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.     

Cytoplasmic distribution of poly(A)-containing RNA in developing Necturus maculosus oocytes with reference to annulate lamellae

The cytoplasmic distribution of poly(A)⁺ mRNA and its relationship to annulate lamellae were examined in developing Necturus maculosus oocytes by in situ hybridization with [³H]poly(U). The specificity of [³H]poly(U) binding was tested by incubating control ovarian sections with either KOH or RNase A before in situ hybridization. In both experiments, the silver grain densities were markedly reduced. Poly(A)⁺ RNA is uniformly distributed in the cytoplasm until the mid-growth phase and then later in vitellogenesis becomes localized in the subcortical ooplasm. The silver grain density in the cytoplasm varied during oogenesis and was greatest in previtellogenic oocytes. Annulate lamellae commonly are observed with the light microscope in oocytes prior to vitellogenesis. In such oocytes, the labeled mRNA probe is observed over cytoplasmic regions of annulate lamellae. The results suggests that a differential localization of messenger RNA occurs during oogenesis in Necturus maculosus. Furthermore, poly(A)⁺ RNA is present in cytoplasmic regions of annulate lamellae.     

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.     

CD5 expression is regulated during human T‐cell activation by alternative polyadenylation,PTBP1, and miR‐204

T lymphocytes stimulated through their antigen receptor (TCR) preferentially express mRNA isoforms with shorter 3´ untranslated regions (3´‐UTRs) derived from alternative pre‐mRNA cleavage and polyadenylation (APA). However, the physiological relevance of APA programs remains poorly understood. CD5 is a T‐cell surface glycoprotein that negatively regulates TCR signaling from the onset of T‐cell activation. CD5 plays a pivotal role in mediating outcomes of cell survival or apoptosis, and may prevent both autoimmunity and cancer. In human primary T lymphocytes and Jurkat cells we found three distinct mRNA isoforms encoding CD5, each derived from distinct poly(A) signals (PASs). Upon T‐cell activation, there is an overall increase in CD5 mRNAs with a specific increase in the relative expression of the shorter isoforms. 3´‐UTRs derived from these shorter isoforms confer higher reporter expression in activated T cells relative to the longer isoform. We further show that polypyrimidine tract binding protein (PTB/PTBP1) directly binds to the proximal PAS and PTB siRNA depletion causes a decrease in mRNA derived from this PAS, suggesting an effect on stability or poly(A) site selection to circumvent targeting of the longer CD5 mRNA isoform by miR‐204. These mechanisms fine‐tune CD5 expression levels and thus ultimately T‐cell responses.     

Drosophila PTB promotes formation of high-order RNP particles and represses oskar translation

Local translation of asymmetrically enriched mRNAs is a powerful mechanism for functional polarization of the cell. In Drosophila, exclusive accumulation of Oskar protein at the posterior pole of the oocyte is essential for development of the future embryo. This is achieved by the formation of a dynamic oskar ribonucleoprotein (RNP) complex regulating the transport of oskar mRNA, its translational repression while unlocalized, and its translational activation upon arrival at the posterior pole. We identified the nucleo–cytoplasmic shuttling protein PTB (polypyrimidine tract-binding protein)/hnRNP I as a new factor associating with the oskar RNP in vivo. While PTB function is largely dispensable for oskar mRNA transport, it is necessary for translational repression of the localizing mRNA. Unexpectedly, a cytoplasmic form of PTB can associate with oskar mRNA and repress its translation, suggesting that nuclear recruitment of PTB to oskar complexes is not required for its regulatory function. Furthermore, PTB binds directly to multiple sites along the oskar 3′ untranslated region and mediates assembly of high-order complexes containing multiple oskar RNA molecules in vivo. Thus, PTB is a key structural component of oskar RNP complexes that dually controls formation of high-order RNP particles and translational silencing.     

Distinct roles for hu li tai shao and swallow in cytoskeletal organization during Drosophila oogenesis

Background: Cytoskeletal organization is essential for localization of developmentally significant molecules during Drosophila oogenesis. Swallow (Swa) and an isoform of Hu li tai shao (Ovhts‐RC) have been implicated in the organization of actin filaments in developing oocytes but their precise roles have been obscured by the dependence of hts RNA localization on swa function. The functional significance of hts RNA localization in the oocyte has not been established. Results: In this study we examine Ovhts‐RC distribution and cytoskeletal organization under conditions in which Swa protein and/or hts RNA localization are perturbed. We find Swa is required for overall actin organization and for the maintenance of a distinct subset of microtubules in the oocyte. hts RNA localization modulates the distribution of Ovhts‐RC in the oocyte and, in turn, local actin filament proliferation. Conclusions: Our results support separate contributions of Swa and hts RNA localization to actin organization during oogenesis. Swa is crucial for the organization of actin networks that lead to the formation of a specialized microtubule population, while Ovhts‐RC acts to modulate spatially restricted actin filament growth at the oocyte cortex. This suggests RNA localization can lead to modifications of both the actin and microtubule cytoskeletons at specific subcellular locales. Developmental Dynamics 243:906–916, 2014. © 2014 The Authors Developmental Dynamics published by Wiley Periodicals, Inc. on behalf of American Association of Anatomists     

Over-expression of Aurora-A targets cytoplasmic polyadenylation element binding protein and promotes mRNA polyadenylation of Cdk1 and cyclin B1

Aurora-A is a centrosomal serine-threonine kinase that regulates mitosis. Over-expression of Aurora-A has been found in a wide range of tumors and has been implicated in oncogenic transformation. However, how Aurora-A over-expression contributes to promotion of carcinogenesis remains elusive. Immunohistochemical analysis of breast tumors revealed that over-expressed Aurora-A is not restricted to the centrosomes but is also found in the cytoplasm. This over-expressed Aurora-A appeared to be phosphorylated on Thr288, which is known to be required for its enzymatic activation. In analogy to Aurora-A's role in oocyte maturation and the early embryonic cell cycle, here we investigated whether ectopically over-expressed Aurora-A can similarly stimulate polyadenylation of mRNA in human somatic cultured cells by interacting with a human ortholog of cytoplasmic polyadenylation element binding protein, h-CPEB. In vitro experiments revealed that Aurora-A binds directly to, and phosphorylates, h-CPEB. We found that polyadenylation of mRNA tails of cyclin B1 and Cdk1 was synergistically stimulated when Aurora-A and h-CPEB were over-expressed, and they were further promoted in the presence of an Aurora-A activator Ajuba. Our results suggest a function of ectopically over-expressed Aurora-A that might be relevant for carcinogenesis.     

Hrp1, a sequence-specific RNA-binding protein that shuttles between the nucleus and the cytoplasm, is required for mRNA 3′-end formation in yeast

In yeast, four factors (CF I, CF II, PF I, and PAP) are required for accurate pre-mRNA cleavage and polyadenylation in vitro. CF I can be separated further into CF IA and CF IB. Here we show that CF IB is the 73-kD Hrp1 protein. Recombinant Hrp1p made in Escherichia coli provides full CF IB function in both cleavage and poly(A) addition assays. Consistent with the presence of two RRM-type motifs, Hrp1p can be UV cross-linked to RNA, and this specific interaction requires the (UA)₆ polyadenylation efficiency element. Furthermore, the CF II factor enhances the binding of Hrp1p to the RNA precursor. A temperature-sensitive mutant in HRP1 yields mRNAs with shorter poly(A) tails when grown at the nonpermissive temperature. Genetic analyses indicate that Hrp1p interacts with Rna15p and Rna14p, two components of CF 1A. The HRP1 gene was originally isolated as a suppressor of a temperature-sensitive npl3 allele, a gene encoding a protein involved in mRNA export. Like Npl3p, Hrp1p shuttles between the nucleus and cytoplasm, providing a potential link between 3′-end processing and mRNA export from the nucleus.     

Topography of polyoma virus-specific giant nuclear RNA molecules containing poly(A) sequences.

The distribution of viral RNA sequences with respect to poly(A) tracts has been determined for giant polyoma virus-specific nuclear RNA molecules. Polyadenylated nuclear RNA sedimenting faster than 30 S was isolated from polyoma virus-infected cells and cleaved into fragments of various sizes by partial alkaline hydrolysis. Fragments containing poly(A) were separated from nonpolyadenylated RNA. The viral sequence composition of polyadenylated or nonpolyadenylated fragments, and of total polyadenylated giant RNA, was determined by hybridization to ³²P-labeled separated strands of specific restriction endonuclease fragments of polyoma virus DNA. The results of this analysis showed that in all polyadenylated giant RNA molecules transcribed from the L DNA strand the portion adjacent to the poly(A) segment hybridizes to the 3′-terminal half of the late region of the viral genome. The next portion of these chains hybridizes to the remainder of the late region, the more distal part hybridizes to the early region, and the portions furthest from the poly(A) contain sequences complementary to the L DNA strand of both early and late regions. This finding shows that polyadenylation of Py giant nuclear RNA is non-random and further suggests that the poly(A)-linked RNA sequence(s) maps within the same region of the viral genome as the poly(A)-linked sequence found in mature cytoplasmic mRNA.