Regulated Polyadenylation Controls Mrna Translation During Meiotic Maturation of Mouse Oocytes

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Regulated Polyadenylation Controls Mrna Translation During Meiotic Maturation of Mouse Oocytes Downloaded from genesdev.cshlp.org on September 23, 2021 - Published by Cold Spring Harbor Laboratory Press Regulated polyadenylation controls mRNA translation during meiotic maturation of mouse oocytes Jean-Dominique Vassalli, 1 Joaquin Huarte, 1 Dominique Belin, 2 Pascale Gubler, 1 Anne Vassalli, 1,4 Marcia L. O'Connell, 3 Lance A. Parton, 3 Richard J. Rickles, 3 and Sidney Strickland 3 1Institute of Histology and Embryology, and 2Department of Pathology, University of Geneva Medical School, CH 1211 Geneva 4, Switzerland; 3Departrnent of Molecular Pharmacology, State University of New York, Stony Brook, New York 11794 USA The translational activation of dormant tissue-type plasminogen activator mRNA during meiotic maturation of mouse oocytes is accompanied by elongation of its 3'-poly(A) tract. Injected RNA fragments that correspond to part of the 3'-untranslated region (3'UTR) of this mRNA are also subject to regulated polyadenylation. Chimeric mRNAs containing part of this 3'UTR are polyadenylated and translated following resumption of meiosis. Polyadenylation and translation of chimeric mRNAs require both specific sequences in the 3'UTR and the canonical 3'-processing signal AAUAAA. Injection of 3'-blocked mRNAs and in vitro polyadenylated mRNAs shows that the presence of a long poly(A) tract is necessary and sufficient for translation. These results establish a role for regulated polyadenylation in the post-transcriptional control of gene expression. [Key Words: Plasminogen activators; meiosis; poly(A); 3'UTR; translational control] Received August 18, 1989; revised version accepted October 13, 1989. A rapid and efficient mechanism to regulate gene ex- in polyadenylation trigger or merely accompany transla- pression involves the post-transcriptional modulation of tion is not known. In fact, since its discovery 20 years mRNA translation or stability. There are many ex- ago (Edmonds and Caramela 1969; Damell et al. 1971; amples of this type of regulation (Ilan 1987), including Edmonds et al. 1971; Lee et al. 1971), it has not been the iron-regulated translation of ferritin mRNA possible to assign a definitive role to the poly(A) tract (Z~ihringer et al. 1976; Shull and Theil 1982) and sta- that is present at the 3' end of most eukaryotic mRNAs bility of transferrin receptor mRNA (Mihllner and Kiihn (Brawerman 1981). Not all mRNAs are polyadenylated, 1988), and the regulation of f~-tubulin mRNA life span indicating that this modification is not an absolute pre- by the protein product (Yen et al. 1988). A striking case requisite for translation. Presence of a poly(A) tract can of post-transcriptional control is provided by the uncou- influence stability and/or translatability of heterologous pling of gene transcription and translation in oocytes: mRNAs injected into Xenopus laevis oocytes or added During oocyte growth, transcription of a number of to a reticulocyte lysate (Huez et al. 1974; Doel and Carey genes yields stable, untranslated mRNAs, whose trans- 1976; Drummond et al. 1985). However, a causal role for lation and degradation are triggered by precisely timed polyadenylation in translation or stability of an endoge- developmental events (Davidson 1986). nous mRNA has not been demonstrated. Studies on the mechanisms controlling activation of The mRNA for tissue-type plasminogen activator dormant mRNAs in oocytes and fertilized eggs from dif- (tPA) accumulates in growing mouse 0ocytes, and it is ferent phyla have revealed a correlation between the ex- stored, stable and untranslated, in the cytoplasm of the tent of polyadenylation of a message and its translation meiotically arrested primary oocyte. Following resump- (Colot and Rosbash 1982; Rosenthal et al. 1983; tion of meiosis, starting -3 hr after dissolution of the Dworkin and Dworkin-Rastl 1985; Huarte et al. 1987b; nuclear membrane (germinal vesicle breakdown, Rosenthal and Ruderman 1987; Goldman et al. 1988; GVBD), tPA mRNA undergoes a progressive 3' polya- Hyman and Wormington 1988; Mutter et al. 1988; denylation; concomitantly, it is translated and then de- Paynton et al. 1988; McGrew et al. 1989). Extension of a graded (Huarte et al. 1985, 1987b). Thus, the extent of pre-existing short poly(A) tail is associated with mRNA polyadenylation of this message is correlated with its activation, and deadenylation is correlated with the ces- translation and subsequent loss of stability. To examine sation of translation. However, whether these changes the possible interrelationship between these phe- nomena, we injected into mouse oocytes various RNA 4Present address: Department of Biology, Massachusetts Institute of fragments and chimeric mRNAs and studied their fate Technology, Cambridge,Massachusetts 02139 USA. under conditions that prevent or allow meiotic matura- GENES & DEVELOPMENT3:2163-2171 © 1989 by Cold Spring HarborLaboratory Press ISSN 0890-9369/89 $1.00 2163 Downloaded from genesdev.cshlp.org on September 23, 2021 - Published by Cold Spring Harbor Laboratory Press Vassalli et al. tion. The results demonstrate that cytoplasmic 3' polya- ~-PA denylation, regulated by sequences present in the 3'-un- i,(A) 5'UTR 3'UTR translated region (3'UTR) of tPA mRNA, is necessary i i and sufficient for translational activation; thus they es- A. RNA FRAGMENTS tablish a role for polyadenylation in the post-transcrip- i I. t-PA 3' END I tional control of gene expression. 2. t-PA 3' END WITHOUT LAST 99 NT i 3. (~-GLOBIN 3' FRAGMENT ..... r Results 4. u-PA 3' FRAGMENT vvv~aNvvO i Regulated polyadenylation of injected RNAs i B. mRNAs Previous experiments demonstrated that the 3'UTR of u-PA (REPORTER RNA) tPA mRNA plays a critical role in its translational acti- 1. vv¢/////////////////~ vation during meiotic maturation. Injection of antisense 5'UTR 3'UTR ' RNAs into mouse oocytes had shown that (1) in contrast t CHIMERIC mRHAs i to other parts of the molecule, the 3'UTR of tPA mRNA 2. ,N~/////////////////~ is accessible to hybrid formation in the primary oocyte; ,5. WC(/////////////////~', and (2) hybrid formation leads to mRNA cleavage, and 4. ,,~T/////////////////A~,w~v,~,w~' amputation of the 3'-terminal sequences of tPA mRNA i prevents its polyadenylation, translation, and degrada- 5. ,,,wr////////////I/I//.~,a, wwwvv,~ tion (Strickland et al. 1988}. t Thus, it appeared that the 3'UTR might contain struc- 6. WVf/////////////////~Ncvvv~,VvVvv.,w i tural determinants that allow recognition of tPA mRNA 7. ,/v~/////////////////d~vww,~v~(A)n during meiotic maturation. To investigate this possi- bility, oocytes were injected with a 487-nucleotide RNA Figure 1. Schematic representation of the transcripts. The fragment that corresponds to the end of the 3'UTR of drawing is to scale; the endogenous tPA mRNA has a length of 2.5 kb. Lines represent UTRs; rectangles represent coding re- tPA mRNA (Fig. 1A). Northern blot analysis of total gions. Small solid rectangles represent a natural AAUAAA se- RNA revealed that the fate of this fragment was similar quence; small open rectangles represent a synthetic AAUAAA to that of the endogenous, 2.5-kb tPA mRNA transcript: sequence. (A) Short poly(A) tail (<40 As); [(A)n] long poly(A) tail Both molecules were essentially stable in primary oo- (200-400 As); IX) cordycepin. cytes and were progressively elongated and degraded in maturing oocytes (Fig. 2A). As shown previously for en- dogenous tPA mRNA (Huarte et al. 1987b), elongation of type PA (uPA) mRNA sequences (Fig. 1A)was not detec- the injected fragment was the result of the addition of a tably elongated in maturing oocytes (Fig. 2D). Finally, poly(A) tract at the 3' end, as treatment with RNase H in transcripts generated from the plasmid used for frag- the presence of oligo(dT) reduced its size to that of the ments A(1) and A(2)(Fig. 1), but linearized at different uninjected transcript (Fig. 2B). The efficiency of pro- sites downstream from the tPA insert and, hence, con- cessing of this RNA fragment was remarkable: in exper- taining up to 180 nucleotides of vector sequences and a iments in which injected oocytes contained up to 300- different nucleotide at their 3' ends, were all elongated fold more fragment than endogenous tPA mRNA, both during meiotic maturation (Fig. 3). The longest fragment species appeared to be completely processed. Thus, in was found to undergo only limited polyadenylation, sug- maturing oocytes, the polyadenylation machinery is gesting that the effect of tPA-derived sequences may de- clearly not present in limiting amount. crease with distance from the end of the transcript. We To determine whether specific features of this 487- conclude that (1) the nature of the 3'-terminal base is nucleotide RNA are required for polyadenylation, other irrelevant, (2) the precise distance from the tPA se- RNA fragments were injected. A fragment lacking the quences to the 3' end of the injected molecule is not crit- terminal 99 nucleotides of tPA sequence, and therefore ical, (3) the presence of tPA 3' sequences, including pos- lacking the 3'-processing signal AAUAAA (Proudfoot sibly AAUAAA, is essential, and (4) the AAUAAA se- and Brownlee 1976), was not polyadenylated (Fig. 2C). quence is not sufficient. However, not all RNAs that contained AAUAAA were elongated during meiotic maturation: A radiolabeled Translation of chimeric mRNAs rabbit ~-globin RNA fragment, containing the 3' AAUAAA sequence of the mRNA (Fig. 1A), was coin- The results obtained with injected RNA fragments im- jected with the radiolabeled 487-nucleotide tPA mRNA plied that a recognition process
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