Regulated CPEB phosphorylation during meiotic progression suggests a mechanism for temporal control of maternal mRNA translation

CPEB is an mRNA-binding protein that stimulates polyadenylation-induced translation of maternal mRNA once it is phosphorylated on Ser 174 or Thr 171 (species-dependent). Disruption of the CPEB gene in mice causes an arrest of oogenesis at embryonic day 16.5 (E16.5), when most oocytes are in pachytene of prophase I. Here, we show that CPEB undergoes Thr 171 phosphorylation at E16.5, but dephosphorylation at the E18.5, when most oocytes are entering diplotene. Although phosphorylation is mediated by the kinase aurora, the dephosphorylation is due to the phosphatase PP1. The temporal control of CPEB phosphorylation suggests a mechanism in which CPE-containing mRNA translation is stimulated at pachytene and metaphase I.     

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.     

CPEB regulation of human cellular senescence, energy metabolism, and p53 mRNA translation

Cytoplasmic polyadenylation element-binding protein (CPEB) stimulates polyadenylation and translation in germ cells and neurons. Here, we show that CPEB-regulated translation is essential for the senescence of human diploid fibroblasts. Knockdown of CPEB causes skin and lung cells to bypass the M1 crisis stage of senescence; reintroduction of CPEB into the knockdown cells restores a senescence-like phenotype. Knockdown cells that have bypassed senescence undergo little telomere erosion. Surprisingly, knockdown of exogenous CPEB that induced a senescence-like phenotype results in the resumption of cell growth. CPEB knockdown cells have fewer mitochondria than wild-type cells and resemble transformed cells by having reduced respiration and reactive oxygen species (ROS), normal ATP levels, and enhanced rates of glycolysis. p53 mRNA contains cytoplasmic polyadenylation elements in its 3′ untranslated region (UTR), which promote polyadenylation. In CPEB knockdown cells, p53 mRNA has an abnormally short poly(A) tail and a reduced translational efficiency, resulting in an ~50% decrease in p53 protein levels. An shRNA-directed reduction in p53 protein by about 50% also results in extended cellular life span, reduced respiration and ROS, and increased glycolysis. Together, these results suggest that CPEB controls senescence and bioenergetics in human cells at least in part by modulating p53 mRNA polyadenylation-induced translation.     

Semaphorin controls epidermal morphogenesis by stimulating mRNA translation via eIF2alpha in Caenorhabditis elegans

Conserved semaphorin-plexin signaling systems govern various aspects of animal development, including axonal guidance in vertebrates and epidermal morphogenesis in Caenorhabditis elegans. Here we provide in vivo evidence that stimulation of mRNA translation via eukaryotic initiation factor 2alpha (eIF2alpha) is an essential downstream event of semaphorin signaling in C. elegans. In semaphorin/plexin mutants, a marked elevation in the phosphorylation of eIF2alpha is observed, which causes translation repression and is causally related to the morphological epidermal phenotype in the mutants. Conversely, removal of constraints on translation by genetically reducing the eIF2alpha phosphorylation largely bypasses requirement for the semaphorin signal in epidermal morphogenesis. We also identify an actin-depolymerizing factor/cofilin, whose expression in the mutants is predominantly repressed, as a major translational target of semaphorin signaling. Thus, our results reveal a physiological significance for translation of mRNAs for cytoskeletal regulators, linking environmental cues to cytoskeletal rearrangement during cellular morphogenesis in vivo.     

An mRNA structure in bacteria that controls gene expression by binding lysine

Riboswitches are metabolite-responsive genetic control elements that reside in the untranslated regions (UTRs) of certain messenger RNAs. Herein, we report that the 5'-UTR of the lysC gene of Bacillus subtilis carries a conserved RNA element that serves as a lysine-responsive riboswitch. The ligand-binding domain of the riboswitch binds to L-lysine with an apparent dissociation constant (KD) of approximately 1 micro M, and exhibits a high level of molecular discrimination against closely related analogs, including D-lysine and ornithine. Furthermore, we provide evidence that this widespread class of riboswitches serves as a target for the antimetabolite S-(2-aminoethyl)-L-cysteine (AEC). These findings add support to the hypotheses that direct sensing of metabolites by messenger RNAs is a fundamental form of genetic control and that riboswitches represent a new class of antimicrobial drug targets.     

Constitutive mTORC1 activation by a herpesvirus Akt surrogate stimulates mRNA translation and viral replication

All viruses require cellular ribosomes to translate their mRNAs. Viruses producing methyl-7 (m⁷) GTP-capped mRNAs, like Herpes Simplex Virus-1 (HSV-1), stimulate cap-dependent translation by activating mTORC1 to inhibit the translational repressor 4E-binding protein 1 (4E-BP1). Here, we establish that the HSV-1 kinase Us3 masquerades as Akt to activate mTORC1. Remarkably, Us3 displays no sequence homology with the cellular kinase Akt, yet directly phosphorylates tuberous sclerosis complex 2 (TSC2) on the same sites as Akt. TSC2 depletion rescued Us3-deficient virus replication, establishing that Us3 enhances replication by phosphorylating TSC2 to constitutively activate mTORC1, effectively bypassing S6K-mediated feedback inhibition. Moreover, Us3 stimulated Akt substrate phosphorylation in infected cells, including FOXO1 and GSK3. Thus, HSV-1 encodes an Akt surrogate with overlapping substrate specificity to activate mTORC1, stimulating translation and virus replication. This establishes Us3 as a unique viral kinase with promising drug development potential.     

麦胚无细胞转译体系在IL—2mRNA研究中的应用

目前,国内尚无将体外转译体系应用于IL 2研究的报道。本文以长春1号小麦种制备麦胚无细胞体系,经与兔网织红细胞、腹水癌细胞两体系比较筛选,结果证明麦胚无细胞转译体系适于实验室应用。通过对ML A 144mRNA作转译活性鉴定,证明该系统可以合成具有生物活性的、完整的IL2分子。     

Cell-free translation of purified avian sarcoma virus src mRNA

Avian sarcoma virus 21 S RNA, purified by hybridization from virus-infected cells, was translated in a cell-free system. The major product of translation was a protein of 60,000 daltons. This protein was the same as authentic pp60^src, the product of the ASV src gene, when compared by electrophoretic mobility in polyacrylamide gels, immunological reactivity and partial protease digestion. These findings confirm that the 21 S ASV RNA serves as mRNA for pp60^src. Furthermore, pp60^src is the only major product of translation of the src gene and is apparently synthesized without a cleavable signal sequence.     

Regulated protein degradation controls PKA function and cell-type differentiation in Dictyostelium

Cullins function as scaffolds that, along with F-box/WD40-repeat-containing proteins, mediate the ubiquitination of proteins to target them for degradation by the proteasome. We have identified a cullin CulA that is required at several stages during Dictyostelium development. culA null cells are defective in inducing cell-type-specific gene expression and exhibit defects during aggregation, including reduced chemotaxis. PKA is an important regulator of Dictyostelium development. The levels of intracellular cAMP and PKA activity are controlled by the rate of synthesis of cAMP and its degradation by the cAMP-specific phosphodiesterase RegA. We show that overexpression of the PKA catalytic subunit (PKAcat) rescues many of the culA null defects and those of cells lacking FbxA/ChtA, a previously described F-box/WD40-repeat-containing protein, suggesting CulA and FbxA proteins are involved in regulating PKA function. Whereas RegA protein levels drop as the multicellular organism forms in the wild-type strain, they remain high in culA null and fbxA null cells. Although PKA can suppress the culA and fbxA null developmental phenotypes, it does not suppress the altered RegA degradation, suggesting that PKA lies downstream of RegA, CulA, and FbxA. Finally, we show that CulA, FbxA, and RegA are found in a complex in vivo, and formation of this complex is dependent on the MAP kinase ERK2, which is also required for PKA function. We propose that CulA and FbxA regulate multicellular development by targeting RegA for degradation via a pathway that requires ERK2 function, leading to an increase in cAMP and PKA activity.     

mTOR phosphorylates IMP2 to promote IGF2 mRNA translation by internal ribosomal entry

Variants in the IMP2 (insulin-like growth factor 2 [IGF2] mRNA-binding protein 2) gene are implicated in susceptibility to type 2 diabetes. We describe the ability of mammalian target of rapamycin (mTOR) to regulate the cap-independent translation of IGF2 mRNA through phosphorylation of IMP2, an oncofetal RNA-binding protein. IMP2 is doubly phosphorylated in a rapamycin-inhibitable, amino acid-dependent manner in cells and by mTOR in vitro. Double phosphorylation promotes IMP2 binding to the IGF2 leader 3 mRNA 5' untranslated region, and the translational initiation of this mRNA through eIF-4E- and 5' cap-independent internal ribosomal entry. Unexpectedly, the interaction of IMP2 with mTOR complex 1 occurs through mTOR itself rather than through raptor. Whereas depletion of mTOR strongly inhibits IMP2 phosphorylation in cells, comparable depletion of raptor has no effect; moreover, the ability of mTOR to phosphorylate IMP2 in vitro is unaffected by the elimination of raptor. Dual phosphorylation of IMP2 at the mTOR sites is evident in the mouse embryo, likely coupling nutrient sufficiency to IGF2 expression and fetal growth. Doubly phosphorylated IMP2 is also widely expressed in adult tissues, including islets of Langerhans.     

Tap and NXT promote translation of unspliced mRNA

Tap has been proposed to play a role in general mRNA export and also functions in expression of RNA with retained introns that contain the MPMV CTE (constitutive transport element). Tap forms a functional heterodimer with NXT/p15. We have previously demonstrated that unspliced intron-containing CTE RNA is efficiently exported to the cytoplasm in mammalian cells. Here we show that Tap and NXT proteins function together to enhance translation of proteins from the exported CTE RNA. Pulse chase experiments show that Tap/NXT significantly increases the rate of protein synthesis. Sucrose gradient analysis demonstrates that Tap and NXT efficiently shift the unspliced RNA into polyribosomal fractions. Furthermore, Tap, but not NXT is detected in polyribosomes. Taken together, our results indicate that Tap and NXT serve a role in translational regulation of RNA after export to the cytoplasm. They further suggest that Tap/NXT may play a role in remodeling of cytoplasmic RNP complexes, providing a link between export pathways and cytoplasmic fate.     

ABC-F proteins in mRNA translation and antibiotic resistance

The ATP binding cassette protein superfamily comprises ATPase enzymes which are, for the most part, involved in transmembrane transport. Within this superfamily however, some protein families have other functions unrelated to transport. One example is the ABC-F family, which comprises an extremely diverse set of cytoplasmic proteins. All of the proteins in the ABC-F family characterized to date act on the ribosome and are translation factors. Their common function is ATP-dependent modulation of the stereochemistry of the peptidyl transferase center (PTC) in the ribosome coupled to changes in its global conformation and P-site tRNA binding geometry. In this review, we give an overview of the function, structure, and theories for the mechanisms-of-action of microbial proteins in the ABC-F family, including those involved in mediating resistance to ribosome-binding antibiotics.     

mRNA stabilization by poly(A) binding protein is independent of poly(A) and requires translation

Translation and mRNA stability are enhanced by the presence of a poly(A) tail. In vivo, the tail interacts with a conserved polypeptide, poly(A) binding protein (Pab1p). To examine Pab1p function in vivo, we have tethered Pab1p to the 3′ UTR of reporter mRNAs by fusing it to MS2 coat protein and placing MS2 binding sites in the 3′ UTR of the reporter. This strategy allows us to uncouple Pab1p function from its RNA binding activity. We show that mRNAs that lack a poly(A) tail in vivo are stabilized by Pab1p, and that the portions of Pab1p required for stabilization are genetically distinct from those required for poly(A) binding. In addition, stabilization by Pab1p requires ongoing translation of the mRNA. We conclude that the primary, or sole, function of poly(A) with respect to mRNA stability is simply to bring Pab1p to the mRNA, and that mRNA stabilization is an intrinsic property of Pab1p. The approach we describe may be useful in identifying and assaying 3′ UTR regulatory proteins, as it uncouples analysis of function from RNA binding.     

Phosphorylation and dephosphorylation events that regulate viral mRNA translation

As they are completely dependent upon the protein synthesis machinery resident in the cells of their host to translate their mRNAs, it is imperative that viruses are able to effectively manipulate the elaborate cellular regulatory network that controls translation. Indeed, this exquisite dependence on host functions has made viral models attractive systems to explore translational regulatory mechanisms operative in eukaryotic cells. Central among these are an intricate array of phosphorylation and dephosphorylation events that have far reaching consequences on the activity of cellular translation factors. Not only do these modulate the activity of a given factor, but they can also determine if the translation of host proteins persists in infected cells, the efficiency with which viral mRNAs are translated and the outcome of a systemic host anti-viral response. In this review, we discuss how various viruses manipulate the phosphorylation state of key cellular translation factors, illustrating the critical nature these interactions play in virus replication, pathogenesis and innate host defense.