-5.8s5.6s-5s

tRNA

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

RNA rom Dictyostelium Urea concentration increasesrom Left toright as ndicated. For a description f the pouring and running ofhegel see "Methods."

does not rule out theossibility of microheterogeneity withinthe sequence f any particula r snRNA, and e present belowevidence for minor heterogeneity in the D2 RNA sequence(see below, Fig. 9). Th e 5.6 S RNA species were fingerprintedas a group for two reasons. Firs t, the bands were so closelyspaced that it was mpossible to dissect out a single species.Second, we initially thought that they might be conforma-tional isomers of a single RNA, analogous to thosedescribedfor bacterial 5 S RNA (31, 32). However, the complexity ofthe fingerprint shown n Fig. 5 demonstrates that5.6 S RNAconsists of many species, and the electrophoreticbehavior of5.6 S RNA on the urea gradient gel (Fig. 4) supports thiscontention: the ladder of 5.6 S RNAs shows no tendency tocoalesce at high urea concentrations, and severalf the bandsactually intersect as though distinct RNA species were re-sponding verydifferently to partial denaturation.Comparison of t he Dl , D2, and D3 fingerprints with thoseof Dictyostelium 5 S and 5.8 S ribosomal RNA demonstratesthat the large (andhence, characterist ic) oligonucleotides ofthese molecules are absent from the snRNA fingerpr ints (datanot shown). Since abundant ribosomal RNAs are the majorpotential contaminant n an snRNA preparation, the absence


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960 - Small Nuclear RNAs of Dictyostelium

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

FIG. . Modified base analysis of D3 RNA by two-dimen-sional chromatography. a , products of complete digestion withnuclease PI ; b . 5-fold longer exposure of a after cutting out the majorspots. Chromatography solvents and other details can be found underMethods.of 5 S and 5.8 S RNA implies that the snRNAs are pure.Furtherexamination of the Dl, D2, andD3 ingerprintsindicates that they share no charac terist ic oligonucleotides.Thus, theDictyostelium snRNAs are unrelated as judgedyfingerprint analysis, and arealso not cross-contaminated.Base Composition a nd Modified Nucleotide Content-ThesnRNAs used in these experiments were purified in two di-mensions as described under Methods since we wanted tobe certain that the resultsould not be attributed to contam-inating species of RNA. D3 snRNA, uniformly labeled with

TABLE 1Base compositions of Dictyostelium small nuclearR NA s

RNA species NucleotidePA PC PUCO c.Dl 31 21 30 18D20 185 17 e1D3 26 19 30 23 =25 s 27 23 24 26

P in vivo, was digested with P1 nuclease and the productswere separated by chromatography in the two-dimensionalsystem introduced by Nishimura (33) and modified by Sil-berklang et al. (27). As shown in Fig. 6, D3 is quite highlymodified. In addit ion to theononucleotides PA, pG, PC, andpU, it contains p$ (identified by co-migration with an unla-beled marker), pCm, and pmA (identified by their positionsrelative to PC and PA). D3 also contains another modifiedbase, marked p X , whose position does not coincide with anyof the nucleotides documented y Silberklang etal. (27). Thespot marked bridge is due toa P1-resistant 5-end structurewhich will be described below. Similar analysis of the otherDictyostelium snRNAs indicates that D2 lso contains pseu-douridine, and D l contains no internal modifications (datanot shown).The base composition of Dl , D2, and D 3 was determinedby quantitation of the excised radioactive spots using Ceren-


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Smalluclear RNAs o f Dictyostelium 961kov radiation. Table1 shows the results obtainedor all threesnRNAs and for 5 S RNA. The modified bases in D3 otherthan pseudouridine were present at less than 176, and, thus,have not been ncluded in th e table.Analysis of 5'-End Structures-Dictyostelium snRNAs la-beled uniformly in vivo were digested to completion with T2RNase, producing3'-mononucleotides and T2-resistant struc-tures which could be separated by polyethyleneimine thinlayer chromatography n the systemof 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 T2RNase in parallel toproduce known capstructures as markers. S ince T2 RNase cannot break pyro-phosphate bonds or phosphodiester bonds bearing a 2'-0-ribose methylation (21), the T2-resistant structures derivedfrom the lime mold snRNAs are most likely to be oligonucle-otides with internal ribose methylations, phosphorylated 5'-terminal nucleotides, or caps. TheT2-resistantstructuresfrom all three snRNAs were eluted from the thin layer plateand found to be partially resistant to both bacterial alkalinephosphatase (which removes all external phosphates) andP1nuclease (which digests RNA to 5'-mononucleotides regard-less of 2"O-ribose methylations and also possesses a 3"phos-phataseactivity 34)); hissuggests hat heTZresistantsnRNA structures contain the internal pyrophosphateinkagecharacteristic of caps (data not shown). The reason for thelow yield of th e D3 5'-end structure in this experiment s

unclear; in other preparations, the yield was similar to thatfound or D l and D2 snRNA. Th e D3 RNAused in thisexperimentmayhave been contaminated with ibosomalRNA breakdown products since it was purified from wholecell RNA rather than rom isolated nuclei.The putative capligonucleotide from D2 RNAwas shownto bea bona fide cap by redigestion in separate experimentswith either nucleaseP1 or nucleotide pyrophosphatase. TheT2-resistant oligonucleotide derived from D2 RNA was redi-gested fvst with P1, and the products chromatographedn thesame two-dimensional thin layer system previously used toidentify modified nucleotides (Fig. 8). Th e T2-resistant oligo-nucleotides of D l and D3 ave apparently identical P1 rediges-tion patterns (data not shown). The positions of unlabeledmarkers are indicated, s well as the approximateocation ofthe P1 bridge structures I (m7GpppAm), 1 (m'GpppA), andI11 (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 determinedby Dottin et al. (21). (Cap structures have been renamed tocorrespond to their mobility in our chromatographic system.)The digestion patternshown in Fig. 8 indicates hatD2snRNA bears a type 0 cap without 2'-O-ribose methylationadjacent to thebridge (21) since no products other than thebridge and phosphate can be detected despite overexposureof the autoradiogram.When the P1-resis tant bridge derived from D2 RNA wasfurther digested with venom nucleotide pyrophosphatase (29)and chromatographed in th e same two-dimensional system,three products were observed: pZ, PA, and phosphate. Thenucleotide designated pZ migrates in a position similar tolabeled pm2**7G obtained by digestionof the T2-resistan t capof mouse Ehrlich ascites U1 snRNA (presumably identicalwith rat Novikoff hepatoma m'. '.'GpppAmpUmpAp (6)),al -though we were unable to obtainufficient unlabeledpm"2. 'Gto prove thi s rigorously.

In order to determine the RNA sequence mmediately ad-jacent to the type 0 cap, an RNase T1 digest of uniformlylabeled D2 RNA was chromatographed on column of dihy-droxyborylaminoethylcellulose, which retains oligonucleo-tides bearing a cis-2',3'-diol by format ion of a cyclic boryl ester(35,36). The capoligonucleotide binds todihydroxyborylam-inoethylcellulose th rough the cis-diol of the inver ted nucleo-tide pZ. Internal T 1 oligonucleotides, bearing a 3'-phosphate,flow through the olumn, while he T1ligonucleotide derivedfrom the 3'-end of D2 hasa free cis-diol and is also retained.Th e 5"and 3'-ends of D2 were eluted from the column withsorbitol and separated by electrophoresis on Cellogel at. pHa b

FIG. . Detection of T2-resistant 5'-end structures py thinlayer chromatography on polyethyleneimine cellulose. T helarge spo ts near the topof the autoradiograp h represent mononuc leo-tides. Arrows mark the position of 5'-end structures. The known FIG. .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, igestion of the T2-resistant capAmpUmpAp (6). Dictyostelium- mRN A caps have the following se- structure with nuclease PI. Position of nonradioactive marker n ucleo-quence? m'GpppAmpAp ( I ) ;m'GpppAp ( I I ) ;m'GpppAmpUp ( I I I ) ; tides, a s well as PI bridges from Dictyosteliurn mRNA (determinedand m'GpppCp ( I V ) (structures as determined in Ref. 21. but re- in separate experiments), are indicated. I , m'GpppAm; I I , m'CpppA;named here according to mobility in thin layer chromatography I I I , m'GpppG; b, digestion of the T2-resistant oligonucleotide of D2rather than electrophoresis onDEAE -cellulose paper at pH 3.5). RNA with P1 nuclease and nucleotide pyrophosphatase.


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962G A>G C>U U

Small Nuclear RN As of Dictyostelium

--U AL-U-G

-G

- C

-u

FIG. . Sequencing gel of 3end-labeled D2 RNA. Productsare displayed on a 25%polyacrylamide gel run in 7 M urea after base-specific chemical modification and cleavage (17). RN A preparationand labeling are described under Methods.3.5 followed by homochromatography on polyethyleneiminethin layers. When the purified 5-end T1 oligonucleotide wasredigested wi th nuclease P1, and the products separated bytwo-dimensional thin layer chromatography, theridge struc-ture ZpppA, together with pU and pG, were obtained (datanot shown). Thus, the 5-end sequence of D2 RNA is Zppp-

Partial Sequence of 0 2 RNA-The free 3-hydroxyl groupof D2 RNA was abeled enzymatically with [5-:P]pCp bybacteriophage T4 RNA ligase (25) and the end-labeled RNAwas hensubjected o apid polyacrylamide gel sequenceanalysisafter base-specific chemicalcleavage 17). Fig. 9shows a 25% polyacrylamide sequencing gel from which 27nucleotides at th e3-end of D2 RNA can be read easily. TheRNA appears toe homogeneous at the 3 terminus andgivesa unique sequence up to osition 21, where both 4 and G arepresent. G appears to predominate overA by a factor of 2 to3, based on a comparison of the intensit ies f these bandswithother s in their respective lanes.

APUPGP.

DISCUSSION

We have isolated and character ized several pecies of smallnuclear RNA from he cellular slime mold D. discoideum.Three lines of evidence indica te that these RNA molecules

represent authentic cellular constituents rather than break-down products of larger RNA pecies such as ribosomal RNA:(a)he snRNAs canbe isolated in identical yield from nucleiprepared using several different detergents and buffers (datanot shown); (6) the same yield of snRNA/nucleus can beobtained by lysing whole cells directly into a mixture ofsodium dodecyl sulfate and phenol (Fig. 2, Lanes c and d ) ;and ( c ) ncubation of nuclei for up to 10 min before additionof sodium dodecyl sulfate and phenol does not increase th eintensity of thesnRNA bands, although severalautolysisproducts of ribosomalRNA do become moreprominent(LanesQ and 6).The existence of specialized 5-terminal capstructures on the snRNAs also argues th at Dl, D2, and D3are mat ure cellular RNA species and not excised portions ofthe 35 S ribosomal RNA precursor (18) or the precursors ofother cellular RNA species such as tRNA.The three most abundant slime mold snRNAs appearsim-ilar insize to the three ost abundant snRNAs f mammaliancells, although none of th e Dictyostelium species actually co-migrate with Ehrlich ascites ell snRNAs. Th e electrophoreticpattern of Dictyostelium snRNAs also correlates well withthat repor ted or two other lower eucaryotes: both A.proteus(13) and Tetrahymena pyriformis (12) poss es three speciesof snRNA which appear larger than.8S ibosomal RNA andresemble slime mold snRNAs in their abundance relative toeach other aswell. Dictyostelium does not appear to containanyabundant low molecular weight nuclear RNAscorre-sponding to 4.5 SI,4.5 SI,,or 4.5 SIIItudied by Ro-Choi andBusch ( l ) ,or to the somewhat smaller 4.5 S RNA recentlycharacterized by Jelinek andLeinwand (37) and sequencedbyHarada and Kat0 (9). Thelime mold nucleus does contain aconspicuous ladder of distinct RNA species centered around5.6 S which may correspond to themuch fainter array of 5 Sspecies recently identified in mammalian cells by antibodyprecipitation using autoimmuneserum from patients withsystemic lupus erythematosus:

The abundance of the snRNAs represents another majordifference between Dictyostelium and higher cells. The mostabundant of the snRNAs n higher eucaryotes, termedU1 (1)or species D (2), is presentn 20% as many copies/cell a s th eribosomal RNAs(2), while the most abundant snRNA inDictyostelium is less than 1%as abundant as the RNAs, andthe otherslime mold snRNAs areproportionally diminished.Thus, quant itation immediately implies tha t the snRNAs donot function in any capacity which is directly related to cellsize or generation time suchs cellular architecture or roteinbiosynthesis. This conclusion is reinforced by knowing thatculturedmammalian cells and vegetative amoebae grownaxenically n shakerculturehavecomparablenumbers ofribosomes/cell (2, 30) and equivalent generation times (- 12h). Since thegenome of Dictyostelium (30) is 50-fold smallerth an th at of mammalian cells (38), one possible explanationfor the relative scarcityf snRNAs in lower eucaryotes is tha tsnRNAs play a role as either primers f DNA replication (fora review, see Ref. 39) or in stabilizing the tertiary structure fchromatin (40, 41). Quantitation of the three major snRNAsin Tetrahymena (12), whose genome is much closer in size tothat of Dictyostelium than to that of mammalian cells (42),supports this notion. Th e cellular abundance of certain sn-RNAs might also correlate with the extent of nuclear RNAprocessing rather than directly with genomic size since theimmediate precursor of cytoplasmic messenger RNA, termedheterogeneousnuclearRNA, s 5- to 10-fold larger thanmRNA in mammalian cells (431, but no more than 0% largerthan mRNA in Dictyostelium (44). In mammalian cells, both

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


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Small Nuclear RN As of Dictyostelium 963U 1 (5) and 4.5 S snRNA (9) have the potential to base pairwith heterogeneous nuclear RNA splice junction sequences.Although the presence of modified bases in snRNAs cannotyet be interpreted in functional terms, t is interesting to notethat the three abundantnRNAs found in slime mold and ratNovikoff hepatoma cells exhibit corresponding patterns ofbase modification. One of the snRNAs, D3 in Dictyosteliumand U2 in the rat 7 ) ,contains many esidues of pseudouridine,as well a s extensive 2'-O-ribose methylations; another snRNA,D2 from the slime mold and U 3 from hepatoma (8), containsonly a few residues of pseudouridine; and the third snRNA,D l from Dictyostelium and U 1 in the rat (6) has no internalmodifications with the exception of a single 2'-0-methylationin U1.Th e functional significance of the 5'-terminal snRNA capstructure is no less mysterious than that f the modified bases.Nearly aU eucaryotic mRNAs possess caps which protect theRNA from at tack by phosphatases and 5'-exonucleases (45),and interact pecifically with a protein initiation factorwhichselectively stimulates ranslation of capped relative to un-capped mRNA (46).By analogy, cap struc tures may increasethe metabolic stability of the snRNAs or berequired forrecognition of snRNAs by various cellular proteins.We have shown here tha t the moeba1 snRNAs are similarin both size, cap structure, and modified base content to thesnRNAs of higher eukaryotes; moreover, by DNA sequenceanalysis of a genomic clone, we have recently found that theprimary sequence of the most abundant DictyosteliumsnRNA, D2, displays extensive homology with the ra tnucleo-lar snRNA U 3 (47). We believe these structural similaritiesimply that the snRNAs of lower and higher eukaryotes arefunctionally analogous as well. In the future e plan to deter-mine whether the synthesisand modification of snRNAsis developmentally regulated in Dictyostelium, as may bethe case in other organisms (481, and whether the slimemold snRNAs are found in discrete small ribonucleoproteinparticles comparable to those characterized in mammaliancells (4 , 5).

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The Small Nuclear RNAs of the Cellular Slime Mold Dictyostelium