CPEB and two poly(A) polymerases control miR-122 stability and p53 mRNA translation

Cytoplasmic polyadenylation-induced translation controls germ cell development, neuronal synaptic plasticity and cellular senescence, a tumour-suppressor mechanism that limits the replicative lifespan of cells. The cytoplasmic polyadenylation element binding protein (CPEB) promotes polyadenylation by nucleating a group of factors including defective in germline development 2 (Gld2), a non-canonical poly(A) polymerase, on specific messenger RNA (mRNA) 3' untranslated regions (UTRs). Because CPEB regulation of p53 mRNA polyadenylation/translation is necessary for cellular senescence in primary human diploid fibroblasts, we surmised that Gld2 would be the enzyme responsible for poly(A) addition. Here we show that depletion of Gld2 surprisingly promotes rather than inhibits p53 mRNA polyadenylation/translation, induces premature senescence and enhances the stability of CPEB mRNA. The CPEB 3' UTR contains two miR-122 binding sites, which when deleted, elevate mRNA translation, as does an antagomir of miR-122. Although miR-122 is thought to be liver specific, it is present in primary fibroblasts and destabilized by Gld2 depletion. Gld4, a second non-canonical poly(A) polymerase, was found to regulate p53 mRNA polyadenylation/translation in a CPEB-dependent manner. Thus, translational regulation of p53 mRNA and cellular senescence is coordinated by Gld2/miR-122/CPEB/Gld4.     

Internal initiation of translation mediated by the 5' leader of a cellular mRNA.

A Robosome-scanning model has been proposed to explain the initiation of eukaryotic messenger RNAs in which binding of the 43S ternary ribosomal subunit near or at the 5' end of the mRNA is facilitated by an interaction between the methylated cap-structure at the end of the mRNA and the cap-binding protein complex eIF-4F. But picornaviral mRNAs do not have a 5' terminal cap structure and are translated by internal ribosome binding. A cellular mRNA, encoding the immunoglobulin heavy-chain binding protein, can be translated in poliovirus-infected cells at a time when cap-dependent translation of host cell mRNAs is inhibited. We report here that the 5' leader of the binding protein mRNA can directly confer internal ribosome binding to an mRNA in mammalian cells, indicating that translation initiation by an internal ribosome-binding mechanism is used by eukaryotic mRNAs.     

Inhibition of mRNA binding to ribosomes by localized activation of dsRNA-dependent protein kinase

The initiation of protein synthesis can be regulated in mammalian cells by protein kinases which phosphorylate the alpha subunit of initiation factor eIF-2. This phosphorylation results in a block in the recycling of eIF-2 and in the inhibition of messenger RNA binding to 80S initiation complexes. After eIF-2 alpha is phosphorylated, the mRNA becomes associated with 48S complexes consisting of a 40S ribosomal subunit, eIF-2 (alpha P), GDP and Met-tRNAf. One of the eIF-2 alpha kinases is activated by low concentrations of double-stranded RNA (dsRNA). This kinase (PKds) is present at a basal level in all mammalian cells investigated and its synthesis is induced in cells treated with interferon. The PKds may be involved in the inhibition of translation of viral mRNA in interferon-treated cells infected with RNA viruses, as it is activated by viral replicative complexes. It is not known, however, if the activated PKds preferentially inhibits the translation of viral mRNA when cellular protein synthesis proceeds at a normal rate in infected cells. We now report that mRNA covalently linked to dsRNA is preferentially inhibited from binding to 80S complexes by a localized activation of PKds. This suggests that in interferon-treated cells the binding of some nascent viral mRNAs to functional initiation complexes may be preferentially inhibited by a similar mechanism.     

重组慢病毒介导的NMD途径因子UPF1和SMG1可诱导干扰细胞株的构建

无义介导的mRNA降解途径是一个比较完善的异常mRNA的降解机制,结合在外显子拼接复合体上的多种蛋白决定NMD途径对异常转录物的识别和降解的启动,其中UPF1和SMG1发挥主要功能.UPF1是一个RNA解旋酶和RNA依赖的ATP酶;而SMG1具有磷脂酰肌醇激酶活性,负责UPF1的磷酸化.本研究构建了含有UPF1和SMG-1基因发夹结构的诱导开关基因表达干扰质粒.利用慢病毒介导转化哺乳动物细胞HEK293T细胞得到重组病毒,经鉴定后感染细胞AD_293,目的基因在细胞中得以高效表达.通过继代培养和单克隆化,得到强力霉素诱导干扰UPF1和SMG-1表达的稳定细胞株.     

Polyamine sensing by nascent ornithine decarboxylase antizyme stimulates decoding of its mRNA

Polyamines are essential organic polycations with multiple cellular functions relevant for cell division, cancer and ageing. Regulation of polyamine synthesis is mainly achieved by controlling the activity of ornithine decarboxylase (ODC) through an unusual mechanism involving ODC antizyme, the binding of which disrupts homodimeric ODC and targets it for ubiquitin-independent degradation by the 26S proteasome. Whereas mammals express several antizyme genes, we have identified a single orthologue, termed OAZ1, in Saccharomyces cerevisiae. Similar to its mammalian counterparts, OAZ1 synthesis is induced with rising intracellular polyamine concentrations, which also inhibit ubiquitin-dependent degradation of the OAZ1 protein. Together, these mechanisms contribute to a homeostatic feedback regulation of polyamines. Antizyme synthesis involves a conserved +1 ribosomal frameshifting (RFS) event at an internal STOP codon during decoding of its messenger RNA. Here we used S. cerevisiae OAZ1 to dissect the enigmatic mechanism underlying polyamine regulation of RFS. In contrast with previous assumptions, we report here that the nascent antizyme polypeptide is the relevant polyamine sensor that operates in cis to negatively regulate upstream RFS on the polysomes, where its own mRNA is being translated. At low polyamine levels, the emerging antizyme polypeptide inhibits completion of its synthesis causing a ribosome pile-up on antizyme mRNA, whereas polyamine binding to nascent antizyme promotes completion of its synthesis. Thus, our study reveals a novel autoregulatory mechanism, in which binding of a small metabolite to a nascent sensor protein stimulates the latter's synthesis co-translationally.     

A conserved mechanism of DEAD-box ATPase activation by nucleoporins and InsP6 in mRNA export

Superfamily 1 and superfamily 2 RNA helicases are ubiquitous messenger-RNA-protein complex (mRNP) remodelling enzymes that have critical roles in all aspects of RNA metabolism. The superfamily 2 DEAD-box ATPase Dbp5 (human DDX19) functions in mRNA export and is thought to remodel mRNPs at the nuclear pore complex (NPC). Dbp5 is localized to the NPC via an interaction with Nup159 (NUP214 in vertebrates) and is locally activated there by Gle1 together with the small-molecule inositol hexakisphosphate (InsP(6)). Local activation of Dbp5 at the NPC by Gle1 is essential for mRNA export in vivo; however, the mechanistic role of Dbp5 in mRNP export is poorly understood and it is not known how Gle1(InsP6) and Nup159 regulate the activity of Dbp5. Here we report, from yeast, structures of Dbp5 in complex with Gle1(InsP6), Nup159/Gle1(InsP6) and RNA. These structures reveal that InsP(6) functions as a small-molecule tether for the Gle1-Dbp5 interaction. Surprisingly, the Gle1(InsP6)-Dbp5 complex is structurally similar to another DEAD-box ATPase complex essential for translation initiation, eIF4G-eIF4A, and we demonstrate that Gle1(InsP6) and eIF4G both activate their DEAD-box partner by stimulating RNA release. Furthermore, Gle1(InsP6) relieves Dbp5 autoregulation and cooperates with Nup159 in stabilizing an open Dbp5 intermediate that precludes RNA binding. These findings explain how Gle1(InsP6), Nup159 and Dbp5 collaborate in mRNA export and provide a general mechanism for DEAD-box ATPase regulation by Gle1/eIF4G-like activators.     

The ribosome uses two active mechanisms to unwind messenger RNA during translation

The ribosome translates the genetic information encoded in messenger RNA into protein. Folded structures in the coding region of an mRNA represent a kinetic barrier that lowers the peptide elongation rate, as the ribosome must disrupt structures it encounters in the mRNA at its entry site to allow translocation to the next codon. Such structures are exploited by the cell to create diverse strategies for translation regulation, such as programmed frameshifting, the modulation of protein expression levels, ribosome localization and co-translational protein folding. Although strand separation activity is inherent to the ribosome, requiring no exogenous helicases, its mechanism is still unknown. Here, using a single-molecule optical tweezers assay on mRNA hairpins, we find that the translation rate of identical codons at the decoding centre is greatly influenced by the GC content of folded structures at the mRNA entry site. Furthermore, force applied to the ends of the hairpin to favour its unfolding significantly speeds translation. Quantitative analysis of the force dependence of its helicase activity reveals that the ribosome, unlike previously studied helicases, uses two distinct active mechanisms to unwind mRNA structure: it destabilizes the helical junction at the mRNA entry site by biasing its thermal fluctuations towards the open state, increasing the probability of the ribosome translocating unhindered; and it mechanically pulls apart the mRNA single strands of the closed junction during the conformational changes that accompany ribosome translocation. The second of these mechanisms ensures a minimal basal rate of translation in the cell; specialized, mechanically stable structures are required to stall the ribosome temporarily. Our results establish a quantitative mechanical basis for understanding the mechanism of regulation of the elongation rate of translation by structured mRNAs.     

靶向沉默人HER2/neu基因的RNA干扰

针对人HER2/neu基因的mRNA序列, 设计并构建shRNA干扰载体, 经聚合酶链式反应(PCR)及测序鉴定后转染SK-BR-3细胞, 并检测HER2/neu mRNA和蛋白抑制情况及SK-BR-3细胞迁移能力的变化. 实验结果表明： 构建了3个HER2/neu shRNA真核表达质粒; 3个重组质粒均可不同程度下调HER2/neu mRNA和蛋白表达水平, 并抑制SK BR 3细胞的迁移; 其中1号重组质粒效应最强, 对mRNA和蛋白的抑制率分别为84.65%和7498%.     

Structural basis for 5'-end-specific recognition of guide RNA by the A. fulgidus Piwi protein

RNA interference (RNAi) is a conserved sequence-specific gene regulatory mechanism mediated by the RNA-induced silencing complex (RISC), which is composed of a single-stranded guide RNA and an Argonaute protein. The PIWI domain, a highly conserved motif within Argonaute, has been shown to adopt an RNase H fold critical for the endonuclease cleavage activity of RISC. Here we report the crystal structure of Archaeoglobus fulgidus Piwi protein bound to double-stranded RNA, thereby identifying the binding pocket for guide-strand 5'-end recognition and providing insight into guide-strand-mediated messenger RNA target recognition. The phosphorylated 5' end of the guide RNA is anchored within a highly conserved basic pocket, supplemented by the carboxy-terminal carboxylate and a bound divalent cation. The first nucleotide from the 5' end of the guide RNA is unpaired and stacks over a conserved tyrosine residue, whereas successive nucleotides form a four-base-pair RNA duplex. Mutation of the corresponding amino acids that contact the 5' phosphate in human Ago2 resulted in attenuated mRNA cleavage activity. Our structure of the Piwi-RNA complex, and that determined elsewhere, provide direct support for the 5' region of the guide RNA serving as a nucleation site for pairing with target mRNA and for a fixed distance separating the RISC-mediated mRNA cleavage site from the anchored 5' end of the guide RNA.     

Interaction of elongation factors EF-G and EF-Tu with a conserved loop in 23S RNA

The elongation factors EF-Tu and EF-G interact with ribosomes during protein synthesis: EF-Tu presents incoming aminoacyl transfer RNA to the programmed ribosome as an EF-Tu-GTP-tRNA ternary complex and EF-G promotes translocation of peptidyl-tRNA and its associated messenger RNA from the A to the P site after peptidyl transfer. Both events are accompanied by ribosome-dependent GTP hydrolysis. Here we use chemical probes to investigate the possible interaction of these factors with ribosomal RNA in E. coli ribosomes. We observe EF-G-dependent footprints in vitro and in vivo around position 1,067 in domain II of 23S rRNA, and in the loop around position 2,660 in domain VI.EF-Tu gives an overlapping footprint in vitro at positions 2,655 and 2,661, but shows no effect at position 1,067. The 1,067 region is the site of interaction of the antibiotic thiostrepton, which prevents formation of the EF-G-GTP-ribosome complex and is a site for interaction with the GTPase-related protein L11 (ref. 3). The universally conserved loop in the 2,660 region is the site of attack by the RNA-directed cytotoxins alpha-sarcin and ricin, whose effects abolish translation and include the loss of elongation factor-dependent functions in eukaryotic ribosomes.     

Human cytomegalovirus induces alteration of β-actin mRNA and microfilaments in human embryo fibroblast cells

Objective: To investigate the infection of human embryo fibroblast cell line HF cells by CMV as well as the effects of CMV on β-actin mRNA and microfilaments. Methods: HF cells shape was observed after the infection of CMV.RT-PCR assay was used to detect the mRNA expression of CMV immediate early (IE) gene, β-actin and GAPDH genes of HF cells infected by CMV. CMV particles and cell microfilaments were detected with electron microscope. Results: Shape of HF cell changed after the infection by CMV. HF cells infected by CMV could express IE mRNA and the expression of β-actin mRNA decreased in a time-and titer-dependent manner compared with the uninfected HF cells whose expression of GAPDH mRNA did not change much. CMV particles were found with electron microscope in the cells. Microfilaments were ruptured and shortened after the infection of CMV. Conclusion: CMV can not only infect human embryo fibroblast cells line HF cells and replicate in the cells, but can also affect the expression of β-actin mRNA and the microfilaments.