AU-rich Sequences Research Articles

AU-A, an RNA-binding activity distinct from hnRNP A1, is selective for AUUUA repeats and shuttles between the nucleus and the cytoplasm.

The 3'-untranslated regions of many labile transcripts contain AU-rich sequences that serve as cis determinants of mRNA stability and translational efficiency. Using a photocrosslinking technique, our laboratory has previously defined three cytoplasmic RNA-binding activities specific for the AUUUA multimers found in the 3'-untranslated regions of lymphokine mRNAs. One of these activities, AU-A, has an apparent molecular mass of 34 kDa, is constitutively expressed in both primary T cells and the Jurkat T cell leukemia line, and binds to a variety of U-rich RNA sequences. Previous studies had shown that AU-A is more prevalent in the nucleus than the cytoplasm, raising the possibility that AU-A is really a nuclear RNA-binding activity that is found in cytoplasmic extracts because of nuclear leakage during cell fractionation. We now show that AU-A shuttles between the cytoplasm and the nucleus. Our results indicate that AU-A is a candidate protein component of ribonucleoprotein complexes that participate in nucleocytoplasmic transport of mRNA and cytoplasmic mRNA metabolism. The properties of AU-A activity are similar to those of heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1). However, using monoclonal antibodies to hnRNP A1 and protease digestion patterns, we show that AU-A activity and hnRNP A1 protein are distinct. These studies have also allowed us to define a fourth RNA-binding activity of apparent molecular mass 41 kDa with specificity for AUUUA multimers. This activity is restricted to the nucleus and contains the hnRNP C protein.

Inducible nitric-oxide-synthase mRNA is transiently expressed and destroyed by a cycloheximide-sensitive process.

Nitric oxide is a mediator of a wide range of physiological processes. It is produced by an enzyme family, the nitric-oxide synthases, one form of which is induced in many cells following stimulation with cytokines and lipopolysaccharide. The aim of the experiments reported in this study was to investigate the regulation of mRNA expression for this inducible nitric-oxide synthase in smooth muscle cells and macrophages. Stimulation of these cells with cytokines and lipopolysaccharide results in a marked elevation of nitric-oxide-synthase mRNA levels, which however do not remain elevated, but reach a maximum at 3-6 h after stimulation before returning to baseline levels over the next 20 h. Enzyme activity, however, remained virtually constant for 48 h following stimulation. Inspection of the 3' untranslated segment of both murine and human inducible nitric-oxide-synthase mRNAs showed the presence of a conserved AU-rich octanucleotide sequence, previously identified in cytokine and oncogene mRNAs and shown to mediate mRNA instability. A particular feature of the breakdown of mRNAs bearing this sequence is that degradation is prevented by protein-synthesis inhibition. We show in this study that the half-life of inducible nitric-oxide-synthase mRNA is 6 h and that in the presence of an inhibitor of protein synthesis this breakdown is prevented. Thus, the mRNA for inducible nitric-oxide synthase shares some features in common with cytokines such as the transient expression and decay of its mRNA which can be prevented by protein-synthesis inhibition.

Induction of cytoplasmic factors that bind to the 3' AU-rich region of human Interferon β mRNA during early development of Xenopus laevis

Certain endogenous Xenopus mRNAs, carrying a destabilizing 3' AU-rich sequence, are unusually very stable in oocytes and become unstable only after fertilization. In addition, heterologous short lived mRNA, containing 3' AU-rich sequences, appear to be very stable when injected into Xenopus oocytes. In the present study, a human interferon β (hu-IFNβ) mRNA, carrying the destabilizing 3' AU-rich element, was used as a probe to identify Xenopus proteins that specifically bind to the 3' AU-rich element as well as to study their relative levels during early embryonic development. While three major proteins that specifically bind to the 3' AU-rich element were detected in human SV80 cells, that naturally express hu-IFNβ (proteins termed AU-F1, F2 and F3), only two proteins, migrating similarly to the SV80 AU-F1 and AU-F3, were detected in cytoplasmic extracts from Xenopus oocytes or eggs. Following fertilization, the intensity of the Xenopus AU-F1 and AU-F3 proteins increased considerably and a new protein, corresponding to SV80 AU-F2, was also detected. Cyclohexamide applied either at the morula or at the early blastula stages reduced the intensity of the AU-binding factors, while actinomycin D did not, indicating that the levels of these factors during these stages are regulated posttranscriptionally. In contrast, application of each of these metabolic inhibitors at the late blastula stage increased the intensity of the AU-binding proteins. The possible function of these AU-binding factors in regulating the expression and half life of AU-rich mRNAs is discussed.

AU-rich intronic elements affect pre-mRNA 5' splice site selection in Drosophila melanogaster.

cis-spliced nuclear pre-mRNA introns found in a variety of organisms, including Tetrahymena thermophila, Drosophila melanogaster, Caenorhabditis elegans, and plants, are significantly richer in adenosine and uridine residues than their flanking exons are. The functional significance of this intronic AU richness, however, has been demonstrated only in plant nuclei. In these nuclei, 5' and 3' splice sites are selected in part by their positions relative to AU-rich elements spread throughout the length of an intron. Because of this position-dependent selection scheme, a 5' splice site at the normal (+1) exon-intron boundary having only three contiguous consensus nucleotides can compete effectively with an enhanced exonic site (-57E) having nine consensus nucleotides and outcompete an enhanced site (+106E) embedded within the AU-rich intron. To determine whether transitions from AU-poor exonic sequences to AU-rich intronic sequences influence 5' splice site selection in other organisms, alleles of the pea rbcS3A1 intron were expressed in Drosophila Schneider 2 cells, and their splicing patterns were compared with those in tobacco nuclei. We demonstrate that this heterologous transcript can be accurately spliced in transfected Drosophila nuclei and that a +1 G-to-A knockout mutation at the normal splice site activates the same three cryptic 5' splice sites as in tobacco. Enhancement of the exonic (-57) and intronic (+106) sites to consensus splice sites indicates that potential splice sites located in the upstream exon or at the 5' exon-intron boundary are preferred in Drosophila cells over those embedded within AU-rich intronic sequences. In contrast to tobacco, in which the activities of two competing 5' splice sites upstream of the AU-rich intron are modulated by their proximity to the AU transition point, D. melanogaster utilizes the upstream site which has a higher proportion of consensus nucleotides. The enhanced version of the cryptic intronic site is efficiently selected in D. melanogaster when the normal +1 site is weakened or discrete AU-rich elements upstream of the +106E site are disrupted. Selection of this internal site in tobacco requires more drastic disruption of these motifs. We conclude that 5' splice site selection in Drosophila nuclei is influenced by the intrinsic strengths of competing sites and by the presence of AU-rich intronic elements but to a different extent than in tobacco.

AU-rich intronic elements affect pre-mRNA 5' splice site selection in Drosophila melanogaster.

cis-spliced nuclear pre-mRNA introns found in a variety of organisms, including Tetrahymena thermophila, Drosophila melanogaster, Caenorhabditis elegans, and plants, are significantly richer in adenosine and uridine residues than their flanking exons are. The functional significance of this intronic AU richness, however, has been demonstrated only in plant nuclei. In these nuclei, 5' and 3' splice sites are selected in part by their positions relative to AU-rich elements spread throughout the length of an intron. Because of this position-dependent selection scheme, a 5' splice site at the normal (+1) exon-intron boundary having only three contiguous consensus nucleotides can compete effectively with an enhanced exonic site (-57E) having nine consensus nucleotides and outcompete an enhanced site (+106E) embedded within the AU-rich intron. To determine whether transitions from AU-poor exonic sequences to AU-rich intronic sequences influence 5' splice site selection in other organisms, alleles of the pea rbcS3A1 intron were expressed in Drosophila Schneider 2 cells, and their splicing patterns were compared with those in tobacco nuclei. We demonstrate that this heterologous transcript can be accurately spliced in transfected Drosophila nuclei and that a +1 G-to-A knockout mutation at the normal splice site activates the same three cryptic 5' splice sites as in tobacco. Enhancement of the exonic (-57) and intronic (+106) sites to consensus splice sites indicates that potential splice sites located in the upstream exon or at the 5' exon-intron boundary are preferred in Drosophila cells over those embedded within AU-rich intronic sequences. In contrast to tobacco, in which the activities of two competing 5' splice sites upstream of the AU-rich intron are modulated by their proximity to the AU transition point, D. melanogaster utilizes the upstream site which has a higher proportion of consensus nucleotides. The enhanced version of the cryptic intronic site is efficiently selected in D. melanogaster when the normal +1 site is weakened or discrete AU-rich elements upstream of the +106E site are disrupted. Selection of this internal site in tobacco requires more drastic disruption of these motifs. We conclude that 5' splice site selection in Drosophila nuclei is influenced by the intrinsic strengths of competing sites and by the presence of AU-rich intronic elements but to a different extent than in tobacco.

Dual Role of the Sequence-specific Bacteriophage T4 Endoribonuclease RegB: mRNA Inactivation and mRNA Destabilization

Gene regB of bacteriophage T4 encodes a sequence-specific endoribonuclease that introduces cuts in early phage messenger RNAs. Cutting takes place specifically in the middle of the tetranucleotide GGAG, as soon as the first minute of infection. Out of the 20 processing sites so far identified, seven are in Shine-Dalgarno sequences. The others are localized in intercistronic regions or within coding sequences. In the latter case, cutting efficiency is much lower. regB-dependent cleavages can occur within AU-rich sequences downstream of processed GGAG motifs that are not in effective translation initiation sites. We looked for possible consequences of regB -dependent cuts on gene expression in two early regions of the T4 chromosome. In the comCα region, none of the three major RegB cleavage sites is in a Shine-Dalgarno sequence, and in the motA region the unique regB-dependent processing site is found within the Shine-Dalgarno sequence of the gene. We find that in the region of gene comC α, RegB decreases two- to threefold the chemical half-life of early transcripts, but does not change the functional half-life of mRNAs coding for protein ComCα. The amount of MotA protein synthesized by the wild-type is half that obtained in a regB mutant infection. We show that this is a direct consequence of mRNA processing by RegB at the Shine-Dalgarno sequence of motA. This regB-mediated translation inhibition is not accompanied by an important modification in motA mRNA chemical half-life. We show that rapid shut-off of MotA protein synthesis that occurs soon after infection results both from RegB processing within the translation initiation region of motA and from early transcription inhibition followed by regB-independent breakdown of the motA mRNA.

Purification of an AU-rich RNA binding protein from Sarcophaga peregrina (flesh fly) and its identification as a Thiolase.

A protein that binds to the AU-rich sequence in the 3'-untranslated region of sarcotoxin IIA mRNA was purified from a Sarcophaga pupal extract to near homogeneity. The molecular mass of this protein was estimated to be 39 kDa by SDS-polyacrylamide gel electrophoresis. The partial amino acid sequences of two peptides obtained from the 39 kDa protein showed striking similarities to partial amino acid sequences of rat and yeast 3-oxoacyl-CoA thiolase, suggesting that this protein is a Sarcophaga thiolase. In fact, the purified 39 kDa protein was found to have thiolase activity. Moreover, rat mitochondrial 3-oxoacyl-CoA thiolase showed affinity to the AU-rich RNA. These suggest that the RNA binding activity is an intrinsic character of thiolase.

3' splice site selection in dicot plant nuclei is position dependent.

In contrast to mammalian and yeast systems, the mechanism for intron recognition and splice site selection in plant pre-mRNAs is poorly understood. Splice site sequences and putative branchpoint sequences are loosely conserved in plant introns compared with other eukaryotes. Perhaps to compensate for these variations, plant introns are significantly richer in adenosine and uridine residues than are their adjacent exons. To define elements critical for 3' splice site selection in dicotyledonous plant nuclei, pre-mRNA transcripts containing intron 3 of the maize Adh1 gene were expressed in Nicotiana benthamiana nuclei by using an autonomously replicating plant expression vector. Using a series of intron rearrangements which reposition the 3' intron-exon border, we demonstrate that the normal 3' splice site is defined in a position-dependent manner and that cryptic 3' splice sites within the intron are masked by the presence of a functional downstream 3' splice site. Disruption of the AU-rich elements upstream from the normal 3' splice site indicates that multiple AU elements between -66 and -6 cooperatively define the 3' boundary of the intron. These results are consistent with a model for plant intron recognition in which AU-rich elements spread throughout the length of the intron roughly define the intron boundaries by generating strong AU transition points. Functional 3' splice sites located downstream from these AU-rich sequences are preferentially selected over sites embedded within them.

3' splice site selection in dicot plant nuclei is position dependent.

In contrast to mammalian and yeast systems, the mechanism for intron recognition and splice site selection in plant pre-mRNAs is poorly understood. Splice site sequences and putative branchpoint sequences are loosely conserved in plant introns compared with other eukaryotes. Perhaps to compensate for these variations, plant introns are significantly richer in adenosine and uridine residues than are their adjacent exons. To define elements critical for 3' splice site selection in dicotyledonous plant nuclei, pre-mRNA transcripts containing intron 3 of the maize Adh1 gene were expressed in Nicotiana benthamiana nuclei by using an autonomously replicating plant expression vector. Using a series of intron rearrangements which reposition the 3' intron-exon border, we demonstrate that the normal 3' splice site is defined in a position-dependent manner and that cryptic 3' splice sites within the intron are masked by the presence of a functional downstream 3' splice site. Disruption of the AU-rich elements upstream from the normal 3' splice site indicates that multiple AU elements between -66 and -6 cooperatively define the 3' boundary of the intron. These results are consistent with a model for plant intron recognition in which AU-rich elements spread throughout the length of the intron roughly define the intron boundaries by generating strong AU transition points. Functional 3' splice sites located downstream from these AU-rich sequences are preferentially selected over sites embedded within them.

CTLA-8, cloned from an activated T cell, bearing AU-rich messenger RNA instability sequences, and homologous to a herpesvirus saimiri gene.

To detect novel molecules involved in immune functions, a subtracted cDNA library between closely related murine lymphoid cells was prepared using improved technology. Differential screening of this library yielded several clones with a very restricted tissue specificity, including one that we named CTLA-8. CTLA-8 transcripts could be detected only in T cell hybridoma clones related to the one used to prepare the library. Southern blots showed that the CTLA-8 gene was single copy in mice, rats, and humans. By radioactive in situ hybridization, the CTLA-8 gene was mapped at a single site on mouse chromosome 1A and human chromosome 2q31, in a known interspecific syntenic region. The CTLA-8 cDNA sequence indicated the presence, in the 3'-untranslated region of the mRNA, of AU-rich repeats previously found in the mRNA of various cytokines, growth factors, and oncogenes. The CTLA-8 cDNA contained an open reading frame encoding a putative protein of 150 amino acids. This protein was 57% homologous to the putative protein encoded by the ORF13 gene of herpesvirus Saimiri, a T lymphotropic virus. These findings are discussed in the context of other genes of this herpesvirus homologous to known immunologically active molecules. More generally, CTLA-8 may belong to the growing set of virus-captured functionally important cellular genes related to the immune system or to cell death and cell survival.

Translational regulation of human beta interferon mRNA: association of the 3' AU-rich sequence with the poly(A) tail reduces translation efficiency in vitro.

The 3' AU-rich region of human beta-1 interferon (hu-IFN beta) mRNA was found to act as a translational inhibitory element. The translational regulation of this 3' AU-rich sequence and the effect of its association with the poly(A) tail were studied in cell-free rabbit reticulocyte lysate. A poly(A)-rich hu-IFN beta mRNA (110 A residues) served as an inefficient template for protein synthesis. However, translational efficiency was considerably improved when the poly(A) tract was shortened (11 A residues) or when the 3' AU-rich sequence was deleted, indicating that interaction between these two regions was responsible for the reduced translation of the poly(A)-rich hu-IFN beta mRNA. Differences in translational efficiency of the various hu-IFN beta mRNAs correlated well with their polysomal distribution. The poly(A)-rich hu-IFN beta mRNA failed to form large polysomes, while its counterpart bearing a short poly(A) tail was recruited more efficiently into large polysomes. The AU-rich sequence-binding activity was reduced when the RNA probe contained both the 3' AU-rich sequence and long poly(A) tail, supporting a physical association between these two regions. Further evidence for this interaction was achieved by RNase H protection assay. We suggest that the 3' AU-rich sequence may regulate the translation of hu-IFN beta mRNA by interacting with the poly(A) tail.

Translational regulation of human beta interferon mRNA: association of the 3' AU-rich sequence with the poly(A) tail reduces translation efficiency in vitro.

The 3' AU-rich region of human beta-1 interferon (hu-IFN beta) mRNA was found to act as a translational inhibitory element. The translational regulation of this 3' AU-rich sequence and the effect of its association with the poly(A) tail were studied in cell-free rabbit reticulocyte lysate. A poly(A)-rich hu-IFN beta mRNA (110 A residues) served as an inefficient template for protein synthesis. However, translational efficiency was considerably improved when the poly(A) tract was shortened (11 A residues) or when the 3' AU-rich sequence was deleted, indicating that interaction between these two regions was responsible for the reduced translation of the poly(A)-rich hu-IFN beta mRNA. Differences in translational efficiency of the various hu-IFN beta mRNAs correlated well with their polysomal distribution. The poly(A)-rich hu-IFN beta mRNA failed to form large polysomes, while its counterpart bearing a short poly(A) tail was recruited more efficiently into large polysomes. The AU-rich sequence-binding activity was reduced when the RNA probe contained both the 3' AU-rich sequence and long poly(A) tail, supporting a physical association between these two regions. Further evidence for this interaction was achieved by RNase H protection assay. We suggest that the 3' AU-rich sequence may regulate the translation of hu-IFN beta mRNA by interacting with the poly(A) tail.

Association of heterogeneous nuclear ribonucleoprotein A1 and C proteins with reiterated AUUUA sequences.

Post-transcriptional regulatory mechanisms have been shown to play a major role in gene expression in eukaryotic cells. The presence of a reiterated pentamer (AUUUA) in the 3'-untranslated region (UTR) of mRNAs encoding lymphokines, cytokines, transcription factors, and proto-oncogenes has been shown to be associated with rapid turnover and translation attenuation. Cytoplasmic proteins (70, 50, 43, 36, and 25 kDa) capable of specifically binding to RNAs containing these AU-rich sequences were identified in human peripheral blood T lymphocytes. Levels of the 36-kDa protein were markedly increased following transcriptional, but not translational inhibition, a feature recently reported for hnRNP A1, a protein of comparable mass. Antibodies directed against heterogeneous nuclear ribonucleoproteins (hnRNPs) A1 and C immunoprecipitated 36- and 43-kDa proteins that had bound the AUUUA-rich region contained in the 3'-UTR of granulocyte-macrophage colony-stimulating factor mRNA. Recombinant hnRNP A1 was shown to preferentially bind to RNAs containing AUUUA sequences in a specific manner, and displayed comparable patterns to the 36-kDa AU-specific binding proteins following partial proteolysis. These data identify for the first time hnRNP A1 and C as cytoplasmic proteins in human lymphocytes that are capable of specifically associating with reiterated AUUUA sequences present in the 3'-UTR of labile mRNAs. As such, they may play a role as trans-acting factors in the modulation of cytoplasmic mRNA turnover and translation, in addition to their previously characterized roles as pre-mRNA binding proteins involved in nuclear mRNA processing.

Expression of maize Adh1 intron mutants in tobacco nuclei

In vivo and in vitro gene transfer experiments have suggested that the elements mediating intron recognition differ in mammalian, yeast and plant nuclei. Differences in the sequence dependencies, which also exist between dicotyledonous and monocotyledonous nuclei, have prevented some monocot introns from being spliced in dicot nuclei. To locate elements which modulate efficient recognition of introns in dicot nuclei, the maize Adh1 gene has been expressed in full-length and single intron constructs in Nicotiana benthamiana nuclei using an autonomously replicating plant expression vector. Quantitative PCR-Southern analyses indicate that the inefficient splicing of the maize Adh1 intron 1 (57% AU) in these dicot nuclei can be dramatically enhanced by increasing the degree of U1 snRNA complementarity at the 5' splice site. This indicates that the 5' splice site plays a significant role in defining the splicing efficiency of an intron in dicot nuclei and that, most importantly, the remainder of this monocot intron contains no elements which inhibit its accurate recognition in dicot nuclei. Deletions in intron 3 (66% AU) which effectively move the 3' boundary between AU-rich intron and GC-rich exon sequences strongly activate a cryptic upstream splice site; those which do not reposition this boundary activate a downstream cryptic splice site. This suggests that 3' splice site selection in dicot nuclei is extremely flexible and not dependent on strict sequence requirements but rather on the transition points between introns and exons. Our results are consistent with a model in which potential splice sites are selected if they are located upstream (5' splice site) or downstream (3' splice site) of AU transition points and not if they are embedded within AU-rich sequences.

Selective Alteration of the Turnover of Interferon β mRNA in Peritoneal Macrophages from LPS-Hyporesponsive Mice and Its Role in the Defective Expression of Spontaneous Interferon

Basal levels of interferon (IFN)-β mRNA transcription were detected in both freshly explanted LPS-responsive (Lpsn) and LPS-hyporesponsive (Lpsd) peritoneal macrophages (PM). In vitro cultivation of PM resulted in a time-dependent reduction in the level of IFN-β mRNA, which was far more rapid in Lpsd than in Lpsn PM. Treatment of Lpsn PM with cycloheximide (CHX) resulted in a marked accumulation of IFN-β mRNA, which was not associated with an increase in IFN-β gene transcription. However, treatment of Lpsd PM with CHX did not induce accumulation of IFN-β mRNA. CHX induced the accumulation of IFN-α-4 mRNA in both Lpsn and Lpsd PM. In Lpsn PM, CHX enhanced the accumulation of two cytoplasmic factors interacting with AU-rich sequences within the 3′ untranslated region of IFN-β mRNA. We conclude that Lpsd PM exhibit an impaired capacity to stabilize IFN-β mRNA that may account for their low expression of IFN-β.

Efficient splicing of an AU-rich antisense intron sequence.

For successful splicing in dicot plants the only recognised intron requirements are 5' and 3' splice sites and AU-rich sequences. We have investigated further the importance of AU-rich elements by analyzing the splicing of an AU-rich antisense intron sequence. Activation of cryptic splice sites on either side of the AU-rich sequence permitted the efficient removal of this essentially non-intron sequence by splicing. This splicing event not only confirms the importance of AU-rich sequences but also has implications for the evolution of interrupted genes and the expression of heterologous genes in transgenic plants.

Binding of sequence-specific proteins to the adenosine- plus uridine-rich sequences of the murine granulocyte/macrophage colony-stimulating factor mRNA.

Adenosine+uridine (AU)-rich sequences in the 3' untranslated region (3'UTR) of the mRNA of many cytokines and oncogenes play an important role in mediating RNA degradation. Among the cytokines containing such AU-rich sequences in their 3'UTR is the hematopoietic growth factor granulocyte/macrophage colony-stimulating factor (GM-CSF). GM-CSF gene expression in T cells is regulated by modulation of mRNA half-life. Transfection studies using murine EL-4 thymoma cells have demonstrated that degradation depends on the presence of specific elements in the 3'UTR, including the AU-rich sequences. A number of AU-binding factors have recently been discovered, suggesting that specific regulation may occur through specific protein-mRNA interaction(s). We present evidence from gel-shift analyses and label-transfer experiments that murine cells contain proteins that bind specifically to AU-rich sequences. Three major proteins of 33, 39.5, and 42 kDa are detected. Phorbol ester treatment of cells does not alter the abundance or apparent binding affinity of the proteins. The 33-kDa protein is present in the cytoplasm of murine and human cells, whereas the 39.5- and 42-kDa proteins are present in murine extracts only. Constitutively expressed AU-binding proteins of the type that we describe may function by directing mRNA degradation in the absence of a stimulus to the contrary.

Alternative 3' splice acceptor sites modulate enzymic activity in derivative alleles of the maize bronze1-mutable 13 allele.

The defective Suppressor-mutator (dSpm)-induced allele bronze1-mutable 13 (bz1-m13) and many of its derivative alleles are leaky mutants with measurable levels of flavonol O3-glucosyltransferase activity. This activity results from splicing at acceptor site-1, one of two cryptic 3' splice sites within the dSpm insertion in bz1-m13. In this study, splicing in bz1-m13 change-in-state (CS) alleles CS-3 and CS-64 was shown to be altered from bz1-m13; previous work found altered splicing in CS-9. CS-64 is a null allele and lacks the acceptor site-1-spliced transcript because this site is deleted. CS-3 and CS-9 had increased levels of the acceptor site-1 transcript relative to bz1-m13 and increased enzymic activities. A deletion in CS-9 altered splicing by eliminating acceptor site-2. Both acceptor sites were intact in CS-3, but a deletion removed most of a 275-bp GC-rich sequence in dSpm. This suggests that GC-rich sequences affect splicing and is consistent with models postulating a role for AU content in the splicing of plant introns. Splicing does not necessarily occur, however, at the junction of AU-rich intron sequences and GC-rich exon sequences.

Evidence for instability of mRNAs containing AUUUA motifs mediated through translation-dependent assembly of a > 20S degradation complex.

Many short-lived mRNAs, including those encoding lymphokines, cytokines, and proto-oncogenes, contain an AU-rich sequence in their 3'-untranslated regions. These AU domains and, more specifically, AUUUA motifs within them, are widely thought to mediate the extreme instability of the corresponding mRNAs. This is most clearly true for granulocyte monocyte colony stimulating factor (GM-CSF) mRNA whose AUUUA motifs are conserved phylogenetically and whose presence in an otherwise stable beta-globin mRNA results in a 50-fold decrease in accumulated mRNA level. We show that RNA instability conferred by the GM-CSF AU motif requires the mRNA to be actively translated and the AU motif to be within the 3'-untranslated region. By analysis of the sedimentation characteristics, we identify a large (> 20S), divalent cation-independent complex found only on unstable RNAs. Like instability, complex formation (1) is dependent on translation of the RNA, (2) requires intact AUUUA motifs, and (3) is blocked by ribosome translocation across the AU-rich motif. We propose that RNA instability mediated by the AU motif is achieved through translation-dependent assembly of this large mRNA-destabilizing complex.

Isolation of novel murine maternal mRNAs regulated by cytoplasmic polyadenylation.

The cytoplasmic polyadenylation element (CPE) is an AU-rich sequence in the 3'-untranslated region of many stored maternal mRNAs. The CPE directs the meiotic maturation-specific cytoplasmic polyadenylation and translational activation of these dormant mRNAs in Xenopus. The work presented here demonstrates that the CPE controls a similar regulation in mouse oocytes and utilizes the information to isolate novel maternal mRNAs by polymerase chain reaction (PCR). A degenerate CPE primer was used in an anchored PCR reaction with cDNAs from primary mouse oocytes. Clones were identified that contained the canonical polyadenylation signal AATAAA. A novel PCR test was then used to determine the polyadenylation state of the respective mRNAs before and after meiotic maturation. Two mRNAs, OM-1 and OM-2, are cytoplasmically polyadenylated upon maturation. Another mRNA is not polyadenylated during maturation, although it contains multiple CPE-like elements, indicating that this sequence element is not sufficient for adenylation during this time. Microinjection into primary oocytes of antisense oligodeoxynucleotides directed against OM-1 destroys the mRNA but does not appear to interfere with maturation in vitro. These experiments identify two novel maternal mRNAs and establish a simple strategy for isolating other maternal messages that control meiotic maturation, fertilization, and early mouse development.