U-rich Element Research Articles

Developmental Regulation of RNA Transcript Destabilization by A + U-rich Elements is AUF1-dependent

The developmental immaturity of neonatal phagocytic function is associated with decreased accumulation and half-life (t((1)/(2))) of granulocyte/macrophage colony-stimulating factor (GM-CSF) mRNA in mononuclear cells (MNC) from the neonatal umbilical cord compared with adult peripheral blood. The in vivo t((1)/(2)) of GM-CSF mRNA is 3-fold shorter in neonatal (30 min) than in adult (100 min) MNC. Turnover of mRNA containing a 3'-untranslated region (3'-UTR) A + U-rich element (ARE), which regulates GM-CSF mRNA stability, is accelerated in vitro by protein fractions enriched for AUF1, an ARE-specific binding factor. The data reported here demonstrate that the ARE significantly accelerates in vitro decay of the GM-CSF 3'-UTR in the presence of either neonatal or adult MNC protein. Decay intermediates of the GM-CSF 3'-UTR are generated that are truncated at either end of the ARE. Furthermore, the t((1)/(2)) of the ARE-containing 3'-UTR is 4-fold shorter in the presence of neonatal (19 min) than adult (79 min) MNC protein, reconstituting developmental regulation in a cell-free system. Finally, accelerated ARE-dependent decay of the GM-CSF 3'-UTR in vitro by neonatal MNC protein is significantly attenuated by immunodepletion of AUF1, providing new evidence that this accelerated turnover is ARE- and AUF1-dependent.

Assembly of AUF1 Oligomers on U-rich RNA Targets by Sequential Dimer Association

Many labile mammalian mRNAs are targeted for rapid cytoplasmic turnover by the presence of A + U-rich elements (AREs) within their 3'-untranslated regions. These elements are selectively recognized by AUF1, a component of a multisubunit complex that may participate in the initiation of mRNA decay. In this study, we have investigated the recognition of AREs by AUF1 in vitro using oligoribonucleotide substrates. Gel mobility shift assays demonstrated that U-rich RNA targets were specifically bound by AUF1, generating two distinct RNA-protein complexes in a concentration-dependent manner. Chemical cross-linking revealed the interaction of AUF1 dimers to form tetrameric structures involving protein-protein interactions in the presence of high affinity RNA targets. From these data, a model of AUF1 association with AREs involving sequential dimer binding was developed. Using fluorescent RNA substrates, binding parameters of AUF1 dimer-ARE and tetramer-ARE equilibria were evaluated in solution by fluorescence anisotropy measurements. Using two AUF1 deletion mutants, sequences C-terminal to the RNA recognition motifs are shown to contribute to the formation of the AUF1 tetramer.ARE complex but are not obligate for RNA binding activity. Kinetic studies demonstrated rapid turnover of AUF1.ARE complexes in solution, suggesting that these interactions are very dynamic in character. Taken together, these data support a model where ARE-dependent oligomerization of AUF1 may function to nucleate the formation of a trans-acting, RNA-destabilizing complex in vivo.

Identification and Characterization of Proteins Binding A + U-Rich Elements

A + U-Rich elements (AREs) have been extensively investigated ascis-acting determinants of rapid mRNA turnover. Recently, a number of RNA-binding proteins interacting with AREs have been described. This article presents strategies and techniques used by our laboratory to identify and characterize a family of ARE-binding proteins collectively termed AUF1. However, these techniques may be applied to the study of any protein displaying sequence-specific RNA binding activity. The techniques described here include the purification of native AUF1 from cultured cells as well as the preparation of recombinant AUF1 proteins using a bacterial expression system. Analyses of RNA–protein interactions are also described, including the use of gel mobility shift assays with synthetic RNA probes to monitor specific RNA binding activity in cell extracts or with recombinant proteins. Variations of this technique are also described to evaluate the RNA binding affinity of recombinant proteins and the use of specific RNA competitors to assess RNA determinants of protein binding specificity. Other techniques presented include the identification of specific proteins in RNA:protein complexes using antibody supershifts and the estimation of molecular weights of RNA-binding proteins by UV crosslinking. Results of individual experiments are presented as examples of some techniques. Throughout the article, suggestions are included to avoid commonly encountered problems and to assist in the optimization of these techniques for the study of other RNA-binding proteins.

The clam 3' UTR masking element-binding protein p82 is a member of the CPEB family.

During early development gene expression is controlled principally at the translational level. Oocytes of the surf clam Spisula solidissima contain large stockpiles of maternal mRNAs that are translationally dormant or masked until meiotic maturation. Activation of the oocyte by fertilization leads to translational activation of the abundant cyclin and ribonucleotide reductase mRNAs at a time when they undergo cytoplasmic polyadenylation. In vitro unmasking assays have defined U-rich regions located approximately centrally in the 3' UTRs of these mRNAs as translational masking elements. A clam oocyte protein of 82 kDa, p82, which selectively binds the masking elements, has been proposed to act as a translational repressor. Importantly, mRNA-specific unmasking in vitro occurs in the absence of poly(A) extension. Here we show that clam p82 is related to Xenopus CPEB, an RNA-binding protein that interacts with the U-rich cytoplasmic polyadenylation elements (CPEs) of maternal mRNAs and promotes their polyadenylation. Cloned clam p82/CPEB shows extensive homology to Xenopus CPEB and related polypeptides from mouse, goldfish, Drosophila and Caenorhabditis elegans, particularly in their RNA-binding C-terminal halves. Two short N-terminal islands of sequence, of unknown function, are common to vertebrate CPEBs and clam p82. p82 undergoes rapid phosphorylation either directly or indirectly by cdc2 kinase after fertilization in meiotically maturing clam oocytes, prior to its degradation during the first cell cleavage. Phosphorylation precedes and, according to inhibitor studies, may be required for translational activation of maternal mRNA. These data suggest that clam p82 may be a functional homolog of Xenopus CPEB.

Auxiliary downstream elements are required for efficient polyadenylation of mammalian pre-mRNAs.

We have previously identified a G-rich sequence (GRS) as an auxiliary downstream element (AUX DSE) which influences the processing efficiency of the SV40 late polyadenylation signal. We have now determined that sequences downstream of the core U-rich element (URE) form a fundamental part of mammalian polyadenylation signals. These novel AUX DSEs all influenced the efficiency of 3'-end processing in vitro by stabilizing the assembly of CstF on the core downstream URE. Three possible mechanisms by which AUX DSEs mediate efficient in vitro 3'-end processing have been explored. First, AUX DSEs can promote processing efficiency by maintaining the core elements in an unstructured domain which allows the general polyadenylation factors to efficiently assemble on the RNA substrate. Second, AUX DSEs can enhance processing by forming a stable structure which helps focus binding of CstF to the core downstream URE. Finally, the GRS element, but not the binding site for the bacteriophage R17 coat protein, can substitute for the auxiliary downstream region of the adenovirus L3 polyadenylation signal. This suggests that AUX DSE binding proteins may play an active role in stimulating 3'-end processing by stabilizing the association of CstF with the RNA substrate. AUX DSEs, therefore, serve as a integral part of the polyadenylation signal and can affect signal strength and possibly regulation.

The Search for Trans-Acting Factors Controlling Messenger RNA Decay

Control of mRNA turnover is an integral component of regulated gene expression. Individual mRNAs display a wide range of stabilities, which in many cases have been linked to discrete sequence elements. The most extensively characterized determinants of rapid constitutive mRNA turnover in mammalian systems are A + U-rich elements (AREs), first identified in the 3' untranslated regions of many cytokine/lymphokine and protooncogene mRNAs. In this article, we describe recent advances in the characterization of ARE-directed mRNA turnover, including links to deadenylation kinetics and functional heterogeneity among AREs from different mRNAs. We then describe strategies employed in the search for trans-acting factors interacting with these elements. Using such techniques, an ARE-binding activity capable of accelerating c-myc mRNA turnover in vitro was identified, and named AUF1. Subsequent cloning and characterization revealed that AUF1 exists as a family of four proteins formed by alternative splicing of a common pre-mRNA and appears to function as part of a multisubunit trans-acting complex to promote ARE-directed mRNA turnover. Investigations using several systems have demonstrated that AUF1 expression and/or activity correlate with rapid decay of ARE-containing mRNAs, and that both expression and activity of AUF1 are regulated by developmental and signal transduction mechanisms.

The Mos pathway regulates cytoplasmic polyadenylation in Xenopus oocytes.

Cytoplasmic polyadenylation controls the translation of several maternal mRNAs during Xenopus oocyte maturation and requires two sequences in the 3' untranslated region (UTR), the U-rich cytoplasmic polyadenylation element (CPE), and the hexanucleotide AAUAAA. c-mos mRNA is polyadenylated and translated soon after the induction of maturation, and this protein kinase is necessary for a kinase cascade culminating in cdc2 kinase (MPF) activation. Other mRNAs are polyadenylated later, around the time of cdc2 kinase activation. To determine whether there is a hierarchy in the cytoplasmic polyadenylation of maternal mRNAs, we ablated c-mos mRNA with an antisense oligonucleotide. This prevented histone B4 and cyclin A1 and B1 mRNA polyadenylation, indicating that the polyadenylation of these mRNAs is Mos dependent. To investigate a possible role of cdc2 kinase in this process, cyclin B was injected into oocytes lacking c-mos mRNA. cdc2 kinase was activated, but mitogen-activated protein kinase was not. However, polyadenylation of cyclin B1 and histone B4 mRNA was still observed. This demonstrates that cdc2 kinase can induce cytoplasmic polyadenylation in the absence of Mos. Our data further indicate that although phosphorylation of the CPE binding protein may be involved in the induction of Mos-dependent polyadenylation, it is not required for Mos-independent polyadenylation. We characterized the elements conferring Mos dependence (Mos response elements) in the histone B4 and cyclin B1 mRNAs by mutational analysis. For histone B4 mRNA, the Mos response elements were in the coding region or 5' UTR. For cyclin B1 mRNA, the main Mos response element was a CPE that overlaps with the AAUAAA hexanucleotide. This indicates that the position of the CPE can have a profound influence on the timing of cytoplasmic polyadenylation.

Structural Determinants in AUF1 Required for High Affinity Binding to A + U-rich Elements

AUF1 is an RNA-binding protein that contains two nonidentical RNA recognition motifs (RRMs). AUF1 binds to A + U-rich elements (AREs) with high affinity. The binding of AUF1 to AREs is believed to serve as a signal to an mRNA-processing pathway that degrades mRNAs encoding many cytokines, oncoproteins, and G protein-coupled receptors. Because the ARE binding activity of AUF1 appears central to the regulation of many important genes, we analyzed the domains of the protein that are important for this activity. Examination of the RNA binding affinity of various AUF1 mutants suggests that both RRMs may be required for binding to the human c-fos ARE. However, the two RRMs together are not sufficient. Highest affinity binding of AUF1 to an ARE requires an alanine-rich region of the N terminus and a short glutamine-rich region in the C terminus. In addition, the N terminus is required for dimerization of AUF1. However, AUF1 binds an ARE as a hexameric protein. Thus, protein-protein interactions are important for high affinity ARE binding activity of AUF1.

RNA recognition by the human polyadenylation factor CstF.

Polyadenylation of mammalian mRNA precursors requires at least two signal sequences in the RNA: the nearly invariant AAUAAA, situated 5' to the site of polyadenylation, and a much more variable GU- or U-rich downstream element. At least some downstream sequences are recognized by the heterotrimeric polyadenylation factor CstF, although how, and indeed if, all variations of this diffuse element are bound by a single factor is unknown. Here we show that the RNP-type RNA binding domain of the 64-kDa subunit of CstF (CstF-64) (64K RBD) is sufficient to define a functional downstream element. Selection-amplification (SELEX) experiments employing a glutathione S-transferase (GST)-64K RBD fusion protein selected GU-rich sequences that defined consensus recognition motifs closely matching those present in natural poly(A) sites. Selected sequences were bound specifically, and with surprisingly high affinities, by intact CstF and were functional in reconstituted, CstF-dependent cleavage assays. Our results also indicate that GU- and U-rich sequences are variants of a single CstF recognition motif. For comparison, SELEX was performed with a GST fusion containing the RBD from the apparent yeast homolog of CstF-64, RNA15. Strikingly, although the two RBDs are almost 50% identical and yeast poly(A) signals are at least as degenerate as the mammalian downstream element, a nearly invariant 12-base U-rich sequence distinct from the CstF-64 consensus was identified. We discuss these results in terms of the function and evolution of mRNA 3'-end signals.

Mouse cytoplasmic polyadenylylation element binding protein: an evolutionarily conserved protein that interacts with the cytoplasmic polyadenylylation elements of c-mos mRNA.

Cytoplasmic polyadenylylation is an essential process that controls the translation of maternal mRNAs during early development and depends on two cis elements in the 3' untranslated region: the polyadenylylation hexanucleotide AAUAAA and a U-rich cytoplasmic polyadenylylation element (CPE). In searching for factors that could mediate cytoplasmic polyadenylylation of mouse c-mos mRNA, which encodes a serine/threonine kinase necessary for oocyte maturation, we have isolated the mouse homolog of CPEB, a protein that binds to the CPEs of a number of mRNAs in Xenopus oocytes and is required for their polyadenylylation. Mouse CPEB (mCPEB) is a 62-kDa protein that binds to the CPEs of c-mos mRNA. mCPEB mRNA is present in the ovary, testis, and kidney; within the ovary, this RNA is restricted to oocytes. mCPEB shows 80% overall identity with its Xenopus counterpart, with a higher homology in the carboxyl-terminal portion, which contains two RNA recognition motifs and a cysteine/histidine repeat. Proteins from arthropods and nematodes are also similar to this region, suggesting an ancient and widely used mechanism to control polyadenylylation and translation.

Increased Granulocyte-Macrophage Colony-Stimulating Factor mRNA Instability in Cord Versus Adult Mononuclear Cells Is Translation-Dependent and Associated With Increased Levels of A + U-Rich Element Binding Factor

The level of granulocyte-macrophage colony-stimulating factor (GM-CSF) mRNA is fourfold lower in phorbol myristate acetate (PMA) + phytohemagglutinin (PHA)-activated mononuclear cells (MNC) from newborns compared with adults. The GM-CSF transcription rate is similar in umbilical cord and adult MNC, but transcript half-life is threefold lower in cord activated MNC. Interaction of RNA binding proteins, such as the cloned adenosine + uridine-rich element, binding factor, AUF1, with eight AUUUA motifs in the human GM-CSF mRNA 3'-untranslated region (GM-3'-UTR) has been implicated in regulating transcript stability. Translational inhibition by cycloheximide (CHX) significantly increased GM-CSF mRNA accumulation and half-life by three-fold in activated cord MNC, but had a minimal effect in activated adult MNC as compared with PMA + PHA alone. Electrophoretic mobility-shift assays with a 32P-labeled, 305-nucleotide RNA comprising the GM-3'-UTR revealed two RNaseT1-resistant, bound complexes that were almost twice as abundant in cord than in adult MNC extracts. Mobility-shift competition assays and RNaseT1 mapping localized the binding site of both complexes to a 52-nucleotide region containing seven of eight AUUUA motifs. Inclusion of AUF1 antiserum produced a supershifted complex at 35-fold higher levels in cord than in adult MNC extracts. Extracts from the carcinoma cell line 5637, with extended GM-CSF mRNA half-life, also had very low levels of anti-AUF1 supershifted complex. Anti-AUF1 immunoblotting showed significantly higher levels of two AUF1 protein isoforms and lower levels of one in cord than in adult MNC or 5637 extracts. These results suggest that destabilization of GM-CSF mRNA in cord MNC is translation-dependent and that increased levels of specific AUF1 isoforms in cord MNC may target transcripts for increased degradation, which could account in part for dysregulation of neonatal phagocytic immunity.

AUF1 Binding Affinity to A+U-rich Elements Correlates with Rapid mRNA Degradation

Rapid degradation of many labile mRNAs is regulated in part by an A + U-rich element (ARE) in their 3'-untranslated regions. Extensive mutational analyses of various AREs have identified important components of the ARE, such as the nonamer motif UUAUUUAUU, two copies of which serve as a potent mRNA destabilizer. To investigate the roles of trans-acting factors in ARE-directed mRNA degradation, we previously purified and molecularly cloned the RNA-binding protein AUF1 and demonstrated that both cellular and recombinant AUF1 bind specifically to AREs as shown by UV cross-linking assays in vitro. In the present work, we have examined the in vitro RNA-binding properties of AUF1 using gel mobility shift assays with purified recombinant His6-AUF1 fusion protein. We find that ARE binding affinities of AUF1 correlate with the potency of an ARE to direct degradation of a heterologous mRNA. These results support a role for AUF1 in ARE-directed mRNA decay that is based upon its affinity for different AREs.

CPEB controls the cytoplasmic polyadenylation of cyclin, Cdk2 and c-mos mRNAs and is necessary for oocyte maturation in Xenopus.

Cytoplasmic polyadenylation is a key mechanism controlling maternal mRNA translation in early development. In most cases, mRNAs that undergo poly(A) elongation are translationally activated; those that undergo poly(A) shortening are deactivated. Poly(A) elongation is regulated by two cis-acting sequences in the 3'-untranslated region (UTR) of responding mRNAs, the polyadenylation hexanucleotide AAUAAA and the U-rich cytoplasmic polyadenylation element (CPE). Previously, we cloned and characterized the Xenopus oocyte CPE binding protein (CPEB), showing that it was essential for the cytoplasmic polyadenylation of B4 RNA. Here, we show that CPEB also binds the CPEs of G10, c-mos, cdk2, cyclins A1, B1 and B2 mRNAs. We find that CPEB is necessary for polyadenylation of these RNAs in egg extracts, suggesting that this protein is required for polyadenylation of most RNAs during oocyte maturation. Our data demonstrate that the complex timing and extent of polyadenylation are partially controlled by CPEB binding to multiple target sites in the 3' UTRs of responsive mRNAs. Finally, injection of CPEB antibody into oocytes not only inhibits polyadenylation in vivo, but also blocks progesterone-induced maturation. This is due to inhibition of polyadenylation and translation of c-mos mRNA, suggesting that CPEB is critical for early development.

Regulation of the mRNA-binding Protein AUF1 by Activation of the β -Adrenergic Receptor Signal Transduction Pathway

In both cell culture based model systems and in the failing human heart, beta-adrenergic receptors ( beta-AR) undergo agonist-mediated down-regulation. This decrease correlates closely with down-regulation of its mRNA, an effect regulated in part by changes in mRNA stability. Regulation of mRNA stability has been associated with mRNA-binding proteins that recognize A + U-rich elements within the 3'-untranslated regions of many mRNAs encoding proto-oncogene and cytokine mRNAs. We demonstrate here that the mRNA-binding protein, AUF1, is present in both human heart and in hamster DDT1-MF2 smooth muscle cells and that its abundance is regulated by beta-AR agonist stimulation. In human heart, AUF1 mRNA and protein was significantly increased in individuals with myocardial failure, a condition associated with increases in the beta-adrenergic receptor agonist norepinephrine. In the same hearts, there was a significant decrease (approximately 50%) in the abundance of beta1-AR mRNA and protein. In DDT1-MF2 cells, where agonist-mediated destabilization of beta2-AR mRNA was first described, exposure to beta-AR agonist resulted in a significant increase in AUF1 mRNA and protein (approximately 100%). Conversely, agonist exposure significantly decreased (approximately 40%) beta2-adrenergic receptor mRNA abundance. Last, we demonstrate that AUF1 can be immunoprecipitated from polysome-derived proteins following UV cross-linking to the 3'-untranslated region of the human beta1-AR mRNA and that purified, recombinant p37AUF1 protein also binds to beta1-AR 3'-untranslated region mRNA.

Cloning of the mink plasminogen activator inhibitor type-1 messenger RNA: An mRNA with a short half life

In mink lung CCL64 epithelial cells the rate of synthesis of plasminogen activator inhibitor type I (PAI-1) increases 10–100-fold within 3 h in response to 12-O-tetradecanoyl phorbol-13-acetate (PMA). The PAI-1 gene is regulated transcriptionally. Parallel studies of the time-courses of PAI-1 synthesis and secretion and of mRNA accumulation indicate that the amount of secreted PAI-1 produced by the cells is tightly coupled to the level of its transcript. The half-life of the PAI-1 mRNA was found to be 25 min which is much shorter than previously reported for PAI-1 in other cells. Actinomycin D, which is commonly used to determine mRNA half-life, stabilized the PAI-1 mRNA. Cycloheximide also stabilized the mRNA. The short half-life and the superinducibility of PAI mRNA are properties shared with rapidly degraded mRNAs encoding protooncoproteins. A 2.97-kb cDNA clone containing the entire coding sequence of PAI-1 was isolated from a cDNA library made from mink lung CCL64 epithelial cells stimulated with PMA. The PAI-1 cDNA contains a long 3′-untranslated region ( UTR) of 1720 bp whose sequence is highly conserved among PAI-1 mRNAs from different species. The PAI-1 mRNA also contains several AUUUA pentamer sequences which are the features of an A + U-rich regulatory element such as is found on the fos protooncogene mRNA. Upstream of one of these AUUUA pentamers are several highly conserved sequences that are also found in the 3′ UTR of the fos and integrin receptor α-subunit mRNAs. These are mRNAs that encode proteins which are involved in the cellular response to tissue wounds. Thus, it is possible that these conserved 3' sequences may be important for regulating the production of Fos, the integrin α-subunit and PAI-1 during wound healing.

Cleavage site determinants in the mammalian polyadenylation signal.

Using a series of position and nucleotide variants of the SV40 late polyadenylation signal we have demonstrated that three sequence elements determine the precise site of 3-end cleavage in mammalian pre-mRNAs: an upstream AAUAAA element, a down-stream U-rich element consisting of five nucleotides, at least four of which are uridine, and a nucleotide preference at the site of cleavage in the order A > U > C >> G. Cleavage occurs no closer than 11 bases, but no further than 23 bases from the AAUAAA element. The downstream U-rich element is usually located 10-30 bases from the cleavage site. The relative position of the AAUAAA and the U-rich elements define the approximate region within a 13 base domain in which cleavage will occur. The exact position of cleavage is then determined by the local nucleotide sequence in the order of preference noted above. This model accounts for nearly three quarters of polyadenylation signals surveyed and is consistent with previous experimental observations.

Translational control by cytoplasmic polyadenylation of c-mos mRNA is necessary for oocyte maturation in the mouse.

The c-mos proto-oncogene product is a key element in the cascade of events leading to meiotic maturation of vertebrate oocytes. We have investigated the role of cytoplasmic polyadenylation in the translational control of mouse c-mos mRNA and its contribution to meiosis. Using an RNase protection assay we show that optimal cytoplasmic polyadenylation of c-mos mRNA requires three cis elements in the 3' UTR: the polyadenylation hexanucleotide AAUAAA and two U-rich cytoplasmic polyadenylation elements (CPEs) located 4 and 51 nucleotides upstream of the hexanucleotide. When fused to CAT coding sequences, the wild-type 3' UTR of c-mos mRNA, but not a 3' UTR containing mutations in both CPEs, confers translational recruitment during maturation. This recruitment coincides with maximum polyadenylation. To assess whether c-mos mRNA polyadenylation is necessary for maturation of mouse oocytes, we have ablated endogenous c-mos mRNA by injecting an antisense oligonucleotide, which results in a failure to progress to meiosis II after emission of the first polar body. Such antisense oligonucleotide-injected oocytes could be efficiently rescued by co-injection of a c-mos mRNA carrying a wild-type 3' UTR. However, co-injection of a c-mos mRNA lacking functional CPEs substantially lowered the rescue activity. These results demonstrate that translational control of c-mos mRNA by cytoplasmic polyadenylation is necessary for normal development.

Characterization of cDNAs encoding the murine A + U-rich RNA-binding protein AUF1

A + U-rich elements ( ARE) serve to control the degradation of some proto-oncogene and lymphokine mRNAs. The protein, AUF1, which consists of two polypeptides of 37 and 40 kDa (p37 and p40, respectively) when purified from cytosol, has been implicated in ARE-directed mRNA turnover due to its binding to ARE. Molecular cloning of a cDNA ( p37 AUF1 ) corresponding to human p37 predicted a polypeptide containing two non-identical RNA recognition motifs (RRM) and a C-terminal Gin-rich domain [Zhang et al. Mol. Cell. Biol. 13 (1993) 7652–7665]. Two cDNAs, designated mu AUF1-3 and mu AUF1-7, were isolated from a murine fetal cDNA library, using as a probe, a fragment of the p37 AUF1 cDNA encoding RRM1 and approximately half of RRM2. The mu AUFl-3 open reading frame (ORF) was very homologous to human p37 AVF1 with the greatest homology between the corresponding RRMs and the C-terminal Gin-rich motif. Clone mu AUFl-7 was highly homologous to mu AUFl-3, but was truncated within the region encoding the RNP-1 box in RRM2. Clone mu AUFl-3 encoded 19 amino acids in RRM1 not encoded by either mu AUFl-7 or human p37 AUF1 . Such alterations in sequence could modify the RNA-binding properties of these proteins and have concomitant effects on ARE-directed posttranscriptional processes.

Polyadenylation and deadenylation of maternal mRNAS during oocyte growth and maturation in the mouse

We previously showed that, during mouse oocyte maturation, specific maternal mRNAs (actins) are deadenylated, while others (hypoxanthine phosphoribosyltransferase:HPRT) are adenylated. As in other systems, these changes can be correlated with changes in translational activities. Maturation-specific polyadenylation in Xenopus depends on the presence of a U-rich cytoplasmic polyadenylation element (CPE) close to the 3' end of the RNA. RNAs that lack CPEs appear to be deadenylated by default when meiosis resumes. We show here that this default program also applies to maturing mouse oocytes. Microinjected beta- and gamma-actin 3' UTR (untranslated region) transcripts lacking CPEs but including polyA tails (100-200 N) behave as endogenous maternal actin mRNAs and are deadenylated by maturing oocytes. "Nonsense" transcripts that do not include CPEs, but that do contain polyA tails, are also deadenylated. beta- and gamma-Actin 3' UTRs with short polyA tails (50-80 N) are stable and exhibit no detectable change in adenylation when injected into growing, full-grown, or maturing oocytes. In contrast, HPRT 3' UTRs, which include the CPE UUUUAAAU and a short polyA tail (50 N), are polyadenylated during maturation. HPRT 3' UTR transcripts with long polyA tails (100-200 N) are more extensively deadenylated by growing and full-grown oocytes that retain germinal vesicles than by maturing oocytes. The presence of CPEs may be required for polyA tail shortening and translational inactivation of stable mRNAs during oocyte growth and subsequent selective readenylation and translation during meiotic maturation.(ABSTRACT TRUNCATED AT 250 WORDS)

Sequence and position requirements for uridylate-rich downstream elements of polyadenylation signals.

We have defined the positional and sequence requirements of U-rich downstream elements using a simian virus 40 late polyadenylation signal containing a substituted downstream region. A UUUUU element will significantly increase the efficiency of 3' end processing when placed between 6 and 25 bases downstream from the cleavage site. Positions in this interval closer than 15 bases from the cleavage site, however, were noticeably less efficient. Placement of the UUUUU element between +20 and +25 caused a partial shift in cleavage site usage to a CA motif at +4. Mutational analysis indicated that the sequence requirements at individual positions of the UUUUU element were somewhat flexible. Changing more than one base of the UUUUU sequence, however, severely diminished the ability of the element to mediate efficient 3' end processing. Finally, although hnRNP C proteins specifically interact with U-rich sequences, this protein--RNA interaction is not required for efficient in vitro polyadenylation.