Development of an in vitro mRNA decay system for Escherichia coli: Poly(A) polymerase I is necessary to trigger degradation

Using a novel Escherichia coli in vitro decay system in which polysomes are the source of both enzymes and mRNA, we demonstrate a requirement for poly(A) polymerase I (PAP I) in mRNA turnover. The in vitro decay of two different mRNAs (trxA and lpp) is triggered by the addition of ATP only when polysomes are prepared from a strain carrying the wild-type gene for PAP I (pcnB+). The relative decay rates of these two messages are similar in vitro and in vivo. Poly(A) tails are formed on both mRNAs, but no poly(A) tails are detected on the 3′ end of mature 23S rRNA. The size distribution of poly(A) tails generated in vitro, averaging 50 nt in length, is comparable to that previously reported in vivo. PAP I activity is associated exclusively with the polysomes. Exogenously added PAP I does not restore mRNA decay to PAP I− polysomes, suggesting that, in vivo, PAP I may be part of a multiprotein complex. The potential of this in vitro system for analyzing mRNA decay in E. coli is discussed.

Effects of Age on the Posttranscriptional Regulation of CCAAT/Enhancer Binding Protein α and CCAAT/Enhancer Binding Protein β Isoform Synthesis in Control and LPS-Treated Livers

The CCAAT/enhancer binding protein α (C/EBPα) and CCAAT/enhancer binding protein β (C/EBPβ) mRNAs are templates for the differential translation of several isoforms. Immunoblotting detects C/EBPαs with molecular masses of 42, 38, 30, and 20 kDa and C/EBPβs of 35, 20, and ∼8.5 kDa. The DNA-binding activities and pool levels of p42C/EBPα and p30C/EBPα in control nuclear extracts decrease significantly whereas the binding activity and protein levels of the 20-kDa isoforms increase dramatically with LPS treatment. Our studies suggest that the LPS response involves alternative translational initiation at specific in-frame AUGs, producing specific C/EBPα and C/EBPβ isoform patterns. We propose that alternative translational initiation occurs by a leaky ribosomal scanning mechanism. We find that nuclear extracts from normal aged mouse livers have decreased p42C/EBPα levels and binding activity, whereas those of p20C/EBPα and p20C/EBPβ are increased. However, translation of 42-kDa C/EBPα is not down-regulated on polysomes, suggesting that aging may affect its nuclear translocation. Furthermore, recovery of the C/EBPα- and C/EBPβ-binding activities and pool levels from an LPS challenge is delayed significantly in aged mouse livers. Thus, aged livers have altered steady-state levels of C/EBPα and C/EBPβ isoforms. This result suggests that normal aging liver exhibits characteristics of chronic stress and a severe inability to recover from an inflammatory challenge.

The quorum-sensing transcriptional regulator TraR requires its cognate signaling ligand for protein folding, protease resistance, and dimerization

Complexes between the quorum-sensing regulator TraR and its inducing ligand autoinducer (AAI) are soluble in Escherichia coli, whereas apo-TraR is almost completely insoluble. Here we show that the lack of soluble TraR is due in large part to rapid proteolysis, inasmuch as apo-TraR accumulated to high levels in an E. coli strain deficient in Clp and Lon proteases. In pulse labeling experiments, AAI protected TraR against proteolysis only when it was added before the radiolabel. This observation indicates that TraR proteins can productively bind AAI only during their own synthesis on polysomes, whereas fully synthesized apo-TraR proteins are not functional AAI receptors. Purified apo-TraR was rapidly degraded by trypsin to oligopeptides, whereas TraR–AAI complexes were more resistant to trypsin and were cleaved at discrete interdomain linkers, indicating that TraR requires AAI to attain its mature tertiary structure. TraR–AAI complexes eluted from a gel filtration column as dimers and bound DNA as dimers. In contrast, apo-TraR was monomeric, and incubation with AAI under a variety of conditions did not cause dimerization. We conclude that AAI is critical for the folding of nascent TraR protein into its mature tertiary structure and that full-length apo-TraR cannot productively bind AAI and is consequently targeted for rapid proteolysis.

Actin Depolymerization Affects Stress-Induced Translational Activity of Potato Tuber Tissue1

Changes in polymerized actin during stress conditions were correlated with potato (Solanum tuberosum L.) tuber protein synthesis. Fluorescence microscopy and immunoblot analyses indicated that filamentous actin was nearly undetectable in mature, quiescent aerobic tubers. Mechanical wounding of postharvest tubers resulted in a localized increase of polymerized actin, and microfilament bundles were visible in cells of the wounded periderm within 12 h after wounding. During this same period translational activity increased 8-fold. By contrast, low-oxygen stress caused rapid reduction of polymerized actin coincident with acute inhibition of protein synthesis. Treatment of aerobic tubers with cytochalasin D, an agent that disrupts actin filaments, reduced wound-induced protein synthesis in vivo. This effect was not observed when colchicine, an agent that depolymerizes microtubules, was used. Neither of these drugs had a significant effect in vitro on run-off translation of isolated polysomes. However, cytochalasin D did reduce translational competence in vitro of a crude cellular fraction containing both polysomes and cytoskeletal elements. These results demonstrate the dependence of wound-induced protein synthesis on the integrity of microfilaments and suggest that the dynamics of the actin cytoskeleton may affect translational activity during stress conditions.

Evidence for a Cytoskeleton-Associated Binding Site Involved in Prolamine mRNA Localization to the Protein Bodies in Rice Endosperm Tissue1

Previous studies have demonstrated that the mRNAs encoding the prolamine and glutelin storage proteins are localized to morphologically distinct membranes of the endoplasmic reticulum (ER) complex in developing rice (Oryza sativa L.) endosperm cells. To gain insight about this mRNA localization process, we investigated the association of prolamine polysomes on the ER that delimit the prolamine protein bodies (PBs). The bulk of the prolamine polysomes were resistant to extraction by 1% Triton X-100 either alone or together with puromycin, which suggests that these translation complexes are anchored to the PB surface through a second binding site in addition to the well-characterized ribosome-binding site of the ER-localized protein translocation complex. Suppression of translation initiation shows that these polysomes are bound through the mRNA, as shown by the simultaneous increase in the amounts of ribosome-free prolamine mRNAs and decrease in prolamine polysome content associated with the membrane-stripped PB fraction. The prolamine polysome-binding activity is likely to be associated with the cytoskeleton, based on the association of actin and tubulin with the prolamine polysomes and PBs after sucrose-density centrifugation.

A highly conserved protein family interacting with the fragile X mental retardation protein (FMRP) and displaying selective interactions with FMRP-related proteins FXR1P and FXR2P

The absence of the fragile X mental retardation protein (FMRP), encoded by the FMR1 gene, is responsible for pathologic manifestations in the Fragile X Syndrome, the most frequent cause of inherited mental retardation. FMRP is an RNA-binding protein associated with polysomes as part of a messenger ribonucleoprotein (mRNP) complex. Although its function is poorly understood, various observations suggest a role in local protein translation at neuronal dendrites and in dendritic spine maturation. We present here the identification of CYFIP1/2 (Cytoplasmic FMRP Interacting Proteins) as FMRP interactors. CYFIP1/2 share 88% amino acid sequence identity and represent the two members in humans of a highly conserved protein family. Remarkably, whereas CYFIP2 also interacts with the FMRP-related proteins FXR1P/2P, CYFIP1 interacts exclusively with FMRP. FMRP–CYFIP interaction involves the domain of FMRP also mediating homo- and heteromerization, thus suggesting a competition between interaction among the FXR proteins and interaction with CYFIP. CYFIP1/2 are proteins of unknown function, but CYFIP1 has recently been shown to interact with the small GTPase Rac1, which is implicated in development and maintenance of neuronal structures. Consistent with FMRP and Rac1 localization in dendritic fine structures, CYFIP1/2 are present in synaptosomal extracts.

A mechanism for intergenomic integration: abundance of ribulose bisphosphate carboxylase small-subunit protein influences the translation of the large-subunit mRNA.

Multimeric protein complexes in chloroplasts and mitochondria are generally composed of products of both nuclear and organelle genes of the cell. A central problem of eukaryotic cell biology is to identify and understand the molecular mechanisms for integrating the production and accumulation of the products of the two separate genomes. Ribulose bisphosphate carboxylase (Rubisco) is localized in the chloroplasts of photosynthetic eukaryotic cells and is composed of small subunits (SS) and large subunits (LS) coded for by nuclear rbcS and chloroplast rbcL genes, respectively. Transgenic tobacco plants containing antisense rbcS DNA have reduced levels of rbcS mRNA, normal levels of rbcL mRNA, and coordinately reduced LS and SS proteins. Our previous experiments indicated that the rate of translation of rbcL mRNA might be reduced in some antisense plants; direct evidence is presented here. After a short-term pulse there is less labeled LS protein in the transgenic plants than in wild-type plants, indicating that LS accumulation is controlled in the mutants at the translational and/or posttranslational levels. Consistent with a primary restriction at translation, fewer rbcL mRNAs are associated with polysomes of normal size and more are free or are associated with only a few ribosomes in the antisense plants. Effects of the rbcS antisense mutation on mRNA and protein accumulation, as well as on the distribution of mRNAs on polysomes, appear to be minimal for other chloroplast and nuclear photosynthetic genes. Our results suggest that SS protein abundance specifically contributes to the regulation of LS protein accumulation at the level of rbcL translation initiation.

Translation initiation of ornithine decarboxylase and nucleocytoplasmic transport of cyclin D1 mRNA are increased in cells overexpressing eukaryotic initiation factor 4E.

The structure of m7GpppN (where N is any nucleotide), termed cap, is present at the 5' end of all eukaryotic cellular mRNAs (except organellar). The eukaryotic initiation factor 4E (eIF-4E) binds to the cap and facilitates the formation of translation initiation complexes. eIF-4E is implicated in control of cell growth, as its overexpression causes malignant transformation of rodent cells and deregulates HeLa cell growth. It was suggested that overexpression of eIF-4E results in the enhanced translation of poorly translated mRNAs that encode growth-promoting proteins. Indeed, enhanced expression of several proteins, including cyclin D1 and ornithine decarboxylase (ODC), was documented in eIF-4E-overexpressing NTH 3T3 cells. However, the mechanism underlying this increase has not been elucidated. Here, we studied the mode by which eIF-4E increases the expression of cyclin D1 and ODC. We show that the increase in the amount of cyclin D1 and ODC is directly proportional to the degree of eIF-4E overexpression. Two mechanisms, which are not mutually exclusive, are responsible for the increase. In eIF-4E-overexpressing cells the rate of translation initiation of ODC mRNA was increased inasmuch as the mRNA sedimented with heavier polysomes. For cyclin D1 mRNA, translation initiation was not increased, but rather its amount in the cytoplasm increased, without a significant increase in total mRNA. Whereas, in the parental NIH 3T3 cell line, a large proportion of the cyclin D1 mRNA was confined to the nucleus, in eIF-4E-overexpressing cells the vast majority of the mRNA was present in the cytoplasm. These results indicate that eIF-4E affects directly or indirectly mRNA nucleocytoplasmic transport, in addition to its role in translation initiation.

Evidence for two protein coding transcripts at the Igf2as locus

The insulin-like growth factor 2 antisense (Igf2as) gene is part of the Ins-Igf2-H19 imprinted gene cluster. The function of the paternally expressed Igf2as is still elusive. In our previous work, we showed that Igf2as transcripts were located in the cytoplasm of C2C12 mouse myoblast cells, associated with polysomes and polyadenylated suggesting that Igf2as is protein coding. In the present work, the protein coding capacity of Igf2as was investigated. We demonstrate for the first time the existence of a polypeptide translated from an Igf2as construct. Furthermore, an RNA-Seq analysis was performed using RNA prepared from skeletal muscles of newborn wild-type and ∆ DMR1-U2 mice to further elucidate the function of Igf2as transcripts. We found no evidence for a regulatory role of Igf2as in the imprinted gene cluster. Interestingly, the RNA-Seq analysis indicated that Igf2as plays a role in the energy metabolism, the cell cycle, histone acetylation and muscle contraction pathways. Our Igf2as investigations further elucidated that there are two distinct Igf2as transcripts corresponding to two putative ORFs.

Pichia pastoris regulates its gene-specific response to different carbon sources at the transcriptional, rather than the translational, level

Background: The methylotrophic, Crabtree-negative yeast Pichia pastoris is widely used as a heterologous protein production host. Strong inducible promoters derived from methanol utilization genes or constitutive glycolytic promoters are typically used to drive gene expression. Notably, genes involved in methanol utilization are not only repressed by the presence of glucose, but also by glycerol. This unusual regulatory behavior prompted us to study the regulation of carbon substrate utilization in different bioprocess conditions on a genome wide scale. Results: We performed microarray analysis on the total mRNA population as well as mRNA that had been fractionated according to ribosome occupancy. Translationally quiescent mRNAs were defined as being associated with single ribosomes (monosomes) and highly-translated mRNAs with multiple ribosomes (polysomes). We found that despite their lower growth rates, global translation was most active in methanol-grown P. pastoris cells, followed by excess glycerol- or glucose-grown cells. Transcript-specific translational responses were found to be minimal, while extensive transcriptional regulation was observed for cells grown on different carbon sources. Due to their respiratory metabolism, cells grown in excess glucose or glycerol had very similar expression profiles. Genes subject to glucose repression were mainly involved in the metabolism of alternative carbon sources including the control of glycerol uptake and metabolism. Peroxisomal and methanol utilization genes were confirmed to be subject to carbon substrate repression in excess glucose or glycerol, but were found to be strongly de-repressed in limiting glucose-conditions (as are often applied in fed batch cultivations) in addition to induction by methanol. Conclusions: P. pastoris cells grown in excess glycerol or glucose have similar transcript profiles in contrast to S. cerevisiae cells, in which the transcriptional response to these carbon sources is very different. The main response to different growth conditions in P. pastoris is transcriptional; translational regulation was not transcript-specific. The high proportion of mRNAs associated with polysomes in methanol-grown cells is a major finding of this study; it reveals that high productivity during methanol induction is directly linked to the growth condition and not only to promoter strength.

CHARACTERIZATION OF TOBACCO MOSAIC VIRUS DIRECTED REPROGRAMMING OF PHLOEM GENE EXPRESSION TO PROMOTE VIRAL PHLOEM LOADING AND SYSTMEIC MOVEMENT

Vascular phloem loading has long been recognized as an essential step in the establishment of a systemic virus infection. Yet little is known about this process and the mechanisms that control it. In this study, an interaction between the replication protein of Tobacco mosaic virus (TMV) and phloem specific auxin/indole acetic acid (Aux/IAA) transcriptional regulators was found to modulate virus phloem loading. Promoter expression studies show TMV 126/183 kDa interacting Aux/IAAs predominantly express and accumulate within the nuclei of phloem companion cells (CC). Furthermore, CC Aux/IAA nuclear localization is disrupted upon infection with an interacting virus but not during infection with a non-interacting virus. In situ analysis of virus spread shows the inability of TMV variants to disrupt Aux/IAA CC nuclear localization correlates with a reduced ability to load into the vascular tissue. Subsequent systemic movement assays also demonstrate that a virus capable of disrupting Aux/IAA localization is significantly more competitive at systemic movement than a non-interacting virus. Similarly, CC expression and over-accumulation of a degradation-resistant-interacting Aux/IAA protein was found to selectively inhibit TMV accumulation and phloem loading. Transcriptional expression studies demonstrate a role for interacting Aux/IAA proteins in the regulation of salicylic acid and jasmonic acid dependent host defense responses as well as virus specific movement factors including pectin methylesterase that are involved in regulating plasmodesmata size exclusion limits and promoting virus cell-to-cell movement. Further characterization of the phloem environment was done using two phloem specific promoters (pSUC2 and pSULTR2;2) to generate epitope-tagged polysomal-RNA complexes. Immuno-purification using the epitope tag allowed us to obtain mRNAs bound to polysomes (the translatome) specifically in phloem tissue. We found the phloem translatome is uniquely altered during TMV infection with 90% and 88% of genes down regulated in the pSUC2 and pSULTR2;2 phloem translatomes, compared to 31% of genes down regulated in the whole plant p35S translatome. Transcripts down regulated in phloem include genes involved in callose deposition at plasmodesmata, host defense responses, and RNA silencing. Combined, these findings indicate TMV reprograms gene expression within the vascular phloem as a means to enhance phloem loading and systemic spread.