Stability of RNA in developing Xenopus embryos and ... · RNA stability in Xenopus embryos 839 analogue cordycepin (3' dATP) to the reactions. The addition of cordycepin alone to

Development 102, 837-852 (1988)Printed in Great Britain © The Company of Biologists Limited 1988

837

Stability of RNA in developing Xenopus embryos and identification of a

destabilizing sequence in TFIIIA messenger RNA

RICHARD HARLAND and LYNDA MISHER

Department of Molecular Biology, University of California, Berkeley, CA 94720, USA

Summary

Synthetic capped RNA transcripts injected into ferti-lized eggs of Xenopus laevis have a half-life of 3—4 h.Addition of a long (~200 nucleotide) poly(A) tailincreases the half-life to 6-8 h which approaches thehalf-life of natural polyadenylated globin RNAinjected into embryos. Since exonucleolytic actionalone could account for the degradation of RNA, wetested whether circular RNA is stable after injectionand find that circles are exceptionally stable (half-lifegreater than 40 h). After the mid blast ula transition,polyadenylated chloramphenicol transferase (CAT)mRNAs transcribed from injected plasmids have ahalf-life of 2 5h. Insertion of a 1000 nucleotide 3'

untranslated region from the Xhox-36 gene into thetranscripts does not affect the half-life. In contrast tothe finding that internal sequences do not affectstability, we find that sequences from the TFIIIAmessage reduce the half-life of CAT mRNA from 2 5 hto less than 30 min. We conclude that most RNAs aredegraded exonucleolytically from the 3' end, butspecialized internal sequences can greatly destabilizethe RNA, possibly by acting as a site for an endonu-clease.

Key words: RNA stability, Xenopus development,TFIIIA, poly(A).

Introduction

The regulation of mRNA stability plays an importantpart in the control of gene expression; the numerousexamples where stability is controlled have beenrecently reviewed (Shapiro et al. 1987). It is ofparticular interest to us to identify mechanisms thatcontrol RNA stability in the developing Xenopusembryo, not only in general, but also in special caseswhere stability of individual RNAs is controlled.

A mechanism for differential RNA stability mustbe in effect throughout early Xenopus developmentsince there are cases of RNAs that were stable in theoocyte being degraded either early in the blastula(King et al. 1986) or at different times around thebeginning of gastrulation (Rebagliati et al. 1985;Dworkin et al. 1985). In these examples, total RNAwas examined, thus excluding the possibility thatdeadenylation led to apparent absence of mRNA onthe basis of oligo(dT) selectability. In Drosophila, ithas been proposed that one mechanism for thegeneration of asymmetry in the homogeneously dis-tributed caudal (cad) mRNA is differential RNA

stability (Macdonald & Struhl, 1986; Mlodzik &Gehring, 1987).

The possibility that differential RNA stability isimportant to regional specification in the amphibianembryo is also attractive. Although prelocalizedRNAs exist along the animal-vegetal axis (Rebagliatietal. 1985; Melton, 1987; Weeks & Melton, 1987), thecapacity for dorsal and ventral development is notprelocalized in the radially symmetrical amphibianegg (reviewed by Gerhart & Keller, 1986). Thetransduction of early cytoplasmic reorganization andinductive interactions into changes in gene expressionmay well involve the differential stabilization ofmaternal RNA in different regions of the egg.

Despite the work that has been done, we still donot understand what RNA sequences and nucleolyticenzymes control RNA stability in Xenopus oocytesand embryos. Interpretations from earlier work,employing natural RNAs, stressed the importance ofa poly(A) tail for stability in oocytes (reviewed byLittauer & Soreq, 1982; Nevins, 1983) though in somecases a fraction of nonadenylated RNA was known tobe stable for an extended period (Woodland & Wilt,

838 R. Harland and L. Misher

1980). Stability of RNA has also been related to itspresence on polysomes (Allende et al. 1974; Richter& Smith, 1981; Audet et al. 1987). More recently,well-defined transcripts synthesized from bacterio-phage SP6 and T7 promoters have been used toaddress questions of stability and translatability. Aconsensus is emerging from these experiments thatnonadenylated RNAs are much more stable inoocytes than formerly believed (Harland & Wein-traub, 1985; Drummond et al. 1985; Kruys et al.1987). Even antisense RNAs, which are poorly trans-lated, are much more stable in oocytes than wouldhave been expected from earlier work (Harland &Weintraub, 1985). Furthermore, a poly(A) tail is notabsolutely required for translation of the RNA (Har-land & Weintraub, 1985; Kruys et al. 1987) though itdoes have an effect on long-term stability and theefficiency of translation of at least some messengerRNAs in oocytes (Drummond et al. 1985).

In this paper, we confine our experiments todeveloping Xenopus embryos. The metabolism ofRNA in the egg is in many respects different fromoocytes; changes in adenylation of endogenous RNAsoccur during maturation (see discussion in Dworkin& Dworkin-Rastl, 1985) and a double-stranded RNAunwinding activity appears (Bass & Weintraub, 1987;Rebagliati & Melton, 1987). There is now a clearconsensus that synthetic RNA injected into embryosis less stable than in oocytes (Rebagliati & Melton,1987; Colman & Drummond, 1986; this work) thoughprevious experiments had suggested that injectedglobin RNA may be stable for several days asmonitored by the continual translation of globinprotein (Gurdon et al. 1974). The role of the poly (A)tail is not clear in embryos and at least one group hasfailed to find any effect on stability (Rebagliati &Melton, 1987).

We have used injection and direct quantification ofRNA to address the effect of short and long poly(A)tails on the stability of RNA. To address the relativeimportance of exo- and endonucleases to degradationwe have injected RNA with no ends; this circularRNA was prepared by exploiting the self-splicingproperties of the Tetrahymena pre-rRNA intron(Been & Cech, 1986).

Our conclusions from RNA injection experimentshave been extended by experiments in which weinjected plasmids encoding defined polyadenylatedtranscripts. We have measured the stability of thesetranscripts and tested whether the presence of a long3' untranslated sequence destabilizes the RNA. Tocomplement our results, which suggest that endonu-cleases are relatively unimportant in degrading mostRNAs, we have asked what is the effect of a sequencefrom an unstable RNA when inserted into another

transcription unit. These experiments identify a se-quence in the TFIIIA mRNA (Ginsberg et al. 1984)which destabilizes RNA and may act as a target for asite-specific endonuclease.

Materials and methods

(A) Plasmid construction and RNA preparationPlasmids with poly(dA) • poly(dT) tracts

In principle, the simplest way to synthesize polyadenylatedRNA is to encode the poly(A) tail in the transcriptiontemplate. We therefore prepared plasmids that could betranscribed to make chloramphenicol acetyl transferase(CAT) mRNA, but which contained a poly(dA) • poly(dT)tract 3' of the CAT coding sequences. This was done bypreparing a CAT fragment with a poly(dA) tail and aplasmid fragment with a poly(dT) tail. The non-tailed endof both fragments was generated by restriction enzymedigestion.

In order to prepare the appropriate DNA fragments, theplasmid pSP65CATS (Harland & Weintraub, 1985) wasdigested with Xbal (3' of the CAT gene relative to the SP6promoter) and this site was filled in with reverse transcrip-tase. The sample was split into two aliquots for tailing withdATP or dTTP and terminal transferase. The reaction wascontrolled by availability of nucleotide to yield tails oflength 100-400 nucleotides. The DNAs were then digested5' of the CAT insert with Sad and both the A-tailed CATfragment and T-tailed plasmid fragments purified from anagarose gel. Following selection of successfully tailed mol-ecules on oligo(dA)- or oligo(dT)-cellulose, the DNAswere annealed and ligated. Transformants were picked andDNA minipreps analysed for poly(A) tail length.

To measure the size of the poly(dA).poly(dT) insert,DNAs were digested on each side of the tail with HindU\and Xbal. The ends were labelled by filling in withradioactive nucleotides and the size of the fragmentsdetermined on a sequencing gel. Out of 24 colonies, 16contained plasmids with tails of greater than 50 nucleotides.The longest tails were of about 400 nucleotides, butconstituted a minority of a population from an individualcolony. Even clonal isolates of such lines derived byrestreaking or retransformation continued to yield hetero-geneous tails. A line with a reasonably homogeneous lengthof poly(A) (80 nucleotides) was amplified from the originalculture to yield a transcription template. Capped transcriptswere prepared as described (Harland & Weintraub, 1985)except that the ATP concentration in the reaction wasincreased to compensate for the increased adenylate con-tent of the RNA. To ensure that only polyadenylatedtranscripts were selected for injection, the RNA waspurified by chromatography on oligo(dT)-cellulose prior toinjection.

Enzymic addition of poly (A) tailsThe enzyme poly(A) polymerase (BRL) was used accord-ing to the manufacturer's instructions to yield RNA withpoly(A) tails of approximately 200 nucleotides. The lengthof the tails was controlled by adding the chain terminating

RNA stability in Xenopus embryos 839

analogue cordycepin (3' dATP) to the reactions. Theaddition of cordycepin alone to RNA has no effect onstability (unpublished results).

Preparation of circular RNAThe plasmid pBGST7 (Been & Cech, 1986) was used as atranscription template under standard conditions but in theabsence of the cap reagent (Harland & Weintraub, 1985).For splicing, the RNA was resuspended in 200mM-NaClwith GTP and MgCl2 and incubated at 42°C for lh(Sullivan & Cech, 1985). The identity of the differentspecies was confirmed by their migration on 4%, 6% or8 % polyacrylamide gels with respect to linear DNAmarkers. Circular RNAs have anomalous mobility underthese conditions. To prepare larger circles containing theCAT sequences, the CAT gene was isolated as a Bamfragment and inserted in both orientations in the BgH\ siteof the intron. In this case, capped transcripts were preparedand spliced prior to injection.

Plasmids with promotersThe plasmid containing a CAT gene and the SV40 promoter(EMSV CATS) has been described previously (Harland &Weintraub, 1985). For testing other promoters, we con-structed a new vector, PT-CAT, which is potentially moreversatile. This exploits the 'bluescript KS+' (Stratageneinc.) polylinker and plasmid. The CAT gene is present as aBamHl fragment and can therefore be removed easily aftera promoter has been tested. Multiple single-cut restrictionsites are present in the polylinker for insertion of promotersequences or cDNAs to be expressed. The SV40 latepolyadenylation signal is located downstream of the CATgene. Single-stranded DNA can be generated by superin-jection with M13 helper phage for confirmation of insertidentity and orientation by sequencing. As a primer forDNA sequencing, we used a synthetic oligonucleotidecomplementary to the region around the CAT gene ATGinitiator codon (gift of H. Weintraub). The derivativecontaining a heat-shock promoter is diagrammed inFig. 3A; the other promoter-containing plasmids are thesame except for the different promoter.

For all the promoters tested here, we inserted promoterfragments into a filled-in Xhol site upstream of the CATgene in order to leave an extensive choice of restriction sitesdownstream of the transcription initiation site. These plas-mids and other details are available on request. Concen-trations of DNA determined from optical density wereroutinely checked by gel analysis to confirm that contami-nating oligonucleotides were not contributing to apparentconcentration. We find that plasmids containing the CATgene, although resistant to chloramphenicol, respond par-ticularly well to amplification by chloramphenicol withyields of 2-10mg plasmid per litre of culture. The followingpromoters have been tested.

Heat-shock promoter. Plasmids were obtained from Mar-iann Bienz (Bienz, 1984) and Jay T'so (pHSTFlO). Apromoter fragment from a deletion endpoint at - 1 % to thePvuII site at +90 was used to make the plasmid HS-CAT.pHS TF10 was modified by digesting the plasmid with BglUwhich removes a part of the heat-shock 5' untranslated

sequence to position +29 (Bienz, 1984) and some of theTFIIIA sequence to position +343 (Ginsberg et al. 1984). ABamHl fragment coding for CAT was inserted in its place tomake the plasmid HS-CAT TF10. These plasmids arefurther diagrammed below in Fig. 3A.

L14 ribosomal protein promoter. A subclone of the L14gene (Bozzoni et al. 1984) containing the promoter and thepromoter sequence were generously provided by ElenaBeccari. A fragment from -880 to +12 was used togenerate L14 CAT. The LI promoter was also tested andhad similar properties (not shown); however, since the LIpromoter initiates at two well-separated positions andyields two distinct RNAs whereas the L14 promoter yieldsone RNA the L14 promoter was used for subsequentexperiments.

Xenopus borealis cytoskeletal actin promoter. The plasmidpSC9 and the sequence around the cap site were generouslyprovided by Gareth Cross and Hugh Woodland (Cross et al.1988). A fragment from the Hindlll site approximately1670 nucleotides upstream of the transcription start site toan Mspl site at +10 was initially tested. Subsequently, wefound that the fragment from the £coRI site (approx.—950) to the Mspl site gave identical transcriptional ef-ficiency. This fragment was used to make CSK-CAT.

(B) Embryo injectionsSome detail is given here since different workers find thatDNA injection yields quite different results with respect tothe amount of amplification of injected DNA.

Eggs were obtained and fertilized by standard pro-cedures. After dejellying in 2 % cysteine dissolved inlOOmM-NaCl, l-8mM-KCl, lmM-MgCl2, 2mM-CaCl2,adjusted to pH8 with NaOH, the embryos were rinsed in4MR (modified Ringer's lOOmM-NaCl, 1-8 mM-KCl, 1 ITIM-MgCl2, 2mM-CaCl2 buffered to pH6-9 with 5mM-Hepes).Batches of 20-60 embryos were transferred to Ficoll(2-5 % Pharmacia) in iMR in plastic dishes to which aNitex grid (lOOO r̂n) had been fixed and just prior toinjection excess buffer was removed. Injection apparatusand needles are described by Gurdon (1974) except that aNarishige MM3 manipulator was used to minimize lateraltearing motion. The surface tension of buffer above theeggs was used to hold the eggs in the grid during injection.For embryo injection, it is important to minimize theinjection volume. The needles that make this possible aresufficiently fine that microforging of the tip is not necessary.5-10nl of solution containing 50-100pg DNA (exceptwhere noted) or up to 20 ng RNA was injected at any timefrom 60min postfertilization (at 22°C) to the 4-cell stage.We have also injected DNA during the first hour afterfertilization (see also Rusconi & Schaffner, 1981; seebelow) and find similar results except that survival of theeggs is reduced. Injection volume was measured as de-scribed by Gurdon (1974). In any one experiment, eggs ofsimilar stage were injected. To provide eggs of differentstages, dishes of dejellied eggs were placed on a largealuminium plate at one end of which was a tray of ice. Thisprovides a temperature gradient over the range 15-22°C.Eggs were injected in a variety of positions, though some


care was taken to avoid known positions of nuclei. Slightpositive pressure was maintained in the needle so thatcontinuous flow of solution occurred after penetration ofthe embryo.

After injection, the dish was refilled with §MR andembryos allowed to develop at either 15°C, 19°C or roomtemperature to enable harvesting of the appropriate stages.Control experiments showed that temperature did notaffect the half-life of RNA with respect to developmentalstage. Throughout this paper, times of incubation havebeen normalized to 23°C in order to simplify comparisonwith Nieuwkoop & Faber (1967) stages.

In the case of DNA injections, we found that the amountof DNA remains approximately constant until it graduallydeclines in the tailbud stage. This result is like that of Krieg& Melton (1985) but quite different to that of others, whohave found amplification of DNA up to the gastrula stagefollowed by degradation of the DNA (Bendig, 1981;Rusconi & Schaffner, 1981; Etkin el al. 1984; Wilson el al.1986). We have deliberately tried to achieve amplificationof injected DNA by following the injection protocols ofSchaffner and colleagues, but so far have not seen grossamplification. However, the amount of DNA injected iscritical to amplification (Rusconi & Schaffner, 1981) and wealways find such doses to be toxic even with highly purifiedDNA. With all of the promoters tested, we found nodifference in transcription efficiency between circular andlinear injected templates (not shown) though we didconfirm that linear DNA is always ligated into long concat-emers in eggs (Harland, 1980; Rusconi & Schaffner, 1981;Bendig, 1981).

(C) RNA preparationBatches of five embryos were homogenized with a Pipet-man in 0-5 ml of 1 % SDS, 20 mM-Tris-HCI pH 7-5,100 mM-NaCI and 30mM-EDTA prior to freezing at -80°C. Em-bryos were thawed at 37°C and supplemented with 0-2 mlproteinase K in the above buffer (250/zgmP1 final). Afterdigestion at 37°C for 30min, the tubes were mildly soni-cated in a cup horn attachment of a Bronwill sonicator. Thisshears the DNA to ease extraction steps. The embryos wereextracted once with aqueous phenol, and nucleic acids wereprecipitated by addition of sodium acetate (pH 5-5) to 0-3 Mand an equal volume of isopropanol. The precipitate wasresuspended in 50/J\ diethyl-pyrocarbonate-treated waterand reprecipitated by addition of 25^1 10M-LiCl for 1 h onice. The RNA pellet was washed with 80% ethanol andresuspended in water. In some cases, the supernatant wassaved and reprecipitated with ethanol for analysis of DNAby Southern blotting. Northern blotting was done asdescribed (Condie & Harland, 1987) using an RNA probegenerated from pSP65CATA (Harland & Weintraub, 1985).Direct autoradiographs were scanned to quantify CATRNA. Control experiments in which known amounts ofsynthetic CAT RNA were mixed with oocyte RNA andsubjected to the above procedure show that this method isquantitative over a range of 100 fg-1 ng CAT RNA. In somecases, bands were excised from the filter and counteddirectly. For removal of the background (as seen in Fig. 3),blots were incubated at room temperature in 2xSSC withl//gml"' RNAse A and 1 unit ml"1 RNAse T! prior to

rinsing and stringent washing to yield clean blots (seeFig. 4).

Radioactive RNAs were visualized by fluorography offixed and dried gels (Harland & Weintraub, 1985). Forquantification, direct autoradiogTaphs were scanned with adensitometer. For these experiments, the amount ofpPJRNA injected into each sample of embryos wasdetermined by Cerenkov counting of the homogenate. Wefind that even though RNA may be degraded in the embryothe radioactivity is not lost and is eventually recycled intonew embryo RNA and DNA. Such quantification revealedsmall variations in the amount of RNA injected. Thesamples loaded onto the gels were adjusted to reflect thesame original amount of radioactivity in the homogenate.

Results

Stability of injected adenylated or nonadenylatedRNA

We wished to assay the stability of various RNAsin the developing Xenopus embryo. Accordingly,capped RNA was synthesized from templates con-taining an SP6 promoter (Harland & Weintraub,1985). The transcripts were radiolabelled during syn-thesis by including 32P-GTP in the reaction mixture sothat their fates could be monitored easily afterinjection into fertilized eggs. After incubation forvarious times the embryos were lysed and RNApurified for electrophoresis on denaturing agarosegels followed by autoradiography. We believe it to beimportant to use such a direct assay for full-lengthRNA since occasional nicks in the starting RNA maynot show up in a nuclease protection assay andthereby would cause difficulties in interpreting exper-iments on stability. The use of a denaturing gel assaymonitors both the amount and the size of the RNA.

The results of an experiment to determine thestability of various mRNAs are presented in Fig. 1. Inaggrement with others, we find that transcripts have amuch shorter half-life in embryos than in oocytes(Colman & Drummond, 1986; Melton & Rebagliati,1986; Bass & Weintraub, 1987). Whereas a transcriptcoding for CAT (chlormaphenicol acetyl transferase)had a half-life of greater than 12 h in oocytes (Harland& Weintraub, 1985), the experiment presented inFig. IB shows that the same transcript is compara-tively short lived in embryos. The synthetic mRNAinjected into fertilized eggs is no longer detectable bythe end of gastrulation. From quantification of theautoradiograph, we estimate the initial half-life of theRNA as 3-4 h.

Gurdon et al. (1974) reported that mammalianglobin RNA is stable after injection. The assayavailable at the time was based on the production oflabelled globin protein at intervals following injec-tion. We examined the stability of natural frog a-globin RNA by a more direct Northern blotting


hours 1-5 2-5 4 7 17 27

ir-

5r

A reticulocyteRNAn-globin

SP6 TRANSCRIPTS

B CAT

C CAT (oligo(A))

D CAT (poly(A))

E human/^-globin(oligo(A))

Stage 2 4 7 9 15 25

Fig. 1. Stability of injected RNA. 10-20 ng RNA wasinjected into embryos at the 2-cell stage. Embryos werehomogenized at the times indicated, RNA extracted andfractionated on denaturing agarose gels. Times are givenas hours after fertilization; the first time point was takenless than 5min after injection. (A) Frog reticulocytemRNA selected by oligo(dT)-cellulose chromatographywas injected into embryos. Adult a--globin RNA wasdetected by hybridizing a Northern blot with an RNAprobe complementary to adult frog cr-globin.(B) Synthetic capped RNA made from pSP65 CAT S wasinjected into embryos and detected by autoradiography.(C) Synthetic RNA made from a template with ahomopolymer tract encoding a poly(A) tail of 80nucleotides. (D) Polyadenylated CAT RNA; the sameRNA as B, but containing an enzymically added poly(A)tail of about 200 nucleotides. (E) Synthetic human /S-globin RNA with RNA with a poly(A) tail of 60nucleotides.

method (Fig. 1A). Essentially we have confirmedthat globin RNA is fairly stable after injection,though with a half-life of 10-15 h rather than greaterthan 8 days as reported by Gurdon et al. (1974). Thediscrepancy is probably due to the assay. Since thepolysome content of early embryos is low (Wood-land, 1974), RNAs may be in competition for trans-lation (Laskey et al. 1977; Richter & Smith, 1981);later in development, polysome content is high andthe competition may not be so severe. Therefore,

early estimates of mRNA amount based on trans-lation are likely to be low and the apparent half-lifewill be overestimated. A second possibility is that asmall proportion of globin RNA was recruited ontopolysomes and thereby stabilized (Richter & Smith,1981; Audet et al. 1987); in this case, the apparenthalf-life of mRNA as judged by the translationproduct would be considerably longer than the half-life of total injected RNA.

In any case, natural globin RNA is considerablymore stable than the CAT transcript described above.The difference in stability could be due to thesequence of the RNA or the difference in polyadenyl-ation. We therefore examined the effect of differentpoly(A) length on CAT RNA stability. Initially wehoped that a poly(A) tail could be encoded in thetranscription template, so we obtained a humanglobin cDNA clone with a long poly(dA) tract (Lang& Spritz, 1985; Lang et al. 1985) and, in addition,constructed plasmids with long poly(dA) tracts. Amajor problem with this approach, however, was theinstability of such long homopolymer tracts duringpropagation in bacterial plasmids. The globinpoly(A) tract was originally reported to be 225nucleotides in length (Lang & Spritz, 1985) but wefound that it had stabilized at approximately 65nucleotides. Furthermore, the new plasmids we con-structed had unstable homopolymer tracts. Althoughmany of the original clonal isolates had homopolymertracts of greater than 100 nucleotides, the size usuallystabilized at 20-30 nucleotides (see Materials andmethods for details). For experiments reported here,we used an isolate in which the bulk of the populationof plasmids still had homopolymer tracts of about 80nucleotides. As is evident from Fig. 1C, the tran-scripts synthesized from such templates and contain-ing a short poly(A) tail are not significantly morestable than nonadenylated RNA.

Because of the limitations of poly(A) length thatcould be encoded in the transcription template, weresorted to enzymic addition of poly(A) tails to thetranscripts using poly(A) polymerase after transcrip-tion. Although the tails added were of variable lengthwe found that long tails of 200-300 nucleotides couldbe added. The main difficulty we encountered waspartial degradation of the RNA in some reactions aswas also observed by Drummond et al. (1986).Extensively degraded preparations were discardedsince the analysis of such RNAs is complex. Fig. IDshows the analysis of RNA with a tail of 200 nucleo-tides. We find that a long poly(A) tail stabilizes thetranscript approximately twofold to yield a half-life of6-8 h. In all other respects, this transcript is identicalto the transcript tested in Fig. IB; therefore, theincrease in stability must be due to the increasedlength of poly(A). Although a doubling of half-life


does not appear dramatic, it is developmentallysignificant; instead of being completely degraded bythe end of gastrulation a significant proportion of theRNA survives through neurulation.

Given that a long poly(A) tail can stabilize CATRNA, it is possible that the stability of injected globinRNA could be accounted for solely by the length ofits poly(A) tail. However, this experiment does notrule out a contribution of globin primary sequence tostability. We therefore synthesized a globin transcriptwith a short poly(A) tail encoded in the template(Lang et at. 1985). In three out of four experiments,this transcript was no more stable than nonadenylatedCAT RNA (Fig. IE). We conclude that the globinprimary sequence does not add greatly to its stability.(This conclusion is supported by experiments pre-sented below which show that endonucleases do notcontribute to the instability of most RNAs).Although three out of four experiments showed that

synthetic globin RNA was no more stable than CATRNA, in one experiment (not shown here) thetranscript was much more stable than CAT RNAinjected into the same embryos and was as stable asnatural globin RNA (surviving until the tailbudstage). This result raises the untidy possibility thatdifferent batches of embryos have different degra-dative activities on poly(A) tails; in this isolated case,the short tail of 65 A residues may have beensufficient to stabilize the globin RNA. Our generalconclusion, however, is that a longer poly(A) tail isrequired to stabilize injected RNA.

One further observation from the gel analysis isthat the polyadenylated RNA starts as a fairly hetero-geneous population but the size distribution sharpensand the size decreases with time. This has beenobserved before, most clearly with transcripts that aretransiently expressed (see for example Restifo &Guild, 1986). The fact that the size diminution does

Minutes

45 90

Hours

5 17

rRNA

Circularintron

Linearintron

Ligatedexon

Unligatedexon

404

309

242

Fig. 2. Stability of Tetrahymena rRNA introncircles and linears. (A) Autoradiograph of a 4%polyacrylamide gel showing the analysis ofradioactive circular and linear RNA injected intoembryos and harvested at the indicated times. Inthis experiment, the RNA was not capped. Theexon fragments show the stability characteristicof uncapped RNA. The circular species is themost stable, but the linear intron is more stablethan expected for uncapped RNA. Theradioactive nucleotide released from degradedRNA is reincorporated into RNA and DNA.The position of rRNA in this gel is indicated.Size markers are pBR322 Mspl fragments.(B) Plot of stability of synthetic TetrahymenaRNAs containing the CAT sequence. Theexperiment is similar to that shown in A, butwas quantified by densitometric scanning of gellanes. In this experiment, splicing is less efficientthan in A because of the CAT insert in theintron. The RNA was capped during synthesis(though only the unspliced linear remainscapped). RNA amount is in arbitrary units. Forplotting the lines decay kinetics were assumed tobe exponential. No detectable unspliced linearRNA was detectable after 12 h.


not continue below the size of full-length CAT RNAsuggests that this may be due to slow deadenylationfollowed by rapid degradation of the nonadenylatedspecies. Evidence will be presented below that thisprocess is fairly synchronous on the members of anRNA population.

Circular RNA is stable in embryosThe finding that a cap at the 5' end and a poly(A) tailat the 3' end increase the stability of RNA raises thequestion of whether cellular RNA is in generaldegraded by endonucleases, or whether all degra-dation can be accounted for by exonucleases. Asimple test of this is to use RNA with no termini,namely circular RNA. Circular RNA was made byexploiting the self splicing pre-rRNA intron of Tetra-hymena thermophila (Been & Cech, 1986). Tran-scripts including the intron will self splice in vitro inthe presence of GTP and Mg2+; subsequently thelinear intron will circularize. The experiment shownin Fig. 2A shows that, indeed, the circular RNA wasextremely stable after injection. The simple con-clusion that this is due to absence of termini wascomplicated by the observation that the linear formwas somewhat stable, even though it contains nocapped 5' end. We suspect that the stability isanalogous to that of tRNA and is due to the extensivesecondary structure that the intron adopts (Price et al.1985). In contrast, the linear exon fragments werequickly degraded, as expected for uncapped linearRNA.

To test the stability of a less-structured region ofRNA within a circle we included the CAT codingsequences in the pre-rRNA intron. This was achievedby cloning the CAT fragment in the BgHl site of theTetrahymena pre-rRNA template. Transcripts of thenew template self splice at reduced efficiency, pre-sumably because the large CAT insert interferes withthe formation of secondary structure necessary to theintron's enzymic activity; nevertheless, sufficientcircles can be generated to test stability. We find thatsuch large circles are stable in embryos, whereas allthe linear species are progressively degraded. Densi-tometric quantification from the gel is presented inFig. 2B. For simplicity, we only present the quantifi-cation of circle and unspliced linear. The unsplicedmolecule, which in this case was capped, has stabilityexpected for a capped but nonadenylated RNA (cf.Fig. 1). The spliced linear (not shown) is intermediatein stability between the circle and unspliced linear,again suggesting that the ends may be protected bysecondary structure of the whole intron. The circularform is extremely stable, with an estimated half-life of40 h. Even this measurable rate of decay of the circlecould be accounted for by slow back reaction of thecircle to a linear form (Sullivan & Cech, 1985) and

instability of this linear form. It is possible, therefore,that no endonucleolytic degradation of circular RNAoccurs at all.

The main result of this experiment is that circularRNA is extremely stable after injection. We takethese results as strong evidence that nonspecificendonucleases do not degrade cellular RNA andsuggest that the primary route of instability is throughdeadenylation followed by 3' exonucleolytic degra-dation.

Use of injected DNA to measure RNA stabilityA different way of testing RNA stability, and onewhich may be considered more physiological, is togenerate transcripts in the embryo from an injectedDNA template. If the promoter in the DNA isinducible, or only transiently active, a pulse ofdefined RNA can be generated whose decay kineticscan be measured. In all the following cases, thetranscribed sequence included a marker CAT geneand the SV40 late polyadenylation signal. The SV40late polyadenylation signal had previously beenshown to function well in oocytes to produce poly-adenylated RNA (Wickens & Gurdon, 1983), and wehave confirmed that it functions well in embryos. TheCAT gene provides a marker for quantitative detec-tion of RNA by Northern blotting. In repeatedreconstruction experiments, we find that CAT RNAyields quantifiable data over the range from 100 fg toIng, a 10000-fold range.

We have tested a variety of promoters for theiractivity in injected embryos and an example of theresults is presented in Fig. 3. The tests were donewith transcription templates similar to HS CAT(shown in Fig. 3A); different promoters wereinserted upstream of CAT as described in Materialsand methods. A more quantitative analysis of tran-scription results is given in Table 1. We estimate thetime of activity of promoters as those stages when thelongest species of RNA is being produced (beforedeadenylation occurs). We have also analysed theamount of DNA in the embryos at different stages bySouthern blotting (not shown). Our results resemblethose of Krieg & Melton (1985), who found thatinjected DNA did not amplify to a great extent, butpersisted into late tailbud stages. The inactivation ofthe promoters at different times cannot therefore beattributed to the gross absence of DNA.

Graves et al. (1985) had previously shown that themurine sarcoma virus (MSV) LTR promoter wasactive in injected oocytes and we expected it to beconstitutively active at all stages of development.Surprisingly, however, it is only transiently active inembryos from the midblastula transition until gastru-lation (Fig. 3B, lanes 9-11; Table 1). The LI and L14ribosomal protein gene promoters are active from the


HS CAT 1077 bases pHSCATTFlO 2000 bases^AAAAAAAA

heat-shockpromoter

B

CAT

SP6RNA

SV40poly(A)signal

con PT-cat MSV L14 CSK

TFIIIA

HS

1 2 3 4 5 6 7 8 9 10 1 1 1 2 13 1 4 - 1 5 1 6 1 7 1 8 1 9 2 0

18S • - . « • • • -

CATRNA

CAT +TF10

Fig. 3. (A) Examples of the transcription units in the injected plasmids. HS CAT is derived by insertion of the heat-shock promoter (from —196 to +90) into the Xhol site of PT-CAT. This plasmid encodes a transcript (excludingpoly(A)) of 1077 nucleotides; the transcript is represented by the wavy line in the figure. Other promoters were testedin a similar template, the heat-shock promoter being replaced by MSV (-381 to +30); L14 (-880 to +12); cytoskeletalactin (-950 to +10). The plasmid pHS CAT TF10 was derived by inserting the CAT gene into Bg/II-digested pHSTFlO.This plasmid retains TFIIIA sequences from nucleotide 343 of the sequence of Ginsberg et al. (1984) to a residualpoly(A) tract in the clone of T'so et al. (1986) at position 1330. The predicted transcript size is 2000 nucleotides(excluding poly(A)). (B) Activity of different promoters in injected embryos at stage 10i (early gastrula), stage 14(neurula) and stage 28 (tailbud tadpole). Autoradiograph of a Northern blot to show CAT RNA expressed frominjected plasmids. This exposure is from a nonstringently washed filter to show the positions of ribosomal RNAs and asmaller species which is often seen as background in blots hybridized with RNA probes; the background shows thatchanges in size of the CAT RNAs are not due to aberrant migration of RNA throughout the gel lane. Lanes 3, 6, 9, 12,15 and 18 are from early gastrula (stage 10i), lanes 4, 7, 10, 13, 16 and 19 are from neurula (stage 14), and lanes 5, 8,11, 14, 17, 20 are from tailbud tadpole (stage 28). Lane 1, 5pg CAT RNA mixed with oocyte RNA; lane 2, 0-5pg CATRNA + oocyte RNA; lanes 3-5, uninjected embryos; lanes 6-8, embryos injected with the PT-CAT vector; lanes 9-11,embryos injected with MSV promoter driving CAT expression; lanes 12-14, ribosomal protein L14 promoter;lanes 15-17, Xenopus borealis cytoskeletal actin promoter; lanes 18-20, a mixture of plasmids was injected, both usingthe heat-shock promoter. One was pHS CAT TF10 which has TFIIIA sequences and the SV40 polyadenylation sitedownstream of the CAT gene, the second was HS CAT. The position of RNA encoded by pHS CAT TF10 is indicatedas CAT + TF10. Embryos were cultured at 20°C except for embryos injected with heat-shock promoters, which wereheat shocked for 30min at 33°C prior to harvesting. One embryo equivalent was loaded in each lane, exposure was for1 day at -80°C with screens.

midblastula transition until neurula stages (Fig. 3,lanes 12-14; Table 1). The X. borealis cytoskeletalactin promoter is active until the latest times, beingon at low levels prior to gastrulation, but reaching apeak of activity from gastrulation until early tailbudstages (Fig. 3, lanes 15-17; Table 1).

The most useful promoter for studying RNA stab-ility is the Xenopus heat-shock promoter. Thisinjected promoter is inactive at 18°C, but is readilyinducible by heat shock in embryos, producing de-tectable transcripts at 25 °C and reaching maximalactivity at 33°C (not shown). Fig. 3 lanes 18-20 show

the inducibility of the promoter at different stages.RNA was harvested after 30min of heat shock at theearly gastrula, neurula or tailbud stages. The injectedpromoter is inducible from the midblastula transitiononward, but its induced activity declines from theneurula onward such that it is only weakly induced inthe tailbud tadpole (Fig. 3, lanes 18-20; Table 1). Theamount of transcript made in a 30min heat shock isrelated to the ratio of DNAs injected; in this casepHS CAT TF10 was injected in excess over HS CAT.

We have done most experiments with the heat-shock promoter and have determined that the half-


Promoter

Table 1.

DNA perembryo

(Pg)

Stage and dose

4-cell

dependence

8-9blastula

of promoterStage

104-11gastrula

efficiency

20neurula

34heartbeat

Key:

MSV

Heat shock

L14

Cytoskeletalactin

100020040

100020040

1000200

40

1000200

40

000

000

000

000

dead

+ 0

+ + + + dead

+ + + dead

+ + + dead

not detected in 15 h exposuredetectable but less than 1 pg

1-5 pg5-25 pg

25-125 pg125-625 pg

dead0

dead0

dead0

dead0

Batches of 40 embryos were injected with the amounts of DNA indicated in a volume of 10 nl. Groups of embryos were harvested atthe indicated times and processed for Northern blotting. Amounts of RNA were estimated compared to a standard curve of syntheticCAT RNA serially diluted in oocyte RNA so that the same amount of total RNA was loaded in each lane. Abnormal embryos wereharvested as long as no signs of cytolysis were present. Embryos injected with 1 ng DNA always cleaved abnormally in the blastulastage. Such embryos do not develop beyond gastrulation. If fewer than five noncytolytic embryos remained the table shows 'dead'. Inour hands, only embryos injected with less than 100 pg DNA are viable indefinitely. The absolute amount of transcript produced by thepromoters is quite respectable when compared to an abundant mRNA like muscle actin, which produces 10-100pg RNA (lO'-lO8

transcripts, Mohun et al. 1984); the heat-shock promoter, which is the most active we have tested, makes up to 25 pg RNA in a 30minheat shock when injected at nontoxic levels. In this context, it is worth noting that the amount of RNA made appears to beproportional to the amount of template injected. In the case of the cardiac actin gene, Wilson et al. (1986) found that ten times moretranscript was made from the injected DNA (which amplified 10-fold) than was made from endogenous genes (i.e. 100-1000pg RNA).

life of the CAT/SV40 hybrid mRNA made from it isabout 3 h when the embryos are returned to 20 °C (seefor example Figs 4, 6 below). From experimentspresented below (Fig. 4B), it is also clear that thepromoter turns off rapidly on return to 20°C. Theheat-shock promoter can therefore be used to tran-scribe a discrete pulse of RNA at any time fromthe midblastula to tailbud stages. Within this range,the half-life of CAT mRNA is not affected by theembryonic stage at which the transcript is made (datanot shown). We do not believe that the heat-shockconditions used here affect RNA metabolism ad-versely since we have found that embryos can surviveextended heat shock at 33°C and splice globin tran-scripts made from injected plasmids normally at 33 °C(not shown).

All of the promoters, when first active, produce atranscript of narrow size range. When the promoter isno longer active, the size of the transcript declines.For the promoters that are active for the shortesttime, it is clear that the decline in RNA size issynchronous. For promoters that are active for longer

periods, such as L14 and cytoskeletal actin, the RNAband is broader (Fig. 3B, lanes 13 and 16). Weattribute this to the presence of transcripts of differ-ent age. The simplest interpretation of these results isthat a newly made transcript has a fairly constant-sized poly(A) tail added; this tail is then synchron-ously removed from all members of a population,leading to a decline in size related to age. Fromanalysis of a more detailed time course than thatpresented in Fig. 3B, we know that the difference insize between the longest and shortest transcriptsdetected can be accounted for by the removal of apoly(A) tail of 250 nucleotides (data not shown). Thisresult resembles that of others (Restifo & Guild,1986) and also that shown in Fig. ID where syntheticCAT RNA with a poly(A) tail declines in size afterinjection into embryos.

One notable feature of the decline in size of RNAsis that the decline does not continue beyond the sizeof nonadenylated RNA, rather the RNA is degradedcompletely. This observation highlights the import-ance of a poly(A) tail for RNA stability; once the tail


Atime afterheat

shock

min hours

0 30 75 3 6 12 20

B min hours

0 30 75 3 6 12 20

CAT+36-1

CAT

•

- i15 19 24 30

-*23-1

•*9-4

-* 6-6

«*4-4

^ 2 - 3-*20 •

«*1-35^1-08 • ••*0-87"*0-6

13

•m

15 19 24 30

CAT+TF10

CAT

Stage 13

Fig. 4. (A) A 1 kb untranslated region has no effect on stability. Northern blot analysis of RNA degradation duringrecovery from heat shock. A mixture of HS-CAT and HS-36CAT plasmids was injected into embryos. The embryoswere heat shocked at the end of gastrulation for 30 min, then returned to 18°C. Groups of five were sampled at theintervals indicated. (B) Sequences from TFIIIA RNA cause instability. As for A except that a mixture of HS-CAT andHS-CAT TF10 was injected into embryos.

is removed degradation is complete. In this paper, wedo not address any connection between the adenyl-ation state of an RNA and its translation. We doknow, however, that all these plasmids transcribetranslatable RNA by performing enzyme assay forCAT activity (not shown).

It is easiest to measure the stability of RNAproduced from the promoters that are most tran-siently active; however, it is clear that even for long-acting promoters that produce RNA of more hetero-geneous size the stability is of the same order.Although we cannot rule out the possibility that apromoter can affect RNA stability, perhaps by modi-fying the transcriptional machinery, the variety ofexamples reported here suggests that the possibility isnot generally true. Most importantly for the sub-sequent experiments, the heat-shock promoter doesnot encode RNA of unusual stability or instability(data presented below).

Long 3' untranslated regions do not affect RNAstabilityResults presented above show that a circular RNA isstable and we concluded that nonspecific endonu-cleases are not active in the embryo even though theRNA is not translated. Another way of testing thispossibility is to examine the stability of RNA with

different-sized 3' untranslated regions. This methoduses substrates with normal 5' and 3' ends, in contrastto the injection of circular RNA. In prokaryotes, it isthought that untranslated RNA is extremely sensitiveto endonucleases (Cannistraro et al. 1986); however,here we show that the presence of a 1 kb untranslatedregion has no effect on RNA stability. We have testedfour different sequences for their effect on RNAstability, the longest of which is from a Xenopushomeobox-containing gene, Xhox-36 (clone 36.1,Condie & Harland, 1987). This cDNA clone spansthe entire 3' untranslated region of 1000 nucleotides.A 1 kb fragment of the cDNA was inserted down-stream of the CAT gene in the heat-shock/CATplasmid. The new DNA construct was coinjected intoembryos with the unmodified CAT construct servingas a control. Because there is experimental variationin the amount of transcript made in different samplesthis control is important; it allows the comparison ofRNA amounts within a lane rather than betweenlanes. At various times after heat shock, RNA wasexamined by Northern blotting and the results arepresented in Fig. 4A. The RNA containing the Xhox-36 3' untranslated region is 1 kb larger than theoriginal CAT transcript and easily distinguished. Twotranscripts of intermediate size appear in the auto-radiograph; the size of these is consistent with poly-adenylation at two AAUAAA sequences that we


have identified in the Xhox-36 sequence. These RNAspecies have consistently been found to be slightlyless stable than the transcripts that terminate at theSV40 polyadenylation sequence but the reasons forthis have not been investigated further. Neither of theXhox-36 polyadenylation signals works quantitativelyin this context thus leaving an ample amount oftranscript which extends all the way to the SV40polyadenylation site. Data presented in Fig. 4A showthat transcripts either containing or lacking the Xhox-36 sequences and that terminate at the SV40 poly-adenylation site have equivalent stability. We there-fore conclude that the 3' untranslated sequence doesnot act as a target for degradation. Inspection of theDNA sequence (not shown) reveals that the Xhox-36

TFIIIA cDNA

3' untranslated sequence contains numerous stopcodons in all three reading frames; therefore, even ifribosomes were to reinitiate translation after the CATopen reading frame, translation would not proceedfar (see review by Kozak, 1986). We therefore assumethat this stretch of RNA cannot be protected fromendonucleases by ribosomes.

Although we initially considered that the Xhox-36transcript might be particularly unstable, it is clearfrom these results that the normal level of instabilityconferred by deadenylation and exonucleolyticdegradation is adequate to account for the disappear-ance of the transcript between the late neurala andtadpole stages (Condie & Harland, 1987).

half-life

pHSCATTFlO

SV40pA

AXba3

AXba4

HSCAT TFSau3A(sense)

<30min

<30min

<30min

<30min

HSCAT TFSau3A

(antisense)~ 3 h

Fig. 5. Plasmids used to locate destabilizing sequences. The TFIIIA cDNA clone used as starting point for theseconstructs is illustrated with relevant sites (Bg, flg/II; S, Sau3A; X, Xbal; B, BamHl). This sequence terminates at thepolyadenylation point identified by Tso et al. (1986) at nucleotide 1328 of the sequence of Ginsberg et al. (1984). Theplasmid pHSCATTFlO contains a heat-shock promoter, 800 nucleotides of CAT sequence and TFIIIA sequences fromnucleotide 342 to 1328. A short polylinker containing BamHl and Xbal sites precedes the late polyadenylation signal ofSV40. The TFIIIA sites shown are at 870 (Sau3A); 1052 and 1212 (Xbal). A 21-nucleotide stretch of AT containing twoATTTA motifs is present from 1088 to 1109. AXba3 and 4 remove sequences from 1212 to 1328 and 1052 to 1328,respectively. The HSCAT TFSau3A constructs were derived from HSCAT by inserting the Sau3A fragment frompHSCATTFlO (a SauSA fragment from 870 to the BamHl site in the linker between TFIIIA sequence and SV40sequence). The overlap of TFIIIA sequence between AXba4 and HSCAT TFSau3A is from nucleotide 870 to 1052.


103!-

hours

Fig. 6. Quantification of stability of RNA expressed frominjected plasmids. Densitometry of the films shown inFig. 4 was used to generate some of the data. Additionaldata came from other injection samples from the samebatch of embryos, using the plasmids diagrammed inFig. 5 coinjected with control HS CAT DNA. Lines weredrawn assuming that the RNA decayed exponentially.Open squares show results with RNA exprsesed fromHS-CAT, triangles show results from RNA expressedfrom HS-CAT TF10. Half-lives are 3h (CAT) and 15min(CATTF10).

The main conclusion of the experiments where thelength of 3' untranslated sequence is altered is thatnonspecific endoribonucleases are not active in theembryo, supporting the conclusions made earlierfrom experiments with circular RNA.

A sequence in TFIIIA mRNA that causes instabilityThe data presented so far suggest that only exonu-cleases act to degrade mRNA and that nonspecificendonucleases are not responsible. However, wehave not yet addressed the question of why sometranscripts are relatively unstable. We thereforetested a likely candidate sequence which may containa destabilizing sequence, in order to find out whethersuch a sequence may act as a site for a sequence-specific endonuclease. An example of a mRNA thatmay be particularly unstable is the mRNA coding forthe transcription factor TFIIIA. This maternal tran-script is degraded some time before the end ofgastrulation (Taylor et al. 1986). We found that, incontrast to other sequences, a model transcript-containing TFIIIA sequence is extremely unstablewhen made in the embryo. We inserted the CAT geneinto the plasmid pHSTFlO (gift of Jay T'so andLaurence Korn) and monitored the fate of the RNAafter injecting this plasmid with the control HS-CATplasmid into embryos. In contrast to the control CAT

mRNA, the RNA containing the TFIIIA sequenceshas a half-life of less than 30min, and in this particu-lar batch of embryos the RNA had a half-life of15min (Figs 4B, 6). The difference cannot simply bedue to size since we have already shown in Fig. 4Athat an extended 3' untranslated sequence does notaffect RNA stability. From the size of the RNAproduced by pHS CAT TF10, we can be sure thatpolyadenylation is occurring at the late SV40 poly-adenylation site. We can therefore rule out thepossibility that the unusual ATTAAA polyadenyl-ation signal of TFIIIA mRNA (T'so et al. 1986) isresponsible for the instability; rather, we concludethat the TFIIIA sequences affect stability when theyare internal in the RNA, possibly by acting as a targetfor a site-specific endoribonuclease.

An obvious candidate for the destabilizing se-quence in a 21-nucleotide stretch of AU whichcontains two repeats of the AUUUA motif identifiedby Shaw & Kamen (1986) in a variety of growth factorRNAs. In order to characterize the destabilizingsequence further, we constructed the plasmids shownin Fig. 5. Using the enzyme Xbal we deleted se-quences from pHSCATTFlO, and found that theRNA from both the deleted plasmids AXba3 andAXba4 was as unstable as RNA from the parentplasmid. Of these AXba4 deletes the 21-nucleotideAU sequence, showing that it is not required in theRNA to mediate instability. Furthermore we took a500-nucleotide Sau3A fragment from pHSTFlO andinserted it in both orientations downstream of theCAT gene in HS CAT. Only one of these plasmids, inwhich the TFIIIA sequences were inserted in thesense orientation, synthesized unstable RNA. Theoverlap of common sequence between AXba3 andHS CAT TFSau3A is only 180 nucleotides which iscontained within the protein-coding sequence forTFIIIA.

In order to obtain a more accurate time course ofdecay of RNA containing or lacking TFIIIA sequencewe quantified the data from the experiment describedabove, where the plasmids diagrammed in Fig. 5 wereassayed (the experiment shown in Fig. 4B is includedin the analysis). The combined results are presentedin Fig. 6. Assuming the RNA decays exponentially,this analysis yields a half-life for the CAT RNA of2-3 h at 23°C; in this batch of embryos inclusion ofTFIIIA sequences reduces the half-life to 15min.Whereas the half-life for CAT RNA is fairly consist-ent, the destabilizing sequence had a particularlydramatic effect in this batch of embryos, the moretypical half-life being closer to 30min.


Discussion

Contribution of polyadenylation to RNA stabilityWe have tested the stability of a variety of injectedRNA sequences and conclude that a long poly(A) tailof 200 nucleotides has a stabilizing effect on RNA inthe developing Xenopus embryo. Without a tail, theRNA has a half-life of 3-4 h and with a tail the half-life is doubled to 6—8h. Thus, if endogenous tran-scripts behave in the same way, we would expectnonadenylated RNAs to be degraded by the end ofgastrulation whereas adenylated transcripts wouldpersist at some level through neurulation. We do notknow to what extent polyadenylation is used by theembryo to effect changes in the stability of maternalmRNA. Clearly many transcripts undergo deadenyl-ation during early development (see discussion inDworkin & Dworkin-Rastl, 1985) but these changesmay not be sufficient to account for the differingstability of different maternal RNAs.

Poly(A)-binding proteins interact with stretches ofpoly(A) of greater than 30 nucleotides (Baer &Kornberg, J980). Poly(A) tails of greater than thislength might therefore be expected to have a stabiliz-ing effect as suggested by the injection of native,deadenylated and readenylated RNAs into oocytes(reviewed by Littauer & Soreq, 1982; Nevins, 1983).However, we find that poly(A) tails of 60-80 nucleo-tides encoded in the plasmid template do not reliablystabilize injected RNAs in embryos. Since we see thatthe poly(A) tails on injected RNAs are progressivelyremoved it may be that short tails are removedsufficiently rapidly that any short-term stabilization isnot seen in the assay. Alternatively, the stabilizingeffect may be cooperative and require severalpoly(A)-binding proteins to affect stability signifi-cantly.

Our results are consistent with results obtained intissue culture cells, where the drug cordycepin(3'dATP) was used to inhibit polyadenylation (Zeeviet al. 1982). Transport of RNA to the cytoplasm wasnot affected, but the half-life of RNA was markedlyreduced. The experiments reported here have theadvantage that the RNA was introduced into anormal, viable cellular environment so that we canexclude any indirect effects that a drug may have.

Stability of endogenously transcribed sequencesWe have tested the stability of RNA produced fromfour different promoters. We can estimate stability inall cases because the promoters are either inducible(heat shock), or only transiently active (MSV LTR,L14 and cytoskeletal actin). The clearest cases are forMSV or the heat-shock promoters which make a briefpulse of RNA. Decay of the transcripts follows a half-life of about 2-5 h (normalized to development at

23°C). We also see that the RNAs produced by theseplasmids are polyadenylated. When first made weestimate the poly(A) tail length to be approximately250 nucleotides; the length progressively declinesuntil the length of nonadenylated RNA is reached. Atthis time no further decrease in size is seen, rather theRNA disappears completely. We take this as support-ing evidence that a poly(A) tail is required forstability. The actin and L14 promoters are active forlonger than MSV and heat-shocked promoters and sodecay kinetics are best estimated when they turn off.We can estimate this time because RNA of greatestpoly(A) tail length is no longer present. We estimatethat these promoters produce RNA of equivalenthalf-life to RNA transcribed from the MSV or heat-shock promoters (Fig. 3; Table 1 and data notshown).

The half-life of these endogenously made RNAs isshorter than that of polyadenylated RNA injectedinto the egg even though its tail is about the same size(200-250 nucleotides). Since injected plasmids areonly expressed after the midblastula transition, it isdifficult to compare stability of injected RNA andRNA expressed from injected plasmids at the samedevelopmental times. The difference might reflect adifference in the metabolism of newly synthesizedRNA compared to RNA that has been in the cyto-plasm since early cleavage stages.

Stability of RNA to endoribonucleasesTwo kinds of experiments support the idea thatinternal sequences in RNA are not susceptible togeneral endonucleolytic cleavage. First, we find thatcircular RNA is quite stable in embryos even when itcontains CAT sequences that are normally degradedas linears (Fig. 2A,B). Second, when a long 3'untranslated sequence (from Xhox-36) is introducedinto a transcription unit driven by the heat-shockpromoter, it has no effect on stability (Fig. 4A).These results confirm that eukaryotic RNA stabiliz-ation is quite different from that of prokaryotes, inwhich untranslated RNA is exquisitely sensitive tonuclease (Cannistraro et al. 1986). Superficially theseresults would indicate that RNA stability would beregulated by the length of the poly(A) tail and thestability of the cap. We have argued that the poly(A)tail is subject to removal and we would thereforeexpect that no RNA would be more stable than any ofthe model CAT transcripts. However, this model isdifficult to reconcile with evidence that some RNAsare unusually stable. In many cases, RNAs arespecifically stabilized (reviewed by Shapiro et al.1987) but there are no clear cases of endogenousRNAs in developing Xenopus embryos that haveunusually high stability. However, inspection of thedata presented by Krieg & Melton (1985) would


suggest that a GS17/globin fusion transcript ex-pressed from an injected plasmid in unusually stable.Although the promoter is only active until gastru-lation, the transcripts persist until the tailbud stage; incontrast, we find that CAT transcripts from thesimilarly active MSV promoter are completely absentat the tailbud stage.

A destabilizing sequence in TFIIIA mRNAAn AU-rich sequence in the 3' untranslated region ofa growth factor RNA has a destabilizing effect onmRNA (Shaw & Kamen, 1986). We have found asimilarly acting sequence in a transcription factorRNA and assayed its activity in the developingembryo. The maternal mRNA, for TFIIIA, does notpersist beyond gastrulation and is thought to bespecifically targeted for degradation (Ginsberg et al.1984; Taylor et al. 1986). We have shown that aspecific mechanism exists to destabilize the RNA. It islikely that the destabilizing activity is induced duringembryogenesis, since the half-life of TFIIIA mRNAis much more than 30min in the oocyte and earlyembryo (Ginsberg et al. 1984; Taylor et al. 1986). Inthis respect, the destabilizing activity resembles thatfor GM-CSF, which is induced in cells by phytohae-magglutinin but not phorbol ester (Shaw & Kamen,1986).

By using the plasmid diagrammed in Fig. 5, wehave shown that a 23-nucleotide AU sequence similarto that shown by Shaw & Kamen (1986) to destabilizegrowth factor RNAs is not required in this example ofan unstable RNA. This sequence is in any casepolymorphic in Xenopus laevis TFIIIA genes and isabsent in the genomic sequence of T'so et al. (1986).From the series of plasmids we have tested (Fig. 5), itappears likely that a 180-nucleotide sequence inTFIIIA mRNA may contain all the informationnecessary to destabilize it. This 180-nucleotide se-quence is contained within the coding region forTFIIIA protein in the normal mRNA, though in theconstructs we have tested, the sequence is down-stream of the CAT open reading frame. We thereforewould expect the 180-nucleotide sequence to bepoorly translated, if, indeed, it is translated at all.Interestingly, a destabilizing sequence in tubulinmRNA is within a protein-coding region of themRNA (Gay etal. 1987). Experiments to characterizethe mechanism of destabilization further are cur-rently in progress.

We thank M. Bienz and J. T'so for heat-shock plasmids,G. Cross and H. Woodland for actin DNA and E. Beccarifor the L14 DNA. We thank C. James for help in construct-ing and testing pHS CAT TF10. We are grateful to ourcolleagues for their comments on the manuscript and inparticular to J. Gerhart and F. Wilt. This work wassupported by grants from the NIH.

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(Accepted 5 January 1988)

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