Ribosomal Protein L32 of Saccharomyces cereuisiae Regulates Both ...

Vol. 268, No. 26, Issue of September 15, pp. 19669-19674, 1993 Printed i n U S A .

THEJOWALOF B I O ~ I C CHEMISTRY AL 8 1993 by The American Society for Biochemistry and Molecular Biology, Inc.

Ribosomal ProteinL32 of Saccharomyces cereuisiae Regulates Both Splicing and Translationof Its Own Transcript* (Received forpublication, April 22, 1993)

Mariana D. Dabeva$ andJonathan R. Warner9 From the Department of Cell Biology, Albert Einstein College of Medicine, Bronx, New York 10461

partialhepatectomy (121, these mRNAs are effectively reRibosomalproteinL32of Saccharomyces cereuisiae regulates the splicingof its own transcript (1,2) appar- cruited into polyribosomes, perhaps by activation of eIF 3 or 4 ently by interacting with a structure composed largely(7, 12). In Dictyostelium the onsetof differentiation causes the of the 5‘ exon. However, even in strains overproducing mRNA for ribosomal proteins to leavethe polyribosomes, probL32 mRNA, e.g. from a cDNA copy of the gene,little’ac- ably due to selective loss of poly(A) tails (13). cumulation of L32 is observed after a brief pulse label. By contrast, inSaccharomyces cerevisiae, the general control When the 5’ leader of the RPL32 mRNA is replaced byan of ribosomal protein synthesis(reviewed in Ref. 14)appears to of the ribosomal protein exogenous leader, the amount of pulse-labeled L32 in- occur through coordinate transcription yield creases severalfold, suggesting that L32 regulates the genes (15-19) whose promoterstrengthsaresuchto translation of its own mRNA, acting through sequences roughly equimolar amounts of mRNA (20). While the coordiin the 5’ region. This conclusion was confirmedby the nate transcription is presumably due in part to the Raplpor observation that in cells carrying a chimeric gene in Abflp binding sites found in the upstream activating sequences which theL32 leader is fused toLacZ coding sequences, of all ribosomal protein genes (21-231, the details are far from the presence of a second gene that overexpresses L32 clear. 500/0,in spite itself reduces the level of p-galactosidase by In addition to this coordinate regulation of ribosomal promRNA, presumably due teins, there are mechanisms to modulate the accumulationof of a doubling of L32-lacZ fusion to stabilizationof the message. Mutations within the5‘ individual proteins.For the most part, this is dueto very rapid leader that abolish the regulation of splicing also abol-degradation of any ribosomal protein synthesized in excess ish the regulation of translation, suggesting that reg the (24-27), a situation that alsooccurs in mammalian cells (28). ulation of translationby L32 involves a structure similar However, we have identified one ribosomal protein, L32, that to that proposed for the regulation of splicing.In cells controls its own synthesis by regulating the splicing of the overproducing L32-mRNA about half the excess mRNA was found in ribonucleoproteins of e25 S , unassociated transcript of its own gene (1).This appears to occur through with ribosomal particles.Much of the rest was found in interaction with a specific structure found in the 5’ exon and the first few nucleotides of the intron (2). We now show that ribonucleoproteinsof 80-120 S. L32 can also regulate the translation of its own mRNA, and

The regulation of the synthesis of ribosomal proteins at the level of translation isa widespread phenomenon. It appears to be the fundamental level of control of ribosomal protein synthesis in Escherichia coli (reviewed in Refs. 3 and 4). The ribosomal protein operons are transcribedconstitutively. For each operon there is one ribosomal protein that,if it does not bind to a molecule of newly formed ribosomal RNA, can bind to its mRNA and prevent translation. Insome but not allcases, it is possible to show that the binding site on the mRNA mimics the binding site for that protein on the rRNA. In the cells of higher eukaryotes, including mammals, Xenopus, Drosophila, and Dictyostelium, regulation of translation plays a key role in the control of ribosomal protein synthesis (reviewed in Refs. 5 and 6 ) . In vertebrates the5’leader of the mRNA contains a polypyrimidine tract that regulates the mobilization of this mRNA onto polyribosomes (6-8). A potential regulatory protein has recently been identified (9). On stimulation of growth by addition of growth factors (10, 11)or by

* This work was supported by National Institutes of Health Grants GM25532 (to J. R. W.) and CA 13330 (to the Albert Einstein Cancer Center). The costsof publication of this article were defrayed inpart by the payment of page charges. This article must therefore be hereby marked “advertisement” inaccordancewith18U.S.C.Section1734 solely to indicate this fact. $’ On leave from the Bulgarian Academy of Sciences. $ To whom correspondence should be addressed: Dept.of Cell Biology, Albert Einstein College of Medicine, 1300Morris Park Ave., Bronx, NY 10461. Tel.: 718-430-3022; Fax: 718-829-7619.

that the sequences involved in the regulation of splicing are also responsible forthe regulationof translation. L32 stabilizes the mRNA, and prevents at least some of it from interacting with ribosomal particles. MATERIALS AND METHODS Yeast Strains-S. cerevisiae strains S150-2B (MATa, trpl-289, his3-1, ura3-52, leu2-3, 11 and W303 (Mat a, ade-2-1, his3-11, 15, leu2-3, 112, trpl-1, ura3-1, canl-100) were used. Plasmids-Five plasmids overexpressing L32 were constructed (Table I): A, a subclone of pYERPL32 (29) of about 1.8 kilobase pairs, carrying the complete RPL32 gene; B,a cDNA clone isolated from a cDNA library (30),modified by fusing the authentic L32 terminator to the coding sequences; C, a chimeric RPSIO-RPL32 gene (described in Ref. 2) in which the fusion pointis in the middle of SlO-L32intron (this gene encodes the complete L32 protein except for an additional lysine residue at the N terminus);D, a chimeric plasmid in which the expression of L32was under GAL10 promoter and the RPL32 leader was substituted by the CYCl leader. For construction of these plasmids,the multicopy vectors Yep351 and Yep352 (31)were used. Plasmids overexpressing P-galactosidase from LacZ of E . coli were based onthe Yep354, Yep357, and Yep367 vectors (32).The expressionof LacZ was drivenby the RPL32 or ADHl promoter. The L32leader was fused to the LacZ gene either 5 nucleotides (plasmid E)or 120 nucleotides (plasmid F)after the beginning of exon 2. The P-galactosidase activity of the transformants was determined as described (33). An integrating version of plasmid E was mutagenized by replacing RPL32 sequences with sequences derived from previously constructed mutants (2) by polymerase chain reaction. One mutant (+2C+T; see Table 111) was newly made. Mutant regions were sequencedto verify the mutation. Southern and NorthernBlot Analysis-Total genomic yeast DNAwas isolated and digested with restriction enzymes and blottedto a nitro-

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cellulose membrane as described before (34). Northern blot analysis was carried out as previously described (1, 2). The blots were probed with RNA or oligonucleotide probesas appropriate. Polyribosome Analysis-'Ib 100 ml of culture at Asoo = 1.0, 150 pg/ml cycloheximide was added. The cells were chilled, poured over ice-cold water, and collected. The pellet was washed 2 times with 20 ml of LHB buffer (10mM Tris-HC1, pH 7.4,lOOmM NaCI, 30mM MgCI2),containing 50 pg of cycloheximide, 200 pg of heparin, and 0.2 pl of DEPC/ml. The final pellet was suspended in 0.5 ml of LHB as above, and 1 ml of glass beads was added. The cells were broken by discontinuous vortexing for 210s, interrupted by coolingin ice. Then 1.5 mlof LHBbuffer containing 0.2% Nonidet P-40was added and the suspension vortexed for another 30 s and centrifuged for 10 min at 10,000 rpm. The supernatant was recentrifuged for 10 min at 12,000 rpm. The supernatant after the second centrifugation (about 1.2-1.3 ml) was overlaid on top of 11.5 mlof 7 4 7 % (w/w)sucrose gradient made in 50 mM Tridacetate, pH 7.0, 50 mM NH,CI, 12 mM MgC12, 1 mM dithiothreitol and centrifuged for 170 min at 39,000 rpm and 4 "C in an SW 4 1 rotor. Thirty-three fractions of 375 plwere collected, and a 2 0 4 aliquot of each was loaded on a native 1.5% agarose gel in 0.5 x TBE buffer, containing 1 mM MgC12.The electrophoresis was run for 4 h at 100 mV with circulating buffer. The gel was blotted to Nytran under denaturing conditions (6%formaldehyde, 10 x SSC) for 16-18 h. The blots were UV cross-linked and baked before hybridization. Synthesis of Ribosomal Proteins-A culture was labeled with either [36Slmethionineor EXPRE?6S36S (Du Pont-New England Nuclear) for the time indicated, and then poured over crushed ice to stop incorporation. The cells were collected by centrifugation and broken with glass beads, and thetotal protein was extracted in 67% glacial acetic acid and analyzed on two-dimensional gels designed to display ribosomal proteins (17).

(Fig. 1,B and C ) .Almost all the spots, other than those in the upper left quadrant, represent ribosomal proteins. L32 is indicated. It is one of the smallest ribosomal proteins and does not run in a compact spot. Nevertheless, it isclear that there isno detectable overexpression of L32 in strainswith excess mature L32 mRNA. Similar results were obtained after a 3-min pulse (data not shown). These results suggest that the translation of L32 is regulated, but cannot exclude the extremely rapid degradation of excess L32. The Leader of RPL32 mRNA Is Involved in the Tkanslational Regulation-To prove that theexpression of L32 is translationally regulated, we constructed plasmids from which L32 is encoded by a chimeric mRNA in which the RPL32 5' leader is replaced by another 5' leader (Table I, constructs C and D). Yeast cells were transformed with either the vector, or plasmid C, overproducing a chimeric SlO-L32 mRNA.RNA was prepared from the cells and subjected to Northern analysis (Fig. ID). Cells were pulse-labeled with [35Slmethioninefor 3 min and the total protein fractionated on two-dimensional gels (Fig. 1, panels E and F). Fig. 1F demonstrates that thepresence of a foreign 5' leader leads to substantially greater production of L32. This could be due either to an intrinsically higher level of translation initiation or to a resistance to the autogenous regulation of translation. Certainly, the result inpanel F suggests strongly that the lack of overexpression of L32 in panel C is not due to extremely rapid turnover of excess L32. However, the protein produced RESULTS from construct C begins M-K-A- instead of the "A- of authenL32 Is Not Overproduced in StrainsSynthesizing Its mRNA tic L32 (Table I); the former might be more stable, although in Excess-The synthesis of ribosomal protein L32 is regulated proteins with lysine at their N terminusare considered to turn in part by a feedback inhibition of splicing (1) in which L32, over more rapidly than those starting with alanine (35). Therefore cells were transformed with plasmid D, in which acting through a structure composed largely of the 5' exon of the RPL32 transcript, blocks the formation of a splicing com- L32 mRNA coding sequences were fused to the CYC15' leader plex (2). Nevertheless, in cells containing several extra copies of and transcription is driven by the conditional GALlO promoter RPL32, some excess splicedmRNA is present, but littledetect- (Table I). This construct codes for authentic L32. It is clear in able accumulation of excess L32 occurs. This observation could Fig. 1 that massive overproduction of this CYCl-RPL32 fusion be due to the rapid degradation of excess ribosomal proteins mRNA (panel G )leads to massive overproduction of L32 (panel observed by many laboratories (24-27). However, since the I ) . The results shown in Fig. 1 suggest that the translation of RNA structure postulated to be involved in the regulation of splicing (2) is largely conserved in the spliced mRNA (see be- the RPL32 mRNA is under autogenous regulation through selow), we asked whether we could detect the regulation of trans- quences within the 5' exon. While they do not rule out the lation of the mRNA for L32. Cells were transformed with a possibility that theinitiation of translation on the 5' leader of multi-copy plasmid carrying a cDNA version of RPL32 (Table I, the L32 mRNA is inherently less efficient than that of S10,we construct B)or with the vector. Fig. lA shows that theamount have shown that generally the mRNAs for different ribosomal of L32 mRNA is increased substantially in the strain bearing proteins are roughly equimolar (20). Since they make equimothe cDNA clone. The proteins of the same cultures were pulse- lar amounts of protein, they are likely to be translated with labeled for 1 min with [36S]methionine,and the total labeled equal efficiency. Banslational Regulation of L32 Involves the 5' Leader of L32 proteins were analyzed on two-dimensional acrylamide gels TABLE I Plasmids overexpressingL32 or p-galactosidase A-D are plasmids overexpressing L32. The vectors Yep351 or Yep352 (31) were used for construction of the following plasmids (see "Materials and Methods.").A is agenomic cloneofRPL32; the size of each element is indicated. B is acDNAclone ofRPL32, lacking the intron. Transcription is driven by the ADHl promoter. C is a chimeric RPSlO-RPL32gene, fused at the middle of the introns of the two genes. The construction of this gene has been described in detail by Ref. 2. The fusion gene encodes a protein identical to L32 except forthe initial amino acid. (The methionine is removed.) The transcript of this gene is not subject to the regulation of splicing (2). For plasmid D, the GAL10 promoter drives transcription of a CYCl5'leader fused to the complete RPL32 coding sequence.E and F are plasmids overexpressingp-galactosidase.Two different constructs were introduced into vectors Yep354 or Yep357 (32).E is a partialcDNA clone of RPL32, fused to LacZ 5 nucleotides (GCCCC) beyondthe exonl-exon2 boundary. Transcription is driven by the RPL32 promoter.F is a partialcDNA clone of RPL32 fusedto LacZ 120 nucleotides after the initiatorto LacZ. Transcription is driven by the ADHl promoter. nt, nucleotide(s). Plasmid Leader

A B C

D

E F

Promoter

RPL32 (500 nt) ADHl RPSlO GALlO RPL32 ADHl

Region

RPL32 (61 nt) RPL32 RPL32 RPL32 RPSlO RPL32 CYC 1 RPL32 RPL32

RPL32 (230 nt) 0 RPSlOBPL32 0 0 0

RPL32 (315 nt) RPL32 RPL32 RPL32 AUGCCCCNLacZ 120 nt from RPL32LacZ

Terminator

RPL32

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FIG.1.Northern blot analysis (A, D, and G ) and pulse labeling of ribosomal proteins in controls (B,E, and H) and i n strains overexpressing L32 transcripts (C, F, a n d I ) . Yeast strain W303a (A-C, G I ) or S150-2B ( S F )was transformed with vectors or with the constructs described in Table I. A-C, construct B, S F ,construct C; G I , construct D. The cells in panels G I were grown in 2% galactose to induce the GA.Ll0 promoter. TheNorthern blot analysis of RNA from thesetransformants was probed with antisense riboprobe from exon 2 of L32. Panels A, D,and G: left lane, control cells; right lane,overexpressingL32 transcripts. Cellswerelabeled invivo with [35Slmethioninefor 1 min ( B and C) or for 3 min ( E and F )or with EXPRE36S36Sfor 3 min (Hand I ) , and total protein was extracted and displayed on a two-dimensional polyacrylamide gel designed to separate ribosomal proteins (17).Panels B, E, and H, cells with the vector; panels C, F, and I , cells with constructs B,C, and D, respectively. See Table I.

TABLEI1 Relative p-galactosidase activityand concentration of fusion mRNA in cellsoverexpressing p-galactosidase fromfusion plasmids E and F (control cells) and afler introduction of plasmid overexpressing L32 Values in parentheses represent number of tested transformants. Plasmid

P-Galactosidase activity" L32 overexpression Control

Concentration of mWAb Control

L32 overexpression

&GalactosidasdmRNA Control L32 overemression

(12) 200 * 92 (12) 1 * 0.30 0.25 i 0.12 (8) 208 i 47 (16) 1 i 0.31 0.23 i 0.08 The values for the p-galactosidaseactivity of the control cellstransformed with plasmid E and plasmid F were 169 and 199 units, respectively, and are taken here as 100. The concentration of the chimeric mRNA in each transformants was determined in relation to the concentration of U1 RNA after scanning of the autoradiograms of the blots probed with appropriate oligonucleotides,and the values in the controls taken as 100.

E F

100 i 9 (10) 100 i (12) 15

50 f 9 (12) 48 f 12 (12)

100 i 24 100 i 27

mRNA--If the 5' leader of L32 mRNAis involved in theautogenous regulation of L32 translation, then mRNA expressing a protein from the L32 leader will be subjected to negative regulation in the presence of excess L32. Two fusion plasmids were constructed expressing P-galactosidase from chimeric genes that include the 5' exon ofRPL32 (see E and F in Table I). In one, the promoter, the 5' exon and five nucleotides of the 3' exon of RPL32 were fused to the coding sequence of LacZ ofE. coli. The second was a cDNA clone of RPL32 (1) containing 120 nucleotides of the coding sequences, driven by the A D H l promoter. The plasmids were introduced in strainsoverexpressing L32 or in strains containing the respective vector. Table I1 shows the level of P-galactosidase activity and the concentration of L32-LacZ mRNA. In strainsoverproducing L32, the activity of P-galactosidase is reduced in both cases by 50%. However, the amount of the L32-LacZ fusion transcript is 2-fold greater, suggesting that L32 enhances the stability of the fusion mRNA, as it did forthe unspliced transcript of RPL32 (2). Thus overproduction of L32

reduced the activity of the mRNA containing the LacZ sequences fused to the 5' sequences of RPL32 by about 75% (Table 11).We conclude that the 5' leader of RPL32 mRNA is involved in the autogenous translational regulation of L32 expression. Sequences more than a few nucleotides downstream of the initiation codon are not involved in this regulation. Similar 5' Structures Regulate Both Splicing and Damlation-The data presented above suggest that the 5' leader of the RPL32 mFtNA plays an important role in the regulation of its translation. The 5' leader also plays an important role in the regulation of splicing of the RPL32 transcript (2). We have proposed a structural model of the 5' leader and shown its involvement in the regulation of splicing (Fig. 2.4). The spliced mRNA can be folded into a structure thatdiffers only slightly from that of the unspliced transcript (Fig. 2 B ) . To ascertain if the structure shown as in Fig. 2B is realistic, we asked whether mutations that abolish regulation of splicing also abolish regulation of translation. The results are shown in Table 111. It is evident that, qualitatively, mutants thatreduce the regulation

L32 Regulates Dunslation Its Own

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J

B

I

5 ‘ splice site

’?

gradient. These molecules are not engaged with ribosomes at all. On the other hand, a substantial proportion migrates between 80 and 150 S. These could represent small polyribosomes, aberrant initiation complexes, or some other form of RNP not associated with ribosomal particles. As a control, we analyzed extractsof cells overproducing the mRNA for ribosomal protein L29. In this case, nearly all the mRNA is found in polyribosomes, which onaverage are slightly larger than those translatingL32, reflecting the difference in length of the two coding regions. These results suggestthat L32 can prevent its mRNA from interacting with ribosomal particles and may inhibit the formation of active polyribosomes even if an initiation complex is formed. DISCUSSION

The data presented above demonstrate that ribosomal proof its own mRNA. I t does tein L32 can regulate the translation 31nt so apparently by interacting witha structure largely within the UON~/U(ONI JUNCTKM 5”untranslated region, a structure nearly identical tothat inFIG.2. A, proposed structure involved in the regulation of splicing of volved in the regulationof splicing of the transcript. Thequesthe RPL32 transcript (2). B,proposed structure involved in the regula- tion may arisethat with the accumulationof L32 regulated at tion of translation of the RPL32 mRNA. the level of splicing, of translation, and of turnover, how does enough excess L32 accumulate to do the regulating? The anTABLEI11 swer is that under normal conditions each L32 mRNA is reCorrelation of the regulation of translation and of splicing by L32 This experiment is similar to that in Table 11, except that the indi- sponsible for the synthesis of about 50 protein molecules (20). cated mutants (see “Materials and Methods”) were constructed and Thus an imbalance of 2% between the molar synthesis of rRNA introduced intothe genome at the URA3 locus. The mutations refer to and L32 would provide enough protein to cause detectable regthe exon the nucleotides in Fig.2B. +2C is the second nucleotide beyond 1-exon 2 junction. The percent reduction in P-galactosidase activity dueulation. Since these experiments have been carried out in vivo, we to the presence of the SL gene on a multicopy plasmid is shown. The right-hand column indicates which transcripts were subject to regula- have no definitive proof that L32 itself interacts with the 5’ tion of splicing (2). NA, not applicable. leader of the mRNA. However, two experiments suggestthat it does. (i) In attempting to epitope-tag L32, we inserted 8 amino I Inhibition of translation Inhibition of splicing Mutation by excess L32 (2) by excess L32 acids at several positions. In each case the modified L32 was inactive, not only in ribosome assembly but alsoin the regula41 Wild type 44 5A+G tion of splicing and of translation. (ii) By fusing L32 to the C 11 9C+G terminus of the maltose-binding protein (361, we have been able 14 10G.A to show that it binds with high affinity to the 5’ end of the 14 14U-A RPL32 transcript.2 It also binds to the spliced form of the 26 15G.C 76 +2C-T transcript, but withmuch lower affinity (data notshown). This is just what we would predict from a comparison of panels A of splicing also reduce the regulation of translation. It is strik- and B of Fig. 2, and from our observation that L32 inhibits ing that the alteration of a single base, more than 50 nucle- splicing far more effectively than it inhibits translation. At present, the caseof L32 seems unique inS. cerevisiae. No otides from the initiation codon, e.g. 9C-G or 10G-.A, can the have such a profound effect on translation. Further confirma- other ribosomal proteins have been shown to regulatespliction that a structure of this type is involved in regulationcomes ing of the transcript of their own gene. No other ribosomal from mutating +2C+T, which would re-establish the left stem proteins have been shown to regulate the translationof their levof transla- own mRNA. However, the measurements to establish such (Fig. 2 B ) and leads to the most extensive inhibition els of regulation areoften difficult,requiring both the accurate tion. of Our working hypothesis, derived from Ref. 2 and from the determination of mRNA concentration and the quantitation labeling of a protein with a very short lifetime. In the caseof data reported above, is that L32 can bind to the structures shown in Fig. 2, stabilizing them to prevent the assembly of L32, the binding of the protein appears to stabilize both the unspliced transcript (2) and the spliced mRNA (Table 11). The spliceosomes or of a translation initiation complex. L32 mRNA Produced in Excess Is Not Engaged in the Poly- RNA is therefore apparent. If unspliceable or untranslatable somes-% determine the distributionof mRNA for L32 in cells RNA were rapidly degraded, it would be nearly impossible to overexpressing L32, we analyzed cell extracts on a 7 4 7 % su- determine whether that were due to some feedback effect. Inof genes withina cell is crose gradient (Fig. 3 A ) . An aliquot of each fraction was then deed, in experiments where the number fractionated on a native RNPi gel (see “Materials and Meth- artificially elevated, e.g. through a multicopy plasmid, rarely ods’’) that was blotted to a Nylon filter and subsequently hy- does the level of mRNA match the numberof additional genes bridized with a probe specific for mature mRNA, i.e. comple- (24-27, 37). Therefore it remains to be seen whether thephenomena described for L32 are representativeof the regulation mentary to the junctionof the two exons (Fig. 3, B and C ) . From comparison of panels B and C, it is evident that in cells of many other ribosomal proteins. A keypoint that we have been unable to concerns address the overexpressing RPL32mRNA its distribution differs from that in normal cells in two respects (Table IV). On the one hand, intracellular location of the untranslated mRNA. It is exceedmore than 30% is in small RNP of