mRNA-dependent Cell-free System Research Articles

Membrane-specific, Host-derived Factors Are Required for US2- and US11-mediated Degradation of Major Histocompatibility Complex Class I Molecules

Human cytomegalovirus encodes two glycoproteins, US2 and US11, that target major histocompatibility complex (MHC) class I heavy chains for proteasomal degradation. We have developed a mRNA-dependent cell-free system that recapitulates US2- and US11-mediated degradation of MHC class I heavy chains. Microsomes support the degradation of MHC class I heavy chains in the presence of US2 or US11 in a cytosol-dependent manner. In vitro, the glycosylated heavy chain is exported from the microsomes. A deglycosylated breakdown intermediate of the heavy chain identical to that generated in intact cells accumulates in soluble form in the presence of proteasome inhibitors. Microsomes derived from the U373 astrocytoma cell line are far more effective than canine-derived membranes in supporting this US2- or US11-dependent reaction. In contrast, the HIV-encoded Vpu membrane protein can cause the destruction of CD4 from either human- or canine-derived membranes. Using the in vitro system, we show that a truncation mutant of US2 that lacks the cytosolic domain is unable to catalyze degradation, whereas a similar truncation of US11 continues to catalyze degradation of class I heavy chains. Therefore, US2 requires both transmembrane and cytosolic interactions to trigger dislocation of heavy chains, whereas US11 relies on the transmembrane domain to target heavy chains. US2 and US11 thus utilize different targeting mechanisms for class I degradation.

In VitroTranslation and Translocation of Apolipoprotein B in a Cell-Free System from HepG2 Cells

An mRNA-dependent cell-free system has been developed from HepG2 cells by hydrolysis of endogenous mRNA with micrococcal nuclease. When supplied with RNA extracted from HepG2 cells, the system synthesized liver specific proteins such as albumin and apolipoprotein B100. Significant amounts of microsomes were also detected in the lysate by measuring NADH-cytochrome c reductase activity and ultracentrifugation. Protease protection assays showed the capability of the HepG2 lysate to translocate newly-synthesized proteins such as apolipoprotein AI, albumin, and apoB into the microsomes as they were protected from digestion with exogenously added protease K, but not protected in the presence of protease K and Triton X-100. The system also proved to be very active toward translation of exogenous mRNAs as evidenced by efficient translation of brome mosaic virus RNA. The HepG2 translation–translocation system appears to provide a unique homologous system for studies on the biogenesis of liver specific proteins, particulary apoB100.

Efficient translation of synthetic and natural mRNAs in an mRNA-dependent cell-free system from the dimorphic fungus Candida albicans

An mRNA-dependent cell-free translation system has been developed from the human pathogenic fungus Candida albicans using either S30 or S100 lysates prepared from glass-bead-disrupted whole cells. Translation of the synthetic template poly(U) in this system is highly efficient at temperatures up to 37 degrees C and is ATP-dependent. Studies using a range of elongation-specific inhibitors suggest that the mechanism of translational elongation in C. albicans is similar to that of another yeast, Saccharomyces cerevisiae. A micrococcal-nuclease-treated C. albicans S100 lysate was able to translate exogenously-supplied homologous mRNAs, and a range of heterologous natural mRNAs, using an initiation mechanism that is inhibited by the antibiotic edeine and the 5' cap analogue 7-methylguanosine 5'-monophosphate (m7GMP). As with cell-free lysates prepared from S. cerevisiae, the C. albicans lysate is unable to initiate translation upon natural mRNAs at temperatures above 20 degrees C.

Actinomycin D Blocks the Hepatic Functional Albumin mRNA Increase in Aminonucleoside-Nephrotic Rats

Hepatic functional albumin-mRNA was measured in the following groups of rats: (a) puromycin aminonucleoside (PAN)-nephrotic rats, (b) PAN-nephrotic rats treated with actinomycin D prior to sacrifice, (c) control rats, and (d) control rats treated with actinomycin D. Albumin mRNA was translated in an mRNA-dependent cell-free system from rabbit reticulocyte lysate. Albumin-mRNA increased about 2-fold in PAN-nephrotic rats. This increase was abolished in vivo in PAN-nephrotic rats treated with actinomycin D. Albumin mRNA was not significantly modified in control rats treated with actinomycin D. These data suggest that the increased level of hepatic functional albumin mRNA observed in PAN-nephrotic rats in vivo was due mainly to the increased rate of albumin gene transcription.

Developmentally regulated alternate modes of expression of the Gpdh locus of Drosophila.

Immunoblot analyses have been performed on extracts prepared from Drosophila melanogaster. Those analyses have revealed two subunit forms of enzyme glycerol 3-phosphate dehydrogenase (GPDH) in larval tissues and in adult abdominal tissues. Thoracic tissue, which accounts for the bulk of the adult GPDH, has only one subunit form, the smaller. The two subunit forms differ by approximately 2400 daltons. In agreement with previous genetic and biochemical data indicating that this enzyme is encoded by a single structural gene, analyses of extracts prepared from a strain carrying a GPDH null mutation detect no GPDH polypeptides in larvae or adults. Similarly, analyses of extracts prepared from a strain carrying a mutation which produces a GPDH polypeptide that differs in size from wild-type reveal a change in the adult thoracic GPDH polypeptide as well as a change in both GPDH polypeptides found in larvae. Total Drosophila RNA prepared from larvae or newly eclosed adults has been translated in a mRNA-dependent cell-free system. GDPH was immunoprecipitated from the translation products and analyzed. Two subunit forms of GPDH were immunoprecipitated from translation products whose synthesis was directed by larval RNA and only one was detected in the polypeptides synthesized from adult RNA. The GPDH polypeptides synthesized in vitro are approximately the same size as the corresponding polypeptides found in vivo. The relative proportion of total GPDH represented by each subunit form synthesized in vitro is similar to those found in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of paromomycin on the fidelity of translation in a yeast cell-free system

The effects of the aminoglycoside antibiotic paromomycin on the fidelity of translation of the synthetic template poly(U), and two natural mRNAs (rabbit globin mRNA and Brome Mosaic virus RNA), were examined in an mRNA-dependent cell-free system from the yeast Saccharomyces cerevisiae. At antibiotic concentrations that did not inhibit translation (100 μM) optimal mistranslation of all three templates was observed, with the effects declining at higher antibiotic concentrations. Synthesis of the opal termination read-through protein of rabbit β-globin mRNA was induced by paromomycin, but only in lysates prepared from a [psi +] strain of yeast. The antibiotic did not induce detectable levels of either ochre or amber read-through, but did induce general misreading of Brome Mosaic virus RNA to the same degree in both [psi +] and [psi −] lysates. This misreading was enhanced by addition of the polyamine spermidine.

Synthesis of S100 protein on free and membrane-bound polysomes of the rabbit brain.

Free and membrane-bound polysomes were isolated from the cerebral hemispheres and cerebellum of the young adult rabbit. The two polysomal populations were translated in an mRNA-dependent cell-free system derived from rabbit reticulocytes. Analysis of the [35S]methionine-labeled translation products on two-dimensional polyacrylamide gels indicated an efficient separation of the two classes of brain polysomes. The relative synthesis of S100 protein by free and membrane-bound polysomes was determined by direct immuno-precipitation of the cell-free translation products in the presence of detergents to reduce nonspecific trapping. Synthesis of S100 protein was found to be twofold greater on membrane-bound polysomes compared with free polysomes isolated from either the cerebral hemispheres or the cerebellum. In addition, the proportion of poly-(A+)mRNA coding for S100 protein was also twofold greater in membrane-bound polysomes compared with free polysomes isolated from the cerebral hemispheres. These results indicate that the cytoplasmic S100 protein is synthesized predominantly on membrane-bound polysomes in the rabbit brain. We suggest that the nascent S100 polypeptide chain translation complex is attached to the rough endoplasmic reticulum by an ionic interaction involving a sequence of 13 basic amino acids in S100 protein.

Translation of tick-borne encephalitis virus (flavivirus) genome in vitro: Synthesis of two structural polypeptides

RNA isolated from tick-borne encephalitis virus (TBEV) was translated in an mRNA-dependent cell-free system derived from Krebs-2 cells, producing a set of polypeptides with M r ranging from ca. 13,000 to 160,000 and higher. Two of these polypeptides with M r of 13,000 (p13) and 53,000 (p53) were identified as TBEV core polypeptide V2 and the polypeptide moiety of envelope glycoprotein, V3, respectively. The amino acid sequences of p13 and p53 were also shown to be contained in a high-molecular-weight polypeptide ( M r ∼ 118,000). The label from the initiator tRNA species, f-[ 35S]Met-tRNA f Met, could be incorporated into p13 but not into p53, suggesting that p13 is encoded in a region of the viral genome immediately adjacent to the site at which the translation in vitro is initiated; on the other hand, the p53 coding segment appears to be located further from the initiation point. The two structural polypeptides of TBEV were accumulated in vitro with dissimilar kinetics and both differed from the majority of other translation products in that their synthesis was relatively resistant to an increase in KCl concentration in the incubation mixture. Possible modes of synthesis of TBEV structural proteins and post-translational processing of their precursor are discussed.

Immunological identification of a common precursor to arginine vasopressin and neurophysin II synthesized by in vitro translation of bovine hypothalamic mRNA.

mRNA from membrane-bound polysomes of bovine hypothalamus was translated in an mRNA-dependent cell-free system from reticulocyte lysate or wheat germ. The translation products were identified by immunoprecipitation with antibodies to either neurophysin II or arginine vasopressin followed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. An immunoreactive polypeptide was obtained with an apparent Mr of 21,000. Sequential immunoprecipitation studies indicated that the Mr 21,000 product is a common precursor to neurophysin II and arginine vasopressin. The specificity of the immunoprecipitation was demonstrated by competition with excess amounts of unlabeled neurophysin II or arginine vasopressin; little or no competition was observed with unlabeled neurophysin II or arginine vasopressin; little or no competition was observed with unlabeled neurophysin I or oxytocin. Processing experiments with microsomal membranes from dog pancreas or tunicamycin-treated ascites tumor cells showed that the Mr 21,000 polypeptide is the prepro form. It was converted to a pro form with Mr 19,000 suggesting a pre sequence of approximately 15 amino acids. The Mr 19,000 polypeptide was coreglycosylated to an apparent Mr of 23,000, indicating that the neurophysin II-arginine vasopressin precursor is a glycopolypeptide.

Faithful and efficient translation of homologous and heterologous mRNAs in an mRNA-dependent cell-free system from Saccharomyces cerevisiae.

A cell-free protein synthesizing system from the yeast Saccharomyces cerevisiae has been optimized for the translation of both homologous yeast mRNA and for a number of heterologous eukaryotic mRNAs. A significant increase in protein synthesis was observed when K(OAc) rather than KCl was used as the source of K+ in the in vitro translation system. This was due primarily to an inhibitory effect oif Cl-. The polyamine putrescine hydrochloride stimulated protein synthesis only at low Mg2+ concentrations. Protein synthesis directed by both yeast mRNA and several eukaryotic mRNAs examined in the system was sensitive to the mRNA 5'-cap analogue, 7-methylguanosine 5'-monophosphate. One-dimensional and two-dimensional polyacrylamide gel analysis of polypeptides synthesized in response to yeast polysomal RNA demonstrated faithful translation in vitro. Translational control and post-translational modifications appear to operate normally in vitro. RNA from several eukaryotic viruses (brome mosaic virus, turnip yellow mosaic virus, and tobacco mosaic virus) were found to be faithfully translated in vitro yielding discrete polypeptides. Reticulocyte polysomal RNA directed the synthesis of a single protein that co-migrated with rabbit globin. The prokaryotic RNAs of Q beta and MS2 were translated with a very low efficiency. The yeast cell-free system programmed with yeast polysomal RNA provides an excellent model for the study of translational control in a eukaryote.

Mouse immunoglobulin mu heavy chain mRNA of Y5781, a high yield myeloma.

The BALB/c myeloma tumor, Y5781, has a high level of mu heavy chain mRNA and kappa light chain mRNA, as suggested by denaturing gel analyses of poly(A)-rich, total polysomal mRNA, and confirmed for the mu heavy chain mRNA by kinetic complexity analyses. Both the mRNA coding for the heavy and light chains appear as very prominent and discrete peaks above the generally polydisperse background of the total polysomal mRNA. This mRNA level appears to be stable through a limited number of subcutaneous passages of this myeloma, providing a potentially useful system for mu heavy chain mRNA synthesis and processing. The mu heavy chain mRNA of this myeloma has been enriched to about 60% homogeneity by physicochemical means. In agreement with a previous report (Faust, C.H., Jr., Heim, I., and Moore, J. (1979) Biochemistry 18, 1106-1119), the following physical and biological properties were observed. The mature cytoplasmic mu heavy chain mRNA is 950,000 daltons, i.e. about 2800 nucleotides, and contains approximately 800 undefined, nontranslated bases. In an mRNA-dependent cell-free system, this mRNA stimulates the synthesis of a single, serologically reactive mu heavy chain-like protein, confirmed by tryptic peptide maps.

Inhibition of translation of lens mRNAs in a messenger dependent reticulocyte lysate by cap analogues

The nuclease treated reticulocyte lysate forms a highly efficient and completely mRNA-dependent cell-free system. In this system the functioning of the cap on eukaryotic mRNAs was explored by blocking cap recognition with cap analogues. Translation of capped mRNAs was severely inhibited, while translation of uncapped mRNAs was unaffected. It is concluded that this cell-free system can be used for screening cap dependence in the translation of specific mRNAs, like calf lens mRNAs. At 1.2 mM m 7G5′p, 0.16 mM m 7G5′pp or 0.16 m 7G5′ppp5′G, translation of all lens mRNAs was totally inhibited. At lower concentrations the sensitivity to cap analogues was different for the various species of lens crystallin messenger. γ-Crystallin mRNA showed relatively the lowest response. The translation of added polyribosomes was also inhibited by the cap analogue. It is concluded that translation of all crystallin messengers is cap-dependent.

Effect of pH on the extraction of polyadenylic acid-containing RNA from soybean seeds.

The effect of pH in the SDS-phenol extraction step on the extractability, messenger activity and the composition of RNA species of the poly (A)-containing RNA from the cotyledons of soybean seeds was examined. The amount of total RNA extracted from the cotyledons was almost the same under neutral and alkaline pH. However the content of the poly (A)-containing RNA was higher in the preparation obtained under alkaline pH than neutral pH. higher in the preparation obtained under alkaline pH than neutral pH. This result shows that the poly (A)-containing RNA was efficiently extracted from soybean seeds under alkaline pH. In a mRNA-dependent cell-free system derived from E. coli, there was no significant difference in the messenger activity of the poly (A)-containing RNA between the two preparations. The patterns of the poly (A)-containing RNAs of the two preparations were very similar to one another in the case of dry soybean seeds, while there was slight difference in the case of green soybean seeds. The pol...