General RNA‐binding proteins have a function in poly(A)‐binding protein‐dependent translation

The interaction between the poly(A)‐binding protein (PABP) and eukaryotic translational initiation factor 4G (eIF4G), which brings about circularization of the mRNA, stimulates translation. General RNA‐binding proteins affect translation, but their role in mRNA circularization has not been studied before. Here, we demonstrate that the major mRNA ribonucleoprotein YB‐1 has a pivotal function in the regulation of eIF4F activity by PABP. In cell extracts, the addition of YB‐1 exacerbated the inhibition of 80S ribosome initiation complex formation by PABP depletion. Rabbit reticulocyte lysate in which PABP weakly stimulates translation is rendered PABP‐dependent after the addition of YB‐1. In this system, eIF4E binding to the cap structure is inhibited by YB‐1 and stimulated by a nonspecific RNA. Significantly, adding PABP back to the depleted lysate stimulated eIF4E binding to the cap structure more potently if this binding had been downregulated by YB‐1. Conversely, adding nonspecific RNA abrogated PABP stimulation of eIF4E binding. These data strongly suggest that competition between YB‐1 and eIF4G for mRNA binding is required for efficient stimulation of eIF4F activity by PABP.     

The proteins of the messenger RNA binding site of Escherichia coli ribosomes.

Oligo(U) derivatives with [14C]-4-(N-2-chloroethyl-N-methylamino)benzaldehyde attached to 3'-end cis-diol group via acetal bond, p(Up)n-1UCHRCl as well as with [14C]-4-(N-2-chloroethyl-N-methylamino)benzylamine attached to 5'-phosphate via amide bond, ClRCH2NHpU(pU)6 were used to modify 70S E. coli ribosomes near mRNA binding centre. Within ternary complex with ribosome and tRNAPhe all reagents covalently bind to ribosome the extent of modification being 0.1-0.4 mole/mole 70S. p(Up)n-1UCHRCl alkylates either 30S (n=5,7) or both subunits (n=6,8). rRNA is preferentially modified within 30S subunit. ClRCH2NHpU(pU)6 alkylates both subunits the proteins being mainly modified. The distribution of the label among proteins differ for various reagents. S4, S5, S7, S9, S11, S13, S15, S18 and S21 are found to be alkylated within 30S subunit, proteins L1, L2, L6, L7/L12, L19, L31 and L32 are modified in the 50S subunit. Most proteins modified within 30S subunit are located at the "head" of this subunit and proteins modified within 50S subunit are located at the surface of the contact between this subunit and the "head" of 30S subunit at the model of Stoffler.     

Facioscapulohumeral muscular dystrophy region gene 1 is a dynamic RNA-associated and actin-bundling protein

FSHD region gene 1 (FRG1) is a dynamic nuclear and cytoplasmic protein that, in skeletal muscle, shows additional localization to the sarcomere. Maintaining appropriate levels of FRG1 protein is critical for muscular and vascular development in vertebrates; however, its precise molecular function is unknown. This study investigates the molecular functions of human FRG1, along with mouse FRG1 and Xenopus frg1, using molecular, biochemical, and cellular-biological approaches, to provide further insight into its roles in vertebrate development. The nuclear fraction of the endogenous FRG1 is localized in nucleoli, Cajal bodies, and actively transcribed chromatin; however, contrary to overexpressed FRG1, the endogenous FRG1 is not associated with nuclear speckles. We characterize the nuclear and nucleolar import of FRG1, the potential effect of phosphorylation, and its interaction with the importin karyopherin α2. Consistent with a role in RNA biogenesis, human FRG1 is associated with mRNA in vivo and invitro, interacts directly with TAP (Tip-associated protein; the major mRNA export receptor), and is a dynamic nuclear-cytoplasmic shuttling protein supporting a function for FRG1 in mRNA transport. Biochemically, we characterize FRG1 actin binding activity and show that the cytoplasmic pool of FRG1 is dependent on an intact actin cytoskeleton for its localization. These data provide the first biochemical activities (actin binding and RNA binding) for human FRG1 and the characterization of the endogenous human FRG1, together indicating that FRG1 is involved in multiple aspects of RNA biogenesis, including mRNA transport and, potentially, cytoplasmic mRNA localization.     

The association of a La module with the PABP-interacting motif PAM2 is a recurrent evolutionary process that led to the neofunctionalization of La-related proteins

La-related proteins (LARPs) are largely uncharacterized factors, well conserved throughout evolution. Recent reports on the function of human LARP4 and LARP6 suggest that these proteins fulfill key functions in mRNA metabolism and/or translation. We report here a detailed evolutionary history of the LARP4 and 6 families in eukaryotes. Genes coding for LARP4 and 6 were duplicated in the common ancestor of the vertebrate lineage, but one LARP6 gene was subsequently lost in the common ancestor of the eutherian lineage. The LARP6 gene was also independently duplicated several times in the vascular plant lineage. We observed that vertebrate LARP4 and plant LARP6 duplication events were correlated with the acquisition of a PABP-interacting motif 2 (PAM2) and with a significant reorganization of their RNA-binding modules. Using isothermal titration calorimetry (ITC) and immunoprecipitation methods, we show that the two plant PAM2-containing LARP6s (LARP6b and c) can, indeed, interact with the major plant poly(A)-binding protein (PAB2), while the third plant LARP6 (LARP6a) is unable to do so. We also analyzed the RNA-binding properties and the subcellular localizations of the two types of plant LARP6 proteins and found that they display nonredundant characteristics. As a whole, our results support a model in which the acquisition by LARP4 and LARP6 of a PAM2 allowed their targeting to mRNA 3′ UTRs and led to their neofunctionalization.     

Externalization of membrane-bound activities during sheep reticulocyte maturation is temperature and ATP dependent

During the maturation of sheep reticulocytes in vitro, there is release of material that can be pelleted from the cell-free incubation medium by centrifugation at 100,000 X g. This pellet contains activities that are derived from both the plasma membrane and lysosomes. No evidence was obtained for the presence of mitochondrial activities or cytosolic enzyme activities. The release of these activities is ATP and temperature dependent, since reduction of either results in a greater retention of the activities by the cells and a lesser amount in the 100,000 X g pellet. The pelleted material is vesicular in nature, and the production and (or) release of the material are reduced upon ATP depletion or lowering of the temperature. It is concluded that the externalization of specific membrane components is a normal metabolic process that occurs during reticulocyte maturation and represents a means by which reticulocytes shed specific types of membrane-associated functions that are known to decrease during reticulocytes maturation.     

A novel family of nuclear transport receptors mediates the export of messenger RNA to the cytoplasm

Fully processed mRNAs are exported to the cytoplasm where they direct protein synthesis. A general feature of mRNA export is that it is an active, receptor-mediated process. The mRNA export receptors are thought to recognize and bind to the mRNA-export cargoes either directly or indirectly (via adaptor proteins) and facilitate their translocation across the central channel of the nuclear pore complex (NPC). On the cytoplasmic side of the NPC, the exported mRNA is released and the receptor returns to the nucleoplasm, without the cargo, to initiate additional rounds of export. Recent, studies in yeast and in higher eukaryotes have led to the elucidation of an evolutionarily conserved pathway for the export of bulk mRNA to the cytoplasm.     

The roles of cytoplasmic poly(A)-binding proteins in regulating gene expression: a developmental perspective.

Poly(A)-binding proteins (PABPs) are central to the regulation of messenger RNA (mRNA) translation and stability; however, the roles and contributions of different PABP family members in controlling gene expression are not yet fully understood. In this paper, the current state of knowledge of the different cytoplasmic PABP proteins and their function in animal cells will be summarised, with particular reference to their roles in development. Possible regulatory mechanisms and potential new roles for these proteins in the control of specific mRNAs are also highlighted.     

Developmental expression of corticotropin releasing hormone messenger RNA and peptide in rat hypothalamus

We have examined corticotropin releasing hormone (CRH), arginine vasopressin (AVP) and somatostatin (SOM) mRNA expression and peptide content in the rat hypothalamus from day 20 of fetal life (F20) to the fifteenth day of postnatal life (P15). During this time, hypothalamic CRH mRNA levels did not change significantly, whereas there was a gradual six-fold rise in CRH peptide levels. AVP mRNA levels fell three-fold between F20 and P1 and increased six-fold between P1 and P15. AVP peptide levels increased three-fold, with most of the rise occurring between P1 and P15. From F20 to P15, SOM mRNA and peptide levels rose four- and eight-fold, respectively. The changes in the levels of these three hypothalamic gene products correlate with the previously described alterations in the responsiveness of the HPA axis observed in fetal and early postnatal rats, suggesting a role for these neuropeptides in the modulation of the HPA axis during this developmental period.     

Identification of several proteins involved in the messenger RNA binding site of the 30 S ribosome by inactivation with 2-methoxy-5-nitrotropone

Chemical modification of unwashed 30 S ribosomal subunits with 2-methoxy-5-nitrotropone causes a rapid loss of their capacity to bind bacteriophage Qβ RNA. Reconstitution experiments show that ribosomal protein is the functionally inactivated species. When purified unmodified ribosomal proteins were included in a mixture of 16 S ribosomal RNA and total protein derived from 2-methoxy-5-nitrotropone-treated subunits, four proteins (S1, S12, S13 and S21) were found to promote the reconstitution of particles capable of binding natural messenger RNA.     

RNA结合蛋白PABPC1的功能及生物学作用

多聚腺苷酸结合蛋白(poly (A) binding protein,PABP)家族通常被认为是mRNA poly (A)尾的一种保护屏障.其中细胞质多聚腺苷酸结合蛋白1 (cytoplasmic poly (A) binding protein-1,PABPC1)在高亲和力作用下能够与mRNA中富含腺苷酸的序列结合,在基因转录后调控中发挥着重要作用.同时PABPC1还参与mRNA的许多代谢通路,包括腺苷酸多聚化/脱腺苷酸化、m RNA转运、m RNA翻译、降解及mircoRNA相关调控.近年来关于PABPC1与生殖细胞的发育、心肌肥大和肿瘤的发生发展的报道屡见不鲜,可见PABPC1与细胞的生长发育有密切联系.本文将主要介绍PABPC1的结构、表达调控、功能及其生物学作用.     

The initiation site for transcription of the TMV 30-kDa protein messenger RNA

The initiation site for transcripotion of the 30-kDa protein mRNA of tobacco mosaic virus was mapped uniquely at residue 1558 from the 3'-terminus on TMV RNA using the primer-extension and the S1-nuclease mapping method.     

Plasminogen activator inhibitor-2 (PAI-2) mRNA is localized in the accumbens nucleus of the mouse brain and is induced in specific brain sites after kainate excitation

Plasminogen activator inhibitor-2 (PAI-2) specifically inhibits plasminogen activators, extracellular fibrinolytic serine proteases that are also implicated in brain plasticity and toxicity. Primarily localized intracellularly, PAI-2 is thought to also counteract apoptosis mediated by a currently undefined intracellular protease. Here we localized PAI-2 mRNA through in situ hybridization in brain cryosections derived from normal adult mice or after kainate excitation. We found that in the normal brain PAI-2 mRNA was confined to an area within the accumbens nucleus shell. After kainate was injected (i.p.), PAI-2 mRNA was substantially and rapidly (within 2 h) induced in neuron-like cells primarily in layers II–III of the neocortex; the cingulate, piriform, entorhinal and perirhinal cortices; the olfactory bulb, nucleus and tubercle; in the accumbens nucleus, shell and core; throughout the caudate putamen and the amygdaloid complex; in the CA1 and CA3 areas of the hippocampus, and in the parasubiculum. These findings suggest that PAI-2 could play a role in the accumbens nucleus as well as in activity-related events associated with olfactory, striatal, and limbic structures.     

RNA结合蛋白调节mRNA稳定性的作用机制

免疫稳态的维持涉及多种细胞因子的基因表达调控,其中在转录后水平对mRNA稳定性的调控起重要作用。ARE(AU-rich element)位于mRNA 3'UTR(非编码区),富含AU碱基,某些RNA结合蛋白通过识别结合ARE影响mRNA的稳定性。本文综合最新研究,概述了TTP、HUR等RNA结合蛋白对mRNA稳定性的调节机制及其在信号通路中的作用。