isolation characterization rna2,rna3, genes of saccharomyces

Vol. 4, No. 11MOLECULAR AND CELLULAR BIOLOGY, Nov. 1984, p. 2396-24050270-7306/84/112396-10$02.00/0Copyright ©D 1984, American Society for Microbiology

Isolation and Characterization of the RNA2, RNA3, and RNAJJGenes of Saccharomyces cerevisiae

ROBERT L. LAST, JEFFREY B. STAVENHAGEN, AND JOHN L. WOOLFORD, JR.*Department of Biological Sciences, Carnegie-Mellon University, Pittsburgh, Pennsylvania 15213

Received 2 July 1984/Accepted 16 August 1984

Temperature-sensitive mutations in the genes RNA2 through RNAJJ cause accumulation of interveningsequence containing precursor mRNAs in Saccharomyces cerevisiae. Three different plasmids have beenisolated which complement both the temperature-sensitive lethality and precursor mRNA accumulation whenintroduced into rna2, rna3, and rnall mutant strains. The yeast sequences on these plasmids have been shownby Southern transfer hybridization and genetic mapping to be derived from the RNA2, RNA3, and RNAJIgenomic loci. Part of the RNA2 gene is homologous to more than one region of the yeast genome, whereas theRNA3 and RNAJJ genes are single copy. RNAs homologous to these loci have been identified by RNA transferhybridization, and the specific RNAs which are associated with the Rna+ phenotype have been mapped. Thiswas done by a combination of transcript mapping, subcloning, and in vitro mutagenesis. The transcripts are

found to be enriched in polyadenylated RNA and are of very low abundance (0.01-0.001% polyadenylatedRNA).

The flow of information from DNA to biologically activeRNA molecules involves a number of RNA processingsteps. These processing steps may include removal andaddition of nucleotides at the ends, chemical modificationsof the heterocyclic base and sugar moieties, and removal ofintervening sequences. RNA processing in eukaryotic organ-isms has received considerable attention since the discoveryof intervening sequences in genes which encode proteins (3,11). The mechanism of removal of these intervening se-quences is just beginning to be explored. Biochemical andgenetic experiments have indicated that sequences at theintron-exon junctions are conserved and necessary for proc-essing of precursor mRNA (pre-mRNA) in vivo (41). Solublecell extracts have been developed which are capable ofsplicing exogenous RNA substrates or pre-mRNAs tran-scribed from a DNA template (14, 20, 28, 30, 46). Studieswith these and improved in vitro processing systems com-bined with a variety of novel RNA substrates should provideuseful information about the properties of RNA processing.As is the case with any in vitro system, one must alwaysconsider the possibility that the components of interest aredisplaying nonphysiological activities under the chosen iso-lation and assay conditions. This possibility argues in favorof using a combination of genetic and biochemical approach-es to study complex cellular processes such as RNA proc-essing.The bakers' yeast Saccharomyces cerevisiae is a unicellu-

lar eukaryote which can be readily manipulated by usingclassical genetics and contemporary molecular genetics (5).Only a few percent of yeast genes examined thus far containintervening sequences, a situation in sharp contrast to that ofhigher eukaryotes. Most of these split yeast genes areribosomal protein genes. These include the genes encodingribosomal proteins L29 (26), 51(55), S10 (35), 52 and 59 (J.Woolford, unpublished data), and 28-a, 28-b, 34-a, and 34-b(D. Donovan and N. Pearson, personal communication).The nonribosomal protein genes which are known to have

Corresponding author.

introns are the actin gene (ACT]) (13, 44) and the mating-type gene MATal (38). The introns of these genes have aninternal conserved sequence, the so-called TACTAAC box,in addition to the 5' donor and 3' acceptor sequences. ThisTACTAAC sequence has been shown to be necessary forcorrect in vivo processing of the actin (31, 32) and rp5l (48)pre-mRNAs. Thus, yeast pre-mRNA processing substrateshave some features in common with those of previouslystudied metazoan organisms (the donor and acceptor do-mains) and have at least one essential element thus far notidentified as essential for pre-mRNA processing in highereukaryotes (the TACTAAC homology). Introduction of rab-bit beta-globin (2) and Drosophila alcohol dehydrogenase(57) genes into S. cerevisiae leads to abnormal processing ofthe primary transcripts of these genes. This is consistentwith yeast RNA processing differing in some manner fromthat of metazoan organisms (33).

Expression of yeast genes containing intervening se-quences is affected by temperature-sensitive lethal muta-tions in the genes RNA2 through RNA]I (12, 34, 50, 54, 55;N. Pearson, personal communication). Cells bearing any ofthese temperature-sensitive mutations accumulate intron-containing transcripts at nonpermissive temperatures. Thesecells eventually die, presumably as a result of reducedsynthesis of essential gene products such as actin andribosomal proteins. Since these Rna- strains are deficient inprocessing mRNA precursors, the RNA gene products mightbe involved in mRNA metabolism. We have chosen to studythese genes and their products to determine their cellularfunctions.To start to understand the roles of these RNA genes we

have isolated plasmids bearing yeast genomic DNAs whichcontain wild-type alleles of the RNA2, RNA3, and RNAIIgenes. The transcripts of these RNA genes were identifiedby RNA blotting, subcloning, and in vitro mutagenesis.These transcripts are polyadenylic acid-containing[poly(A)+] RNAs of low abundance in glucose-grown, expo-nential-phase cells. There is no noticeable accumulation oflarger pre-mRNA species homologous to these RNA genesin RNA prepared from rna2, rna3, or rnall temperature-

2396

RNA2, RNA3, AND RNAII GENES OF S. CEREVISIAE 2397

sensitive mutants shifted to 36°C. These results are consis-tent with the RNA2, RNA3, and RNA]J genes producingprimary transcripts which do not require removal of intronsand which are translated into proteins of relatively lowabundances. We are interested in understanding the relation-ships of these genes and of their products to each other andwhether they are directly involved in pre-mRNA processing.

MATERIALS AND METHODS

Genetic methods and culture conditions. Standard yeastgenetic manipulations were performed as described by Mor-timer and Hawthorne (39). The Rna- strains used in thisstudy were derived by crossing our laboratory strains withstrains characterized by Hartwell and colleagues (16, 17).These strains were obtained from the Yeast Genetic StockCenter, Berkeley, Calif. Relevant strains are listed in Table1. Cells were grown vegetatively either in YEPD (1% yeastextract, 2% peptone, 2% glucose) or in defined drop-outmedium with 2% glucose as the carbon source (25). a/a

diploids were sporulated by incubation on agar plates con-

taining either 1% potassium acetate-0.8% nutrient broth-1%yeast extract or 1.5% potassium acetate-0.25% yeast ex-

tract-0.1% glucose. Auxotrophic diploids were sporulated inmedium supplemented with the required nutrient.

Materials. T4 DNA ligase and [a-32P]dCTP were pur-

chased from New England Nuclear Corp.; deoxynucleosidetriphosphates, HindIII linkers and ATP were from P-LBiochemicals; BamHI, Sall, and calf alkaline phosphatasewere from Boehringer-Mannheim Biochemicals. All otherrestriction endonucleases and BamHI and XhoI linkers were

from New England Biolabs. Escherichia coli DNA polymer-ase holoenzyme and Klenow fragment were kind gifts fromWilliam E. Brown, Carnegie-Mellon University. T4 polynu-cleotide kinase was a kind gift from W. McClure, Carnegie-Mellon University. DNase-free bovine serum albumin was

from Bethesda Research Laboratories. DEAE-nitrocellulose(NA 45) was from Schleicher & Schuell.

Nucleic acid electrophoresis, transfer, and hybridization.DNA restriction fragments were electrophoresed and trans-

ferred to nitrocellulose as previously described (34, 58).RNA electrophoresis and transfer were done as previouslydescribed (34) with minor modifications. Poly(A)+ RNA was

electrophoresed on 1% agarose-formaldehyde gels. The gelswere treated with two changes of 100 mM NaCI-50 mMNaOH for a total of 45 min before equilibration with 20xSSPE (1 x is 180 mM NaCI-10 mM NaH2PO4-8 mM NaOH-1

TABLE 1. Strain list"Strain designation Genotype

RL2 ............ a rnall his4-519 leu2-3,2-112RL4 ............ arnall trpl ura3-52RL4-a .............arnall tpl ura3-52 + pRN11 (Ylp5)RL5............ arnall trpl ura3-52RL25............ a Ade- leu2-3,2-112 tvrl ura3-52RL68.............a rna3 his3Al leu2-3,2-112 ura3-52

+ pRN3-1 (YIp5)RL74 (DBY 1033)..a...a

ura3-52 SUC2RL78............ a ura3-52RL89.............a rna3 his3Alleu2-3,2-112 ura3-52RL91.............a rna3 his3A leu2-3,2-112lys2 trpl tyrl

ura3-52RL92............ a rna2leu2-3,2-112 ura3-52RL96.............a rna2 ade6leu2-3,2-112 ura3-52RL99............ a rna2leu2-3,2-112 ura3-52 + pRN2 (Ylp5)

mM EDTA). After transfer, the filters were immediately airdried and baked in vacuo at 80°C for 2 h. The blots werewashed with boiling water as described by Silverman et al.(53) before hybridization. Hybridizations were done in 50%formamide as described by Davis et al. (9), using DNAprobes labeled in vitro to 1 x 108 to 10 x 108 cpm/,ug with [a-32P]dCTP (4, 49). Approximate RNA sizes were determinedby using DNA restriction fragments as standards.

Yeast transformation. Yeast transformation were done asdescribed by Sherman et al. (51) with modifications de-scribed by Orr-Weaver et al. (45). Transformants containingintegrating plasmids were tested for stability as follows.Transformants were grown on nonselective plates, singlecolonies were suspended in sterile water and cells werespread plated at 100 to 500 colonies per plate on nonselectivemedium. The resultant colonies were assayed for the pres-ence of the integrated plasmid-borne LEU2 or URA3 genesby replica plating onto selective medium. True integrativetransformants yielded 100% stability by this assay. Thisplasmid stability assay was also used to monitor cosegrega-tion of Ts' and prototrophic phenotypes in plasmid-lossexperiments.

Isolation of DNA fragments from agarose gels. After re-stricted DNA was electrophoresed under appropriate condi-tions, the gel was stained by addition of ethidium bromide to0.5 ,ug/ml. Bands were identified by using long-wave UVlight. A razor blade was used to cut slits in front and back ofthe bands of interest. Strips of DEAE-nitrocellulose werehydrated in 10 mM EDTA (pH 7.5) for 10 min, activated in0.5 N NaOH for 5 min, and washed well with water. Theactivated membrane was then carefully inserted into theslits. The DNA was electrophoresed into the membrane. Themembrane strips were then washed vigorously with 0.15 MNaCI-10 mM Tris (pH 7.5)-l mM EDTA and eluted in either1 M NaCI-10 mM Tris (pH 7.5)-i mM EDTA (for fragments.7 kilobases [kb]) or 2 M NaCl-10 mM Tris (pH 7.5)-i mMEDTA (for fragments -7 kb) at 55°C for 1 to 2 h. Ethidiumbromide was removed by extraction with water-saturatedbutanol, and the aqueous phase was precipitated by additionof 2.5 volumes of ethanol.

Isolation of DNA and RNA. Bacterial plasmid DNA wasprepared as previously described (58). Yeast RNA wasprepared as described by Hereford and Rosbash (19) fromstrains grown to late-log phase at 230C on yeast syntheticmedium. When the accumulation of pre-mRNA characteris-tic of rna2 throughrnall mutant strains was being assayed,cells were grown to mid-log phase at 230C and shifted to 36°Cfor 1 h. Strains which contained a replicating plasmid weregrown on drop-out medium appropriate for selection ofplasmid-bearing strains. Yeast genomic DNA was preparedby a modification of the method of Davis et al. (10). Theethanol-precipitated nucleic acid was suspended for 1 h in200,lI of 10 mM Tris (pH 7.5)-i mM EDTA-1,ug of RNaseA per ml. Next, 20,ul of 3 M sodium acetate and 220,ul of-20°C isopropyl alcohol were added. The sample was mixedwell by inversion and centrifuged at room temperature for 2min in an Eppendorf microfuge. The pellet was dried invacuo and suspended in 50,ul of 10 mM Tris-1 mM EDTA(pH 7.5). After cleavage with restriction enzymes,S to 10,u1of this nucleic acid was used per gel lane for genomeSouthern experiments.

Plasmid constructions. Ligation of DNA fragments withcohesive ends was done at 12 to 140C overnight in 100-,ulreactions at a total DNA concentration of 1 to 20,ug/ml,using 0.2 to 0.4 Weiss units of T4 DNA ligase. The ligationreaction contained 50 mM Tris (pH 7.8)-20 mM dithiothrei-

"All strains were obtained during these experiments. except for RL74(DBY1033), which was obtained from D. Botstein.

VOL. 4, 1984

2398 LAST, STAVENHAGEN, AND WOOLFORD

tol-1.0 mM ATP-10 mM MgCl2-50 ,ug of DNase-free bovineserum albumin per ml. Blunt-end ligations were done in 10-to 20-pl reactions, using 2 to 10 Weiss units of T4 DNAligase. The reactions were terminated either by heating at65°C for 10 min or phenol-chloroform extraction, followedby ethanol precipitation before use in E. coli transforma-tions. Replacement of a staggered end with a linker was doneas described by Maniatis et al. (37). Ligated DNAs weretransformed into E. coli HB101 (6) as described by Davis etal. (9).

All RNA2 region subclones were derived from pRN2 (seeFig. 1). pRN2-1 (YIp5) and pRN2-1 (pBR322) were derivedby ligating the 4.4-kb BamHI-CiaI fragment into BamHI-ClaI-cut YIp5 and pBR322, respectively. pRN2-1 (pJDB207)was created by replacing the ClaI site of pRN2-1 (YIp5) witha BamHI linker and ligating this fragment into BamHI-cutpJDB207. pRN2-2 (YRp17) was made by ligating the 2.1-kbBamHI-HindIII fragment from pRN2-1 (pBR322) intoBamHI-HindIII-cut YRp17.

All RNA3 region subclones were derived from pRN3 (seeFig. 1). pRN3-1 (YEp24) and pRN3-1 (YIp5) were construct-ed by ligating the 6-kb SalI-EcoRI fragment, which con-tained the 275-base-pair BamHI-SalI region from pBR322,into SalI-EcoRI-cleaved YEp24 and YIp5, respectively.pRN3-2 was constructed by cloning the BamHI-ClaI frag-ment which contained a part of YEp24, including the URA3gene, into BamHI-ClaI-cut pBR322. pRN3-3 (pJDB207) wasconstructed by replacing the EcoRI site of the 2.4-kbBamHI-EcoRI fragment with a BamHI linker and cloningthis modified fragment into BamHI-cleaved pJDB207.pRN3-4 (YEp13) was made by replacing the NdeI site of the1.6-kb BamHI-NdeI fragment with a HindlIl linker andcloning this fragment into BamHI-HindIII-cleaved YEp13.

All RNAJJ region subclones were derived from pRN11(see Fig. 1). pRN11 (YIp5) was constructed by cloning the 9-kb SalI-EcoRI genomic fragment, which included the 275-bpSaII-BamHI region from pBR322, into SalI-EcoRI-cleavedYIp5. pRN11-1 (YIp5) was constructed by cloning the 2.6-kbBamHI-EcoRI fragment into BamHI-EcoRI-cleaved YIp5.pRN11-1 (pJDB207) was constructed by replacing the EcoRIsite with a BamHI linker and cloning this fragment intoBamHI-cleaved pJDB207. pRN11-2 (YEp13) was construct-ed by replacing the NruI site of the 2.1-kb NruI-BamHIfragment of pRN11-1 (pJDB207) with a HindIII linker and

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l,1ul is presumably a 4-base-pair insertion mutation. It wasconstructed from pRN2-1 (pBR322) by cleaving the plasmidwith XbaI, filling in the ends with the Klenow fragment anddNTPs, and blunt-end ligating. This destroyed the XbaI site.pRN2-1,u2 (pBR322), a plasmid deleted for the XbaI-HindIIIregion, was constructed by replacing the XbaI site of pRN2-1 (pBR322) with a Hindlll linker, cutting the plasmid withHindIII, and ligating under conditions which favor uni-molecular, cohesive-end ligation. Plasmid pRN2-1,u3(pBR322) was made by cleaving pRN2pu2 (pBR322) withHindIII and ligating in the presence of the URA3-containingHindIII fragment from YEp24. pRN2-1,l1 (YRp17) andpRN2-1,u2 (YRp17) were constructed by cloning BamHI-ClaI fragments from the appropriate plasmids into BamHI-ClaI-cleaved YRp17. pRN2-1,u3 (pJDB207) was made byreplacing the ClaI site with a BamHI linker and cloning thepRN2-1,3-containing fragment into BamHI-cleavedpJDB207. XhoI linker-insertion mutations were isolated inpRN3-3 (pJDB207) and pRN11-1 (pJDB207) by the methodof Heffron et al. (18), with the modifications of K. Tatchell(personal communication). The URA3 gene-containingHindIII fragment from YEp24 was converted into an XhoIfragment using XhoI linkers. This fragment was cloned intothe linker-insertion mutation plasmids to create rna3° andrnall° URA3 insertion mutations.

RESULTSIsolation of the RNA2, RNA3, and RNAJ1 genes. Recombi-

nant plasmid DNAs containing wild-type alleles of theRNA2, RNA3, and RNAJJ genes were identified by theirability to complement temperature-sensitive mutations in therespective RNA genes upon transformation of mutant strainsof yeast (RL92, RL91, and RL2). A library of yeast genomicDNA cloned into YEp24 (8) was used to isolate the rna2-complementing plasmid pRN2 and a similar yeast genomicDNA library cloned in YEp13 (43) yielded the RNA3- andRNAJJ-containing plasmids pRN3 and pRN11. Yeast strainswere transformed with these libraries, incubated at 23°C for40 h, and then shifted to 37°C. This preincubation at 23°Callowed expression of plasmid-borne wild-type RNA genesbefore exposure of the cells to nonpermissive temperatures.Omission of this step decreased the number of CFU greater

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MOL. CELL. BIOL.

RNA2, RNA3, AND RNAIH GENES OF S. CEREVISIAE 2399

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than 10-fold when the purified RNAII-conwas used in subsequent reconstruction e)

analogous result had been obtained in clortransformation of a temperature-sensitivestrain (42).

Colonies which survived these doubleanalyzed in two ways to determine whethertemperature-sensitive phenotype was associamid. These transformants were grown nonseto 25 generations to allow loss of the episomnplasmids. The cells were then spread plated 4grown at 23°C, and the resultant colonies wepresence of the plasmid-borne URA3 or LELgrowth at 36TC. Leu- or Ura- colonies, i.

had lost the recombinant plasmids, were alsensitive (data not shown). These results arethe rna mutation-complementing activity besociated. To purify the recombinant plasmthese putative RNA genes, total DNA wasthese yeast transformants and used to transampicillin resistance. Plasmid DNAs prepabacterial transformants were used to retransfrna mutant yeast strains. These DNAs tiappropriate rna mutants to growth at 36°C wicy. Restriction mapping of these "shuttledvealed that several overlapping fragments (DNA were obtained containing each of thegenes (data not shown). One plasmid frooverlapping clones was chosen for further artion maps of the inserts from pRN2, pRN3,shown in Fig. 1.Complementation of the mutant biochem

Rna- mutant strains accumulate intervening sequence con-taining pre-mRNAs under nonpermissive conditions (36°C),rna 11- whereas Rna+ strains do not (12, 34, 50, 54, 55). We askedwhether introducing the cloned RNA genes into rna mutant361 230 360 strains would relieve this pre-mRNA metabolism defect.

m c c Figure 2 shows accumulation of precursor RNA homologousz X eD to the intervening sequence containing the CR YJ (rp59) gene- O O (34) at 36°C in rna mutants transformed with vector DNA.

Little or no CR YJ pre-mRNA accumulates in the samestrains transformed with plasmids bearing the putative wild-type RNA genes. This complementation of the pre-mRNAaccumulation phenotype is seen whether the genes arepresent on these multicopy replicating plasmids or as a singlecopy integrated at the respective mutant rna loci (data not

* shown). This complementation is consistent with theseDNAs containing wild-type RNA2, RNA3, and RNAJHalleles.

* * Subcloning the rna mutation-complementing regions. Thesecloned DNAs were subcloned to determine which regionswere capable of complementing the temperature-sensitivemutations when present on replicating plasmids. The resultsof these experiments as well as RNA blot experimentsdescribing the mapping of RNA gene transcripts (discussedbelow) are shown in Fig. 3. The plasmid pRN2-1 (YIp5), a4.4-kb BamHI-ClaI subclone, complements the rna2 mutant

utant pre-mRNA phenotype, whereas pRN2-2 (YRp17), a subclone whichnts (RL92. RL89, contains the 2.3-kb BamHI-HindIII fragment, does not. Thisnds containing the is consistent with sequences necessary for complementationd with the intron of the rna2 mutant phenotype spanning the HindIII sitee (34). The upper within pRN2-1. pRN3-4 (YEp13), a subclone containing thetranscript is 630 1.6-kb NdeI-EcoRI region, and pRN11-3 (YEp13), a sub-intron containing clone containing the 2.1-kb NruI-EcoRI region, contain

sequences sufficient for complementation of rna3 and rnallmutant phenotypes, respectively. These results agree with

itaining plasmid those of transcript mapping and in vitro mutagenesis experi-Kpernments. An ments presented below.ning CDC28 by Mapping the cloned DNAs to the RNA loci. Although theselethal mutant recombinant plasmids were capable of eliminating the tem-

perature-sensitive lethality and pre-mRNA accumulation ofselections were the appropriate rna mutants, it was possible that rather thanthe relief of the containing the respective RNA genes, they contained extra-ited with a plas- genic suppressors which were relieving the rna mutant,lectively for 20 phenotypes. We tested whether the cloned DNAs originatedial transforming from the RNA2, RNA3, and RNAJIJ genomic loci by makingonto YEPD and use of yeast integrative transformation (22). Yeast strainsre tested for the were isolated which contained the RNA gene bearing plas-/2 genes and for mids stably integrated into the nuclear genome. Genomice., those which DNA from integrative transformants RL99, RL68, andso temperature- RL4a, their untransformed parental strains RL92, RL89, andconsistent with RL4, and the Rna+ strain RL74 were cut with Sall restric-ing plasmid as- tion endonuclease, electrophoresed on agarose gels, andnids containing subjected to Southern transfer analysis. These blots wereprepared from probed with purified DNA fragments from the cloned RNA

sform E. coli to genes. Figure 4 diagrams the expected integration events ifred from these the plasmids recombined with their homologous loci andorm the original shows the Southern blot results which indicated that this wasransformed the indeed the case. The probes are homologous to one SailIith high efficien- fragment in untransformed yeast DNA. Each integratingI" plasmids re- plasmid contains one Sall site within the YIp5 vector DNA.of cloned yeast As a result of plasmid integration, the RNA gene-specificputative RNA probes now hybridize to two new recombinant genomicm each set of fragments in Sall-cut integrant DNAs. Each of these frag-nalysis. Restric- ments has homology to YIp5, as expected (data not shown).and pRN11 are The Southern transfer results shown in Fig. 4 indicated

that the plasmids containing the putative RNA genes hadical phenotype. integrated into their homologous locations. To prove that

VOL. 4, 1984


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these regions correspond to the RNA2, RNA3, and RNAJJloci, the integrant strains were crossed to appropriatelymarked strains, and the diploids were sporulated. Tetradanalyses indicated linkage of the integrated pRN2-1 (YIp5) in

RL99 to PET8 and RNA2, linkage of the integrated pRN3-1(YIp5) in RL68 to the RNA3 gene, and linkage of theintegrated pRN11 (YIp5) in RL4a to TRPI and RNAII(Table 2). These results are consistent with the cloned DNAsoriginating from the RNA2, RNA3, and RNAJJ loci (40) andstrongly suggest that we have isolated wild-type alleles ofthese genes.RNA2 region contains a repeated sequence. When the

pRN2-1 fragments A (EcoRV-EcoRV), B (EcoRV-HindIII),and C (HindIII-ClaI) were used as genome Southern hybrid-ization probes, hybridization to the expected RNA2 genomefragments was seen (Fig. 5). Probe B (EcoRV-HindIII) alsohybridizes weakly to other genomic fragments (arrowheads,

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Fig. 5). Much weaker hybridization signalsnear the top of the blot in the strips probedM(EcoRV-EcoRV) and B (EcoRV-HindIII).were obtained by using genomic DNA cutHindlIl. Since the HindIll digest generatehybridizing fragments smaller than the stronfragment, this observation is not an artifact cadigestion of the genomic DNA. Similar I

hybridization of the RNA3 and RNAJJ regi(

JA 11 Probe Southern blots showed hybridization to the homologousregions only (Fig. 4A and unpublished results). These resultsindicate that sequences homologous to the RNA2 gene are

Efi repeated in the yeast genome. The weak hybridization+ signals do not appear to be due to contamination of fragment

-,,== B by pBR322 DNA as probing genome Southern blots with< nick-translated pBR322 does not yield a similar hybridiza-

Clzl tion pattern (unpublished results). The RNA3 and RNAJJ7 8 9 ?genes appear to be single copy when high-stringency hybrid-78~ ization and wash conditions are used.

Transcript mapping of the RNA gene clones. RNA blotanalysis was used to determine the location, size, and

* b abundance of transcripts homologous to the cloned DNAsfrom the RNA2, RNA3, and RNAJJ loci. Poly(A)+ RNAwas fractionated on agarose-formaldehyde gels and trans-ferred to nitrocellulose. These blots were probed with radio-actively labeled restriction fragments spanning the clonedregions. Figure 3 shows the results of these experiments andour interpretations of these results. This type of experimentaffords a low-resolution view of transcript locations. Theresolution is sufficiently good to determine which transcriptshave homology with the smallest subclones which still haverna mutation-complementing activities. The data shown inFig. 3 indicate the locations of the transcripts from the RNAgenes. These data are consistent with the conclusions thatthe RNA2 transcript is ca. 2,500 nucleotides long, the RNA3transcript is ca. 1,500 nucleotides long, and the RNAJJtranscript is ca. 1,050 nucleotides long. These transcripts arepresent at steady-state levels of one-tenth or less of ribosom-al protein 59 transcript (data not shown). This indicates thatthese RNAs are present at 0.01 to 0.001% of the poly(A)+population (27). Approximately equivalent quantities of eachof these RNA gene transcripts are present in poly(A)+ RNAfrom rna2, rna3, and rnall strains grown at 23°C or shiftedto 36°C for 1 h (data not shown).

s_______ In vitro mutagenesis of the cloned regions. Noncomple-menting "null" alleles of these cloned genes were construct-ed to test our assignments ofRNA gene transcripts described

their homologous in the previous section. The structures of these mutatedigests of genomic DNAs are shown in Fig. 3. Two mutations were introducedment from pRN2- into pRN2-1 to determine whether a functional RNA2 genent from pRN3-3, requires wild-type sequences in the XbaI to HindlIl interval.

it from pRN11-1' A small insertion mutation (pRN2-1l,u) was created by filling2; 4, DNA from in the XbaI site in pRN2-1 (pBR322), using the Klenowand 8, DNA from fragment of E. coli DNA polymerase I and religating the74. (B) Schematic blunt ends. A deletion mutation (pRN2-1,u2) was construct-INA gene into the ed by removing the 0.6-kb region between the XbaI andtegrating plasmid HindlIl sites of pRN2-1 (pBR322) and replacing it with aridization probes HindIll linker. The URA3 gene was cloned into the resultingames of untrans- HindIll site of pRN2-1,u2 to create pRN2-113. The pRN2-Lion probe identi- 1A.l and pRN2-1Fl2 mutant DNAs are incapable of comple-ormed strain (the menting the rna2 mutation when present at 3 to 10 copies perfragments in the cell in YRp17 (5). The pRN2-1R3 mutant DNA does not:han the doublet complement the rna2 mutation when present at 50 to 200

copies per cell in pJLSB207 (1, 7). These mutations lie withinDNA homologous to the 2,500-nucleotide transcript de-scribed above and interrupt a long open reading frame which

are also seen is presumably part of the RNA2-coding region (J. Beggs,vith fragment A personal communication). The combination of subcloning,These results transcript mapping, and mutagenesis indicates that the

with BamHI or 2,500-nucleotide transcript shown in Fig. 3A is responsiblees two weakly for complementation of the rna2 mutant phenotype.gly hybridizing Random linker-insertion mutagenesis (18) was used tomused by partial create mutations which abolish the ability of pRN3-3high-stringency (pJDB207) and pRN11-1 (pJDB207) to complement the re-cns to genomic spective rna3 and rnall temperature-sensitive mutations.

ARNA 2 Probe

-0-cXE0

I-

VOL. 4, 1984


TABLE 2. Genetic mapping of integrated cloned RNA genes

Cloned Cross analyzed Markers of interest Map units Total no. Ascus type'gene (cMorgans) of tetrads PD NPD T

RNA2 RL99 a rna2 ura3-52 leu2-3,2-112 + URA31RNA2 and PET8 0 15 15 0 0pRN2-1 (YIp5) x RL96 a rna2 ura3-52 pet8 ade6

RL99 a rna2 ura3-52 leu2-3,2-112 + RNA2 0 8 8 0 0pRN2-1 (YIp5) x RL74 a ura3-52SUC2+

RNA3 RL68 a rna3-ura3-52 his3Al leu2-3,2-112 RNA3 0 14 14 0 0+ pRN3-1 (YIp5) x RL78 a ura3-52

RNAJJ RL4-a a rnall ura3-52 + pRN11 (YIp5) URA31RNAJl and TRP1 13 38 28 0 10x RL5 a rnall trpl ura3-52

RL4-a a rnall ura3-52 + pRN11 (YIp5) RNAJJ 0 11 11 0 0x RL25 a ura3-52 Ieu2 adel tyri

a PD, Parental ditype; NPD, nonparental ditype; and T, tetratype.

The rna3° and rnall mutations which were isolated (pRN3-3I,2, pRN3-3,u4, and pRN11-l,ul) are shown in Fig. 3B andC. The URA3 gene was cloned into the resultant XhoI sitesof pRN3-3,u4 and pRN11-1,l1 to create the plasmids pRN3-

Wholensert

(Brm-Cla I)

E -n.

co

A;(Rv-RV)

FRAGMENT

B(RV- Hind)

C

H ind -CIaI)

E = E I E =0 c)CE I m I I

-4

> rla:-rn1

0 0 C

F _w_ A W B0

2,500 NTr

cI_cN)

1,0

FIG. 5. The RNA2 gene contains repeated DNA. GenomeSouthern analysis of BamHI and Hindlll digests of RL74 DNAprobed with subfragments derived from pRN2-1 (pBR322). IdenticalSouthern blot strips were probed with the indicated purified frag-ments. Several of the hybridization signals of low intensity are

indicated with arrowheads.

3,lO and pRN11-11x4 (Fig. 3). The locations of these muta-tions are consistent with the RNA3 and RNAJ I genes codingfor the previously identified 1,500- and 1,050-nucleotidepoly(A)+ RNAs.

DISCUSSIONThe rna mutants were first identified by Hartwell (16) in a

screen for temperature-sensitive mutants of yeast whichwere defective in synthesis of macromolecules. The rnalthrough rnall mutants were all shown to be defective inRNA metabolism, although the rnal mutant had characteris-tics quite different from those of rna2 through rnall strains(16, 17, 23). The rnal mutant had reduced levels of tRNA,rRNA, and mRNA, and as a result, protein synthesis ceasedquite rapidly upon a shift of the mutant to nonpermissivecondition (23). In contrast, strains containing temperature-sensitive lesions in the RNA2 through RNAJI genes wereshown to synthesize greatly reduced quantities of 5S, 5.8S,17S, and 25S rRNAs at 36°C, whereas tRNA, mRNA, andprotein synthesis were not dramatically affected (17).Warner and Gorenstein (15, 56) showed that the synthesis ofmost ribosomal proteins and ribosomal protein mRNAs wasgreatly reduced upon shifting these mutants from the permis-sive temperature to the nonpermissive temperature. Thesynthesis of many other yeast proteins and mRNAs wasunaffected for at least 2 h at the nonpermissive temperatureof 36°C. Shulman and Warner (52) later showed that al-though synthesis of mature rRNAs was inhibited in the rna2through rnal l mutants at 36°C, 35S pre-rRNA was producednormally. This deficiency in 35S pre-RNA processing couldbe a result of the lack of synthesis of most ribosomal proteinsin rna2 through rnall mutants at the nonpermissive tem-perature. Since intervening sequence containing pre-mRNAhomologous to most characterized ribosomal protein genesaccumulate in rna2 through rnall mutant strains at 360C (26,35, 55; J. Woolford, unpublished results; D. Donovan and N.Pearson, personal communication), it seems likely that thedeficiency in translatable ribosomal protein mRNA synthe-sis in rna2 through rnall mutants described by Warner andGorenstein (56) is due to the inability of the rna2 throughrnall mutants to process ribosomal protein pre-mRNA.The RNA genes are good candidates for genes whose

products are involved in pre-mRNA processing, as tempera-ture-sensitive mutations in these genes prevent yeast cellsfrom processing pre-mRNA at wild-type levels. Althoughthe products of these RNA genes might not facilitate theprocessing of pre-mRNA molecules by directly interactingwith them, wild-type RNA gene expression is probably

MOL. CELL. BIOL.

RNA2, RNA3, AND RNAJJ GENES OF S. CEREVISIAE 2403

required for normal processing of those yeast pre-mRNAsreported to date. An understanding of the physical proper-ties of the RNA gene products, their relationships to eachother and pre-mRNA, and their subcellular locations arerequired to elucidate their roles in pre-mRNA processing.We have isolated wild-type copies of three of these genes

(RNA2, RNA3, and RNAJI) as the first step in an analysis oftheir role in RNA processing. Plasmids were isolated whichcontain yeast genomic DNA sequences with the ability tocomplement the temperature-sensitive lethality and accumu-lation of pre-mRNAs in appropriate rna mutants. Thesecloned sequences have been shown genetically to be ho-mologous to sequences tightly linked to the RNA2, RNA3,and RNAJJ genetic loci. Therefore, we conclude that theseplasmids contain wild-type alleles of the RNA genes ortightly linked suppressors of these temperature-sensitivemutations. The latter possibility is unlikely since theseplasmids still complement when only one integrated copy ispresent in haploid or diploid mutant cells. This type ofplasmid-associated extragenic suppression would be differ-ent from published examples (21, 24, 36), all of which aremediated by high-copy number of the suppressor-containingplasmids or higher than normal production of plasmid-bornegene products.The RNA3 and RNAJJ clones have been shown to hybrid-

ize to only one genomic region, whereas at least part of theRNA2 gene appears to be duplicated under normal stringen-cy hybridization conditions. It is possible that an RNA2-likepseudogene exists, or there might be another yeast polypep-tide which has amino acid sequence homology to the RNA2polypeptide. The latter hypothesis suggests many potentiallyinteresting and testable scenarios. For example, does thisregion of homology correspond to a functional domainshared by proteins with a common cellular role. It will be ofinterest to ask whether other RNA genes, including RNA3and RNAJJ, are repeated in the genome by performingrelaxed stringency hybridization experiments, and whethertheir protein gene products share antigenic determinants.

It was necessary to use a combination of techniques toidentify transcripts homologous to the RNA2, RNA3, andRNAJJ genes because RNA blot analyses indicated thateach of the originally isolated clones containing an RNAgene was homologous to more than one poly(A)+ mRNAspecies. Transcript mapping and subcloning results correlat-ed the detectable transcripts associated with the rna mutantcomplementation activities. In vitro mutagenesis of thesesubclones has provided further evidence that the RNA2 geneencodes a 2,500-nucleotide RNA, the RNA3 gene encodes a1,500-nucleotide RNA, and the RNAJI gene encodes a1,050-nucleotide RNA. The mutations which were used mapentirely within the transcript boundaries as defined by RNAblot analyses. Unless there are overlapping transcripts froma region, and we are only able to detect one of thesetranscripts, our transcript assignments are correct. Steady-state levels of the RNA2, RNA3, and RNAIJ transcripts donot change dramatically when rna2, rna3, and rnall mutantsare shifted to 36°C (unpublished results). These results implythat these mutations do not dramatically alter synthesis orstability of their own mRNAs. They are also consistent withthese genes having no intervening sequences, interveningsequences too small to detect by RNA blot analysis, orintervening sequences whose removal is not affected in rna2,rna3, and rnall mutant strains.The RNA gene transcripts we have identified are enriched

in poly(A)+ RNA (unpublished results) consistent with theirencoding protein gene products. RNA blot analyses with

preparations of total RNA did not show any other homolo-gous transcripts (data not shown). Control experimentsindicated that we should have been able to identify nonpoly-adenylic acid-enriched transcripts as small as tRNAs (80nucleotides) and of as low abundance as small nuclear RNAs(ca. 200 copies per cell). Dot-blot hybridizations with a 32plabeled fraction enriched in RNAs of 150 to 300 nucleotidesas probes showed no hybridization to these cloned RNAgenes under conditions which permitted identification ofyeast small nuclear RNA genes (J. Thompson, R. Last, J.Woolford, and M. Fournier, unpublished data). Thus, we areconfident that these RNA genes code for poly(A)+ RNAs ofvery low abundance [0.01 to 0.001% of poly(A)+ RNA]. Alikely interpretation is that these RNAs are translated intopolypeptides of relatively low abundances in wild-type yeastcells. Recent experiments in this laboratory with antiserawhich recognize the RNA2 and RNA3 polypeptides areconsistent with this interpretation (R. Last and J. Woolford,unpublished data).Dominant and recessive suppressors of the temperature

sensitivity of a large number of rna mutants, including thephenotypically distinct rnal, have been isolated by workersat several laboratories (47; S. Nolan and A. Hopper, person-al communication). A dominant suppressor allele of SRNI(47) has the ability to suppress a number of rnal throughrnall mutants singly and in a variety of pair-wise combina-tions. This SRNI allele partially reverses the depletion ofrpSl and rp52 mRNA without greatly decreasing the accu-mulation of pre-mRNA in an SRNlIsrnl + rna2lrna2 strain at34°C (29). Suppression of the temperature-sensitive lethalitycaused by mutations in such a large number of RNA genesby a single SRNI mutation implies that the RNA genes havesome functional relationships to each other. However, anal-ysis of SRNI mutants has yielded no specific informationabout these relationships. We hope to use the cloned genesto more accurately define these relationships and the roles ofthe RNA2 through RNAJJ genes in yeast metabolism andgrowth.

ACKNOWLEDGMENTS

We thank John Larkin, Charles Moehle, Craig Peebles, and MitchRotenberg for critical reading of the manuscript and Jean Beggs forcommunicating unpublished results. Thanks go to David Botsteinfor the YEp24 yeast genomic library and strain DBY1033 and to JimHaber for the YEp13 yeast genomic library. We are grateful toKathy Galligan for typing the manuscript.

This work was supported by Public Health Service grant GM-28301 from the National Institutes of Health. R.L.L. was supportedby a National Institutes of Health National Research Service Award(GM-08067) awarded to the Department of Biological Sciences.

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