RINGO/cdk1 and CPEB mediate poly(A) tail stabilization and translational regulation by ePAB

One activity that controls mRNA translation in vertebrate oocytes, embryos, and neurons is cytoplasmic polyadenylation. In Xenopus oocytes, where much of the biochemistry of this process has been elucidated, nuclear pre-mRNAs containing a cytoplasmic polyadenylation element (CPE) in their 3' untranslated regions (UTRs) have long poly(A) tails; once the RNAs are spliced and transported to the cytoplasm, the tails are shortened. Following the resumption of meiosis, the poly(A) tails are lengthened and translation ensues. CPEB is a sequence-specific RNA-binding protein that coordinates these events and does so by binding to the CPE as well as several factors including Gld2, a poly(A) polymerase, and PARN [poly(A)-specific ribonuclease], a deadenylase. Here, we show that ePAB, embryonic poly(A)-binding protein, transiently associates with the polyadenylation complex; it initially interacts with CPEB, but after polyadenylation, it binds the poly(A) tail. ePAB dissociation from CPEB is regulated by RINGO (Rapid Inducer of G(2)/M progression in Oocytes), a cyclin B1-like cofactor that activates cdk1, a protein kinase that phosphorylates CPEB. Subsequent ePAB binding to the poly(A) tail is necessary to protect the homopolymer from degradation by deadenylating enzymes. Poly(A)-bound ePAB also interacts with eIF4G, which instigates translation initiation of CPEB-bound mRNAs.     

Tethering of eIF4G to adenoviral mRNAs by viral 100k protein drives ribosome shunting

Although most mRNAs initiate translation by 5' ribosome scanning, some small fraction of mammalian and viral mRNAs utilize either of two alternate mechanisms, known as internal ribosome entry and ribosome shunting. Ribosome shunting is a poorly understood form of initiation in which 40S ribosome subunits are loaded onto mRNA through interactions with the m7GTP cap, but then bypass large segments of the mRNA as directed by cis-acting RNA shunting elements and trans-acting protein factors. Here, we describe the molecular mechanism by which ribosome shunting occurs with high efficiency on adenovirus late mRNAs. We show that the viral 100k protein possesses a selective binding element for the 5' noncoding region (5'NCR) of viral late mRNAs (known as the tripartite leader), forms a complex with initiation factor eIF4G and poly(A)-binding protein (PABP), and strongly and selectively enhances the level of both factors and 40S ribosome subunits on viral mRNAs in polysomes. Mutational and biochemical studies demonstrate that the ability of 100k protein to bind both the tripartite leader and eIF4G are critical to promote a high level of ribosome shunting. A molecular mechanism for ribosome shunting is described by which enhanced binding of eIF4G and possibly PABP with 100k protein, and simultaneous interaction with the tripartite leader 5'NCR, drives 40S ribosome recruitment and initiation on mRNAs.     

Epidermal growth factor enhances preimplantation developmental competence of maturing mouse oocytes

The objective of this study was to determine whether epidermal growth factor (EGF) promotes nuclear and cytoplasmic maturation of mouse oocytes grown in vivo or in vitro. In-vivo-grown oocytes were isolated at the germinal vesicle (GV) stage from gonadotrophin-primed (PR) or -unprimed (UPR) 22-day-old mice before in-vitro maturation (IVM). In-vitro-grown (IVG) oocytes were isolated from preantral follicles of 12-day-old mice and grown in vitro without gonadotrophins for 10 days before maturation (IVG/IVM oocytes). IVM and IVG/IVM oocytes were matured in medium supplemented with either EGF (10 ng/ml), follicle stimulating hormone (FSH) (100 ng/ml), EGF plus FSH, or with neither ligand (control). When oocyte-cumulus cell complexes were isolated from PR and UPR mice, IVM with EGF (10 ng/ml), alone or in combination with FSH (100 ng/ml), increased (P < 0.05) the incidence of nuclear maturation to metaphase II. Cytoplasmic maturation of oocytes from PR females, manifested as increased frequency of cleavage to the 2-cell stage and development to the blastocyst stage, was also enhanced with EGF (P < 0.05). Moreover, EGF increased the number of cells per blastocyst, but only in the absence of FSH (P < 0.01). In contrast, EGF, FSH, or EGF plus FSH did not affect the percentage of oocytes from UPR mice completing preimplantation development, but did increase the number of cells per blastocyst. These ligands also increased the proportion of IVG oocytes reaching metaphase II (53-57%) compared with controls (25%; P < 0.05). EGF alone or in combination with FSH increased (P < 0.05) the frequency of blastocyst formation (23% and 28%, respectively) compared with controls (13%). EGF treatment of maturing IVG oocytes produced blastocysts with more cells than other IVG groups (P < 0.05). It is concluded that gonadotrophins in vivo increase the sensitivity or responsiveness of cumulus cell-enclosed oocytes to EGF, thereby promoting both nuclear and cytoplasmic maturation. However, oocyte-granulosa cell complexes grown in vitro become responsive to EGF without gonadotrophin treatment. Thus, nuclear and cytoplasmic maturation of IVG oocytes is promoted by EGF treatment during meiotic maturation.     

Four factors are required for 3'-end cleavage of pre-mRNAs

We reported previously that authentic polyadenylation of pre-mRNAs in vitro requires at least two factors: a cleavage/specificity factor (CSF) and a fraction containing nonspecific poly(A) polymerase activity. To study the molecular mechanisms underlying 3' cleavage of pre-mRNAs, we fractionated CSF further and show that it consists of four separable subunits. One of these, called specificity factor (SF; Mr, approximately 290,000), is required for both specific cleavage and for specific polyadenylation and thus appears responsible for the specificity of the reaction. Although SF has not been purified to homogeneity, several lines of evidence suggest that it may not contain an essential RNA component. Two other factors, designated cleavage factors I (CFI; Mr, approximately 130,000) and II (CFII; Mr, approximately 110,000), are sufficient to reconstitute accurate cleavage when mixed with SF. A fourth factor, termed cleavage stimulation factor (CstF; Mr, approximately 200,000), enhances cleavage efficiency significantly when added to a mixture of the three other factors. CFI, CFII, and CstF do not contain RNA components, nor do they affect specific polyadenylation in the absence of cleavage. Although these four factors are necessary and sufficient to reconstitute efficient cleavage of one pre-RNA tested, poly(A) polymerase is also required to cleave several others. A model suggesting how these factors interact with the pre-mRNA and with each other is discussed.     

Uncoupling of GTP hydrolysis from eIF6 release on the ribosome causes Shwachman-Diamond syndrome

Removal of the assembly factor eukaryotic initiation factor 6 (eIF6) is critical for late cytoplasmic maturation of 60S ribosomal subunits. In mammalian cells, the current model posits that eIF6 release is triggered following phosphorylation of Ser 235 by activated protein kinase C. In contrast, genetic studies in yeast indicate a requirement for the ortholog of the SBDS (Shwachman-Bodian-Diamond syndrome) gene that is mutated in the inherited leukemia predisposition disorder Shwachman-Diamond syndrome (SDS). Here, by isolating late cytoplasmic 60S ribosomal subunits from Sbds-deleted mice, we show that SBDS and the GTPase elongation factor-like 1 (EFL1) directly catalyze eIF6 removal in mammalian cells by a mechanism that requires GTP binding and hydrolysis by EFL1 but not phosphorylation of eIF6 Ser 235. Functional analysis of disease-associated missense variants reveals that the essential role of SBDS is to tightly couple GTP hydrolysis by EFL1 on the ribosome to eIF6 release. Furthermore, complementary NMR spectroscopic studies suggest unanticipated mechanistic parallels between this late step in 60S maturation and aspects of bacterial ribosome disassembly. Our findings establish a direct role for SBDS and EFL1 in catalyzing the translational activation of ribosomes in all eukaryotes, and define SDS as a ribosomopathy caused by uncoupling GTP hydrolysis from eIF6 release.     

Protein quality control systems associated with no‐go and nonstop mRNA surveillance in yeast

Quality control systems eliminate aberrant proteins derived from aberrant mRNAs. Two E3 ubiquitin ligases, Ltn1 and Not4, are involved in proteasomal protein degradation coupled to translation arrest. Here, we evaluated nonstop and translation arrest products degraded in a poly(A) tail‐independent manner. Ltn1 was found to degrade aberrant nonstop polypeptides derived from nonstop mRNA lacking a termination codon, but not peptidyl‐tRNA, even in the absence of the ribosome dissociation complex Dom34:Hbs1. The receptor for activated C kinase (RACK1/ASC1) was identified as a factor required for nascent peptide‐dependent translation arrest as well as Ltn1‐dependent protein degradation. Both Not4 and Ltn1 were involved in the degradation of various arrest products in a poly(A) tail‐independent manner. Furthermore, carboxyl terminus‐truncated degradation intermediates of arrest products were stabilized in a cdc48‐3 mutant defective in unfolding or the disassembly related to proteasomal degradation. Thus, we propose that stalled ribosomes may be dissociated into subunits and that peptidyl‐tRNA on the 60S subunit is ubiquitinated by Ltn1 and Cdc48 is required for the degradation following release from tRNA.     

Polyadenylation of SV40 late pre-mRNA is dependent on phosphorylation of an essential component associated with the 3' end processing machinery.

We investigated whether phosphorylation of the essential components involved in the 3' end processing of mRNAs was required for mRNA polyadenylation. The proteins in HeLa nuclear extract were dephosphorylated with alkaline phosphatase, which is known to remove the phosphate moieties from serine and tyrosine. The dephosphorylated extract was used for analyzing cleavage-dependent polyadenylation of SV40 late pre-mRNA. The phosphatase treatment of the extract completely blocked the polyadenylation reaction, whereas dephosphorylation of the extract did not inhibit the cleavage reaction. Since the cleavage depends upon functional integrity of the specificity factor, it is unlikely that the phosphorylated state of the latter factor is required for the 3' end processing. Sodium vanadate, a potent inhibitor of alkaline phosphatase, markedly reduced the inhibitory effect of the phosphatase on the polyadenylation reaction. Dephosphorylation of the extract also prevented formation of the polyadenylation-specific complex with pre-mRNA, whereas the cleavage-specific complexes were formed under this condition. The Mn-dependent polyadenylation, which is largely poly(A) extension reaction, was relatively resistant to the phosphatase treatment. These data indicate that phosphorylation of a key factor is essential for the 3' end processing of pre-mRNA, and suggest that the factor may be poly(A) polymerase.     

Maturation-specific polyadenylation: in vitro activation by p34cdc2 and phosphorylation of a 58-kD CPE-binding protein.

During Xenopus oocyte maturation, poly(A) elongation controls the translational recruitment of specific mRNAs that possess a CPE (cytoplasmic polyadenylation element). To investigate the activation of polyadenylation, we have employed oocyte extracts that are not normally competent for polyadenylation. Addition of cell lysates containing baculovirus-expressed cyclin to these extracts induces the polyadenylation of exogenous B4 RNA. The involvement of p34cdc2 kinase in cyclin-mediated polyadenylation was demonstrated by p13-Sepharose depletion; removal of the kinase from oocyte extracts with this affinity matrix abolishes polyadenylation activation. Reintroduction of cell lysates containing baculovirus-expressed p34cdc2, however, completely restores this activity. To identify factors of the polyadenylation apparatus that might be responsible for the activation, we employed UV cross-linking and identified a 58-kD protein that binds the B4 CPE in oocyte extracts. In polyadenylation-proficient egg extracts, this protein has a slower electrophoretic mobility, which suggests a post-translational modification. A similar size shift of the protein is evident in oocyte extracts supplemented with lysates containing baculovirus-expressed cyclin and p34cdc2. This size shift, which is reversed by treatment with acid phosphatase, coincides temporally with cyclin-induced polyadenylation activation. We propose that p34cdc2 kinase activity leads to the phosphorylation of the 58-kD CPE-binding protein and that this event is crucial for the cytoplasmic polyadenylation that occurs during oocyte maturation.     

The tRNA-like structure of Turnip yellow mosaic virus RNA is a 3'-translational enhancer

Many positive stand RNA viral genomes lack the poly(A) tail that is characteristic of cellular mRNAs and that promotes translation in cis. The 3′ untranslated regions (UTRs) of such genomes are expected to provide similar translation-enhancing properties as a poly(A) tail, yet the great variety of 3′ sequences suggests that this is accomplished in a range of ways. We have identified a translational enhancer present in the 3′ UTR of Turnip yellow mosaic virus (TYMV) RNA using luciferase reporter RNAs with generic 5′ sequences transfected into plant cells. The 3′ terminal 109 nucleotides comprising the tRNA-like structure (TLS) and an upstream pseudoknot (UPSK) act in synergy with a 5′-cap to enhance translation, with a minor contribution in stabilizing the RNA. Maximum enhancement requires that the RNA be capable of aminoacylation, but either the native valine or engineered methionine is acceptable. Mutations that decrease the affinity for translation elongation factor eEF1A (but also diminish aminoacylation efficiency) strongly decrease translational enhancement, suggesting that eEF1A is mechanistically involved. The UPSK seems to act as an important, though nonspecific, spacer element ensuring proper presentation of a functional TLS. Our studies have uncovered a novel type of translational enhancer and a new role for a plant viral TLS.