Improved translation efficiency of injected mRNA during early embryonic development

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<ul><li><p>TECHNIQUES</p><p>Improved Translation Efciency of InjectedmRNA During Early Embryonic DevelopmentMaria Fink, Gabriele Flekna, Alfred Ludwig, Thomas Heimbucher, and Thomas Czerny*</p><p>Injection techniques are a powerful approach to study gene function in sh and frog model systems. Inparticular, in vitro transcribed mRNA is broadly used for such misexpression experiments. Sequenceelements anking the coding region, such as untranslated repeats and polyadenylation sequences, areknown to affect the stability and the translation efciency of mRNA. Here we show that in early embryos,poly(A) signals strongly contribute to the activity of the injected mRNA. Of interest, they only marginallyaffect mRNA stability, whereas the translation efciency is dramatically enhanced. Combination of apoly(A) tail and an SV40 late poly(A) signal leads to highly synergistic effects of the two elements forinjected mRNA. Compared with established vector systems, we detected a 20-fold improvement for mRNAderived from the novel transcription vector pMC. Developmental Dynamics 235:33703378, 2006. 2006 Wiley-Liss, Inc.</p><p>Key words: mRNA injection; UTR; -globin; SV40 late poly(A); T7TS; pCS2; pMC</p><p>Accepted 18 September 2006</p><p>INTRODUCTION</p><p>Ectopic gene expression is an impor-tant method to study gene function,complementing the analysis of mu-tants. It allows investigators to testfor sufciency of gene action and,through the application of antimor-phic molecules, also for loss-of-func-tion experiments. In sh and frogmodel systems, transient misexpres-sion techniques based on microinjec-tion have been developed. mRNA in-jected at the one or two-cell stage isreadily distributed and translatedwithin the embryo and allows the ex-pression of products even before mid-blastula transition (Vize et al., 1991;Hyatt and Ekker, 1999). This tech-nique is, therefore, ideal to analyze</p><p>effects of misexpression on early de-velopment of the embryos (Harlandand Misher, 1988).</p><p>The stability and the translationefciency of mRNA represents animportant control of gene expression(reviewed in Wilusz and Wilusz,2004). During processing, a 7-methylguanosine cap-structure and a poly(A)tail are added to the transcript in thenucleus. After nuclear export, themRNA molecule is present in the cy-toplasm as a circular complex, due tointeractions between poly(A) bindingproteins and the elF4F-cap complex(Sachs et al., 1997; Mazumder et al.,2003). Bridging the mRNA termini en-hances the recruitment of new ribo-somal subunits by elF4F, resulting in</p><p>enhanced translation. In addition,this complex inhibits the action of thedecapping enzyme, which destabilizesmRNAs by removing the cap (Dehlinet al., 2000; Pickering and Willis,2005). Furthermore, poly(A) se-quences block mRNA exonucleases(Ford et al., 1997). The poly(A) tail,therefore, represents a key factor forboth mRNA stabilization as well astranslation efciency (de Moor et al.,2005). Cytoplasmic activities thatmodify the length of this tail, there-fore, critically inuence gene expres-sion. Both cytoplasmic deadenylationas well as cytoplasmic adenylation actupon mRNA molecules and are knownto be regulated during embryonic de-velopment (Huarte et al., 1992; Rich-</p><p>The Supplementary Material referred to in this article is available at of Animal Breeding and Genetics, University of Veterinary Medicine, Vienna, AustriaGrant sponsor: Austrian Science Fund (FWF); Grant number: P13138; Grant number: P15185.Dr. Fleknas present address is Institute of Meat Hygiene, Meat Technology and Food Science, University of Veterinary Medicine,Veterinarplatz 1, A-1210 Vienna, Austria.*Correspondence to: Thomas Czerny, Institute of Animal Breeding and Genetics, University of Veterinary Medicine, Veteri-narplatz 1, A-1210 Vienna, Austria. E-mail:</p><p>DOI 10.1002/dvdy.20995Published online 26 October 2006 in Wiley InterScience (</p><p>DEVELOPMENTAL DYNAMICS 235:33703378, 2006</p><p> 2006 Wiley-Liss, Inc.</p></li><li><p>ter, 1999; de Moor et al., 2005). Dead-enylation is the default pathway formRNA molecules in the cytoplasm.Stepwise reduction of the poly(A) tailup to a certain limit eventually endsin a rapid degradation by exonucle-ases (Copeland and Wormington,2001; Wilusz and Wilusz, 2004). Thedecay rate of mRNA is further regu-lated by AU-rich sequence elementsmainly located in the 3 untranslatedregion (UTR; de Moor et al., 2005).</p><p>The UTRs can inuence both thestability and the translatability of themRNA. Untranslated AUGs and sta-ble secondary structures in the 5UTR affect translation initiation(Pickering and Willis, 2005), and se-quence-specic binding of proteins toelements in both 5 and 3 UTRs canaffect degradation, polyadenylation,and mRNA localization (Wickens etal., 1997; Kloc et al., 2002). Especiallyduring embryonic development, thereis a wealth of examples of 3UTRs andpolyadenylation events inuencingtranslation efciency (Seydoux, 1996;Kuersten and Goodwin, 2003).</p><p>During early development, mater-nal mRNAs have to direct develop-ment before the onset of zygotic tran-</p><p>scription. A mechanism has, therefore,evolved that translationally masks apool of maternal mRNAs duringoozyte maturation, which are then ac-tivated during early development(Paillard and Osborne, 2003). Inacti-vation involves deadenylation of themRNAs, whereas cytoplasmic adeny-lation reactivates the dormant tran-scripts (Richter, 1999). Two discretesignals in the 3 UTR are necessaryfor this process, the nuclear polyade-nylation site AAUAAA and a U-richcytoplasmic polyadenylation element(CPE) with a consensus sequence ofUUUUUAU (Wormington, 1993). Thesequence of the CPE and its positionrelative to AAUAAA may control thetiming and extent of polyadenylation(Richter, 1999).</p><p>We tested different UTRs andpoly(A) sequences for their efciencyin mRNA injection experiments. Sur-prisingly variation of the UTRs hadlittle effect, contrary to the poly(A) se-quences. Both articial poly(A)stretches as well as the SV40 latepoly(A) signal strongly improvedtranslation activity, but not the stabil-ity of the RNA. Combined applicationof these elements in a new transcrip-</p><p>tion vector resulted in highly syner-gistic activity of the elements for in-jected mRNA in early embryos.</p><p>RESULTS</p><p>Pax2 UTRs Reduce theActivity of Injected mRNA</p><p>The Pax2 gene is a key player in mid/hindbrain development and conse-quently leads to strong phenotypeswhen misexpressed in early embryos(Kelly and Moon, 1995; Okafuji et al.,1999; Ye et al., 2001). We tried to re-peat these experiments in medakash by injecting in vitro transcribedcapped mRNA of zebrash Pax2 intoone-cell stage embryos. Contrary toour expectations, we did not observeany phenotypes in the mid/hindbrainregion (data not shown). When weraised the amount of injected mRNA,increasing numbers of embryos werearrested during gastrulation, but thesurviving embryos still appeared un-affected during somitogenesis. We as-sumed, that rapid degradation of theinjected mRNA in combination withthe slow development of medaka couldcause these unexpected results. Be-cause mRNA stability is mainly regu-lated by untranslated regions (UTR),we tested the effects of the Pax2 UTRsin a construct containing the reyluciferase reporter gene anked bythese UTRs (Fig. 1; Pax2 luc). Wemaintained the endogenous transla-tion start signals of Pax2 by fusing theluciferase coding region directly to theATG of the Pax2 cDNA. In vitro tran-scribed mRNA of this construct wasthen injected into medaka embryos.Luciferase activity measurements af-ter 24 hr revealed sevenfold reducedvalues when compared with a refer-ence group of embryos injected withmRNA containing luciferase togetherwith articial UTRs derived from thecell culture expression vector pKW10(construct KW luc, Table 1; Fig. 1).At 48 hr post-fertilization (hpf), thedifference in luciferase activity be-tween the two constructs was evenmore pronounced (38-fold, Table 1).Because the context of the start codonfor both constructs was similar(CCCATGG for Pax2 luc and AC-CATGG for KW luc), these data sug-gest a destabilizing effect of the Pax2UTR sequences. These results were</p><p>Fig. 1. Schematic presentation of the injected mRNAs. All mRNAs contain the rey luciferasecDNA. KW luc, CS luc, and CSas luc mRNA contain articial untranslated regions (UTRs) derivedfrom pKW10 (KWluc) and pCS2 (CS luc and CSas luc). The 5 and the 3 UTRs of the zebrash Pax2gene are part of Pax2 luc mRNA and the Xenopus -globin UTRs of T7TS luc, T7TS luc pA, and MCluc. Except for T7TS luc, all mRNAs contain either the early (e-pA) or the late (l-pA) SV40polyadenylation signal. In addition, T7TS luc, T7TS luc pA, and MC luc mRNAs contain an articialA30 poly(A) sequence (AAAA).</p><p>IMPROVED TRANSLATION EFFICIENCY OF mRNA 3371</p></li><li><p>further supported by cell culture ex-periments. The two constructs, bothcontaining a CMV promoter, wereused for transient DNA transfection ofNIH 3T3, HeLa, and U2-OS cells. Theexperiments revealed a 10- to 25-foldreduced luciferase activity for thePax2 UTR-containing construct, de-pending on the cell line used (Table 1).Therefore, Pax2 UTR sequences nega-tively inuence mRNA activity, possi-bly by enhancing the degradation rateboth in embryonic and in cell culturecells.</p><p>Contrary to Pax2, UTRs of othergenes have been shown to increase theactivity of injected mRNA. The -glo-bin UTRs present in the pT7TS con-struct are widely used for misexpres-sion experiments (Hyatt and Ekker,1999). Indeed when we applied thesesequences we could improve markergene expression compared with thepKW10-derived mRNA (Table 1; T7TSluc pA), but the effects were only two-to fourfold. In cell culture experimentsaddition of the -globin sequences af-fected the luciferase activity twofoldat most, both positively and nega-tively, depending on the cell line (Ta-ble 1).</p><p>SV40 PolyadenylationSignals Strongly AffectmRNA Activity in EarlyEmbryos</p><p>The pCS2 vector (Rupp et al., 1994;Turner and Weintraub, 1994) repre-sents another backbone frequentlyused for misexpression experiments.Of interest, we observed an almost 20-fold increase in luciferase activity formRNA derived from this constructcompared with the KW luc reference(Table 2). The pCS2 vector containsarticial UTRs and an SV40 poly(A)signal. Such a signal is also present inthe T7TS luc pA construct and could,therefore, not explain the dramatic in-crease of reporter gene activity forpCS2. However, we realized that thetwo constructs differ in the orienta-tion of the inserted SV40 fragment,which contains poly(A) signals in bothorientations. Thus in pCS2, the latepoly(A) signal is used, whereas inT7TS luc pA, the oppositely orientedearly poly(A) signal of the SV40 virusis used. To test whether one of the twopoly(A) signals preferentially affects</p><p>mRNA activity, we generated a pCSconstruct with a reversed poly(A) se-quence. mRNA derived from this con-struct (CSas luc, Table 2) indeed re-sulted in strongly reduced luciferaseactivities, comparable to that forT7TS luc pA. Of interest, these manip-ulations of the expression vectors didnot affect luciferase activity in cell cul-ture cells (Table 2), indicating that theeffects of the poly(A) signals are re-stricted to embryonic development.Therefore, viral SV40 poly(A) se-quences specically affect the activityof injected mRNA in early embryos.</p><p>Combined SequenceElements SynergisticallyAffect mRNA Activity inEarly Embryos</p><p>Because both pT7TS, as well as pCS2vectors, positively affected the activityof injected mRNA, we tested a combi-nation of their elements. The result-</p><p>ing vector pMC contains the backboneof pCS2, including the CMV promoterand the SV40 poly(A) signal and the 5and 3 UTRs of the -globin gene to-gether with a poly(A) tail of the pT7TSconstruct. When we tested mRNA de-rived from this construct, we sawstrong synergistic effects of the com-bined sequences. Compared with theisolated elements in pT7TS or pCS2,pMC-derived mRNA resulted in al-most 20-fold higher luciferase activityin the embryos (Table 3). Again thecorresponding cell culture experi-ments did not reveal similar effects.</p><p>The results for all constructs arecompared in Figure 2. In cell culture(mean values for all three cell lineswere calculated), -globin UTRs andthe orientation of the SV40 poly(A)sequences showed almost no effects,only the Pax2 UTRs signicantly re-duced luciferase activity. Contrary tocell culture cells, the different ele-ments strongly affected the results for</p><p>TABLE 1. Pax2 UTRs Reduce the Activity of Injected mRNAa</p><p>Construct</p><p>Embryo Cell culture</p><p>24 hpf 48 hpf NIH HeLa U2-OS</p><p>KW luc 100 (9) 205 (3) 100 100 100100 (13)</p><p>Pax2 luc 8.8 (9) 5.4 (4) 10.0 4.0 4.616 (16)</p><p>T7TS luc pA 378 (7) 298 (7) 202 58 101201 (9)</p><p>aThe data represent relative luciferase activities of the indicated constructs obtainedafter mRNA injection into medaka embryos or DNA transfection into the indicated celllines. Values for the KW luc construct were set at 100. The numbers of the injectedembryos are given in brackets. Two series of injections were performed, each includingall three mRNAs. The results of the two series are presented in the rst and secondrows, respectively. The cell culture results are based on four independent transfectionsexperiments. UTRs, untranslated regions; hpf, hours postfertilization</p><p>TABLE 2. Orientation of the SV40 Sequences Affects the Activity ofInjected mRNAa</p><p>Construct</p><p>Embryo Cell culture</p><p>24 hpf NIH HeLa U2-OS</p><p>KW luc 100 (11) 100 100 100100 (17)</p><p>CSas luc 233 (10) 198 54 98149 (14)</p><p>CS luc 1944 (10) 110 46 1021859 (17)</p><p>aFor details, see Table 1.</p><p>3372 FINK ET AL.</p></li><li><p>mRNA injections in embryos. ThuspMC-derived mRNA was 385-foldmore active than KW luc mRNA, andcompared with the established tran-scription vector pCS2 and pT7TS, ledto a 20-fold improvement in earlymedaka embryos.</p><p>The cell culture experiments sug-gested that the activating potential ofpMC might be restricted to embryonicdevelopment. To compare differentconstructs during later stages of de-velopment, we performed in ovo elec-troporation of chick embryos. With</p><p>this technique, we transferred DNAinto neural tube cells during somito-genesis (Hamburger and Hamiltonstage [HH] 1214, 1620 somites). Wetested KW luc, CS luc, and MC lucconstructs, all containing a CMV pro-moter. Experimental variations werecorrected by coelectroporation of a Re-nilla luciferase reference construct.The electroporated embryos were cul-tured for 24 hr (HH1820), dissected,and lysed to measure luciferase activ-ity. The corrected values did not showany benecial effects for -globin or</p><p>SV40 late poly(A) sequences. MC lucvalues were slightly below that for CSluc and for KW luc (1.6-fold and 1.5-fold,respectively). The improved expressionof pMC-derived mRNA, therefore,seems to be restricted to the earlieststages of embryonic development.</p><p>-globin and SV40 SequencesOnly Marginally AffectmRNA Stability</p><p>The simplest way to mechanisticallyexplain the enhancing effects of the-globin and poly(A) sequences wouldbe a prolonged stability of the injectedmRNA. To te...</p></li></ul>


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