Cell-free translation of silkmoth chorion mRNAs: Identification of protein precursors and characterization of cloned DNAs by hybrid-selected translation

The secretory silkmoth chorion proteins are synthesized as precursors bearing signal peptides. Precursors are detected upon cell-free translation of chorion mRNAs in the wheat germ system; they are processed into products identical in size to authentic chorion proteins when translation is performed in the presence of microsomal membranes from dog pancreas. Precursors corresponding to specific protein size classes and subclasses are identified by three approaches: comparison of precursors and products encoded by stage-specific mRNAs, comparison of precursors and products encoded by mRNAs specifically hybridizing to individual chorion cDNA clones, and comparison of relative amino acid compositions of precursors and authentic chorion proteins. Translation of stage-specific mRNA preparations indicates that, in general, the developmental changes of in vivo chorion protein synthesis are based on changes in concentrations of the corresponding mRNAs. Characterization of the precursors makes it possible to identify, for any chorion DNA clone, the protein subclass, a member of which is encoded by the clone sequence.     

Identification and time of synthesis of chorion proteins in Drosophila melanogaster.

The chorion of Drosophila melanogaster consists of proteins secreted by the follicular epithelium during late oogenesis. Petri, Wyman and Kafatos (1976) have described six major protein components of the Drosophila chorion and reported the synthesis of these proteins in vitro by mass-isolated egg chambers. We have used two-dimensional gel electrophoresis to identify approximately twenty components in highly purified chorion preparations. The synthesis patterns of these proteins in vivo were determined by isolating egg chambers of different developmental stages from flies injected with 14C amino acids. Chorion proteins constitute a large fraction of the protein synthesized by ovarian egg chambers in stages 12--14. The sizes and times of synthesis of the chorion proteins correlate closely with the production of poly(A)-containing RNAs by the follicle cells (Spradling and Mahowald, 1979).     

Effects of testosterone on messenger ribonucleic acid and protein synthesis in rat seminal vesicle

In a previous report [Higgins et al. (1976) Biochem. J. 158, 271–282] we described the effects of alterations in androgen status on the synthesis of two basic secretory proteins of the rat seminal vesicle. In the present paper we examine the effects of testosterone on the activity of mRNA in the seminal vesicle. Total cellular poly(A)-rich RNA was isolated and translated in a cell-free system prepared from wheat germ. Translation products were separated on denaturing polyacrylamide gels and the protein bands corresponding to the two basic secretory proteins were identified immunologically. Incorporation of radioactive methionine into these bands was taken as a measure of the individual mRNA activities. Total mRNA activity was estimated by radioactivity in total acid-precipitable material. The results show that 1 to 2 weeks after castration the activities of mRNA molecules for the basic secretory proteins were decreased 10–20-fold on a tissue basis. Testosterone given in vivo rapidly and substantially restores mRNA activity to normal. Since these changes correlate closely with variations in the rates of synthesis of the secretory proteins in whole cells it suggests that androgenic steroids control protein synthesis chiefly via mRNA availability. In this respect their action resembles those of other steroid hormones acting in other systems. However, these effects of testosterone on the mRNA molecules for the major secretory proteins could not be distinguished from those on total mRNA. Thus the proportion of the total mRNA population accounted for by the two specific mRNA molecules showed less than a 2-fold variation with androgen status. Similarly the two secretory proteins always accounted for 25–33% of general protein synthesis. This is in sharp contrast with the markedly differential effects of other steroid hormones controlling synthesis of major proteins in other well-studied systems. We interpret our results as indicating that testosterone regulates the mRNA population of the seminal vesicle as a whole.     

Specific protein synthesis in cellular differentiation: III. The eggshell proteins of Drosophila melanogaster and their program of synthesis

The eggshell of Drosophila melanogaster is composed of a set of proteins synthesized by the follicular epithelium during the last third of oogenesis and organized into an inner zone (vitelline membrane) and an outer zone (chorion). To study these proteins, the authors developed techniques for mass-isolating follicles of mixed stages, mature (stage 14) follicles, chorion from stage 14 follicles, and chorion and vitelline membrane from laid eggs. The eggshell is composed mainly of protein and is unusually rich in proline and alanine. Six proteins of the chorion have been identified on polyacrylamide gels. The program of synthesis of these proteins was studied by incubating follicles of different developmental stages in culture with ³H-labeled amino acids and displaying the labeled proteins on gels with the aid of autofluorography. The proteins are synthesized in a specific overlapping sequence during stages 10–14, a period when chorion deposition is known to occur. In addition, putative vitelline membrane proteins have been identified by their preferential incorporation of [³H]proline and [³H]alanine during stages of active vitelline membrane synthesis.     

INCREASED RATE OF ACETYLCHOLINESTERASE SYNTHESIS IN DIFFERENTIATING NEUROBLASTOMA CELLS

When neuroblastoma cells (N18) in vitro are maintained in the absence of serum, the specific activity of AChE begins to rise rapidly after an initial lag period of about 2–3 days, reaching a maximum level (10–20-fold increase) by 7 days after induction. In order to clarify the mechanism of induction, it was necessary to measure the rate of AChE synthesis and its sensitivity to metabolic inhibitors. Return of enzymatic activity after irreversible inhibition of AChE in "differentiated" cells was blocked by cycloheximide, but not by cordycepin or actinomycin D, suggesting that protein but not mRNA synthesis was required for replacement. By using the initial rate of this replacement as a measure of the rate of synthesis of the enzyme, it was shown that cells which had differentiated in the absence of serum synthesized AChE 50-fold faster on a specific activity basis than their undifferentiated counterparts. In contrast, cordycepin effectively blocked the increase in the rate of AChE synthesis that occurs as a result of serum deprivation, indicating that the induction process itself requires the synthesis of new mRNA. Axonation, another index of differentiation, was not completely blocked by inhibition of RNA or protein synthesis and presumably utilizes only pools of pre-existing structural proteins.     

Translational control by RGS2

The regulator of G protein signaling (RGS) proteins are a family of guanosine triphosphatase (GTPase)–accelerating proteins. We have discovered a novel function for RGS2 in the control of protein synthesis. RGS2 was found to bind to eIF2Bϵ (eukaryotic initiation factor 2B ϵ subunit) and inhibit the translation of messenger RNA (mRNA) into new protein. This effect was not observed for other RGS proteins tested. This novel function of RGS2 is distinct from its ability to regulate G protein–mediated signals and maps to a stretch of 37 amino acid residues within its conserved RGS domain. Moreover, RGS2 was capable of interfering with the eIF2–eIF2B GTPase cycle, which is a requisite step for the initiation of mRNA translation. Collectively, this study has identified a novel role for RGS2 in the control of protein synthesis that is independent of its established RGS domain function.     

Cancer‐secreted miRNAs regulate amino‐acid‐induced mTORC1 signaling and fibroblast protein synthesis

Metabolic reprogramming of non‐cancer cells residing in a tumor microenvironment, as a result of the adaptations to cancer‐derived metabolic and non‐metabolic factors, is an emerging aspect of cancer–host interaction. We show that in normal and cancer‐associated fibroblasts, breast cancer‐secreted extracellular vesicles suppress mTOR signaling upon amino acid stimulation to globally reduce mRNA translation. This is through delivery of cancer‐derived miR‐105 and miR‐204, which target RAGC, a component of Rag GTPases that regulate mTORC1 signaling. Following amino acid starvation and subsequent re‐feeding, ¹³C‐arginine labeling of de novo synthesized proteins shows selective translation of proteins that cluster to specific cellular functional pathways. The repertoire of these newly synthesized proteins is altered in fibroblasts treated with cancer‐derived extracellular vesicles, in addition to the overall suppressed protein synthesis. In human breast tumors, RAGC protein levels are inversely correlated with miR‐105 in the stroma. Our results suggest that through educating fibroblasts to reduce and re‐prioritize mRNA translation, cancer cells rewire the metabolic fluxes of amino acid pool and dynamically regulate stroma‐produced proteins during periodic nutrient fluctuations.     

Effect of the pleiotropic GrB mutation of Bombyx mori on chorion protein synthesis

The Gr^B mutation has a profound pleiotropic effect, leading in the homozygous state to the absence or extreme reduction of a substantial number of chorion proteins. The effect shows developmental specificity: most of the proteins normally synthesized beginning with stage III of choriogenesis or later, but possibly none of these normally beginning with stage II, are eliminated in the mutant. More subtle quantitative effects on certain other proteins are also observed, including prolongation of synthesis of some proteins which normally terminate at stage VIII. The proteins eliminated in the mutant are present in the heterozygote at intermediate levels, quantitatively close to those in the wild-type. The differences in chorion protein composition result from correspondingly altered protein synthesis rather than from post-translational degradation or modification. The missing proteins also fall to be synthesized in vitro when total RNA from mutant follicles is translated in the wheat germ system. It appears that as a consequence of the mutation, a set of mRNAs fails to be synthesized or accumulated. These results are consistent with the possibility that Gr^B is a regulatory mutation, or a deletion eliminating multiple chorion genes, clustered predominantly according to the developmental period of their expression.     

Specific protein synthesis in cellular differentiation: V. A secretory defect of chorion formation in the Grcol mutant of Bombyx mori

When homozygous, the Gr^col mutation of Bombyx mori causes the production of an eggshell in which most proteins are underrepresented to varying degrees. Neither the relative rates nor the timing of chorion protein synthesis appear to be affected; instead, the mutant phenotype results from the post-translational loss of normally synthesized proteins. The extent of loss of each protein correlates with its developmental timing, being maximal at early to middle stages. At the same stages, secretion appears to be deficient: chorion proteins overaccumulate within mutant cells, and slowly disappear. A preliminary electron microscopic examination has revealed the presence of mutant-specific cytoplasmic vesicles. The deficient complement of secreted proteins fails to form the highly ordered structure characteristic of normal chorion.     

Specific protein synthesis in cellular differentiation: II. The program of protein synthetic changes during chorion formation by silkmoth follicles,and its implementation in organ culture

During their differentiation, the follicular epithelial cells of the silkmoth, Antheraea polyphemus, produce the extracellular proteinaceous eggshell or chorion. Choriogenesis entails continuous changes in cell-specific protein synthesis; the various chorion proteins are synthesized with distinct kinetics. On the basis of protein synthetic profiles, 17 stages of choriogenesis are defined. The average duration of the stages is 3.0 hr, and thus choriogenesis lasts a total of approximately 51 hr. This program of protein synthetic changes is autonomous; i.e., it is implemented with normal kinetics by follicles cultured in isolation in a defined tissue culture medium.     

Differential synthesis in vitro of barley aleurone and starchy endosperm proteins

To widen the selection of proteins for gene expression studies in barley seeds, experiments were performed to identify proteins whose synthesis is differentially regulated in developing and germinating seed tissues. The in vitro synthesis of nine distinct barley proteins was compared using mRNAs from isolated endosperm and aleurone tissues (developing and mature grain) and from cultured (germinating) aleurone layers treated with abscisic acid (ABA) and GA₃. B and C hordein polypeptides and the salt-soluble proteins β-amylase, protein Z, protein C, the chymotrypsin inhibitors (CI-1 and 2), the α-amylase/subtilisin inhibitor (ASI) and the inhibitor of animal cell-free protein synthesis systems (PSI) were synthesized with mRNA from developing starchy endosperm tissue. Of these proteins, β-amylase, protein Z, and CI- 1 and 2 were also synthesized with mRNA from developing aleurone cells, but ASI, PSI, and protein C were not. CI-1 and also a probable amylase/protease inhibitor (PAPI) were synthesized at high levels with mRNAs from late developing and mature aleurone. These results show that mRNAs encoding PAPI and CI-1 survive seed dessication and are long-lived in aleurone cells. Thus, expression of genes encoding ASI, PSI, protein C, and PAPI is tissue and stage-specific during seed development. Only ASI, CI-1, and PAPI were synthesized in significant amounts with mRNA from cultured aleurone layers. The levels of synthesis of PAPI and CI-1 were independent of hormone treatment. In contrast, synthesis of α-amylase (included as control) and of ASI showed antagonistic hormonal control: while GA promotes and ABA reduces accumulation of mRNA for α-amylase, these hormones have the opposite effect on ASI mRNA levels.     

Growth-regulated expression and G0-specific turnover of the mRNA that encodes URH49, a mammalian DExH/D box protein that is highly related to the mRNA export protein UAP56

URH49 is a mammalian protein that is 90% identical to the DExH/D box protein UAP56, an RNA helicase that is important for splicing and nuclear export of mRNA. Although Saccharomyces cerevisiae and Drosophila express only a single protein corresponding to UAP56, mRNAs encoding URH49 and UAP56 are both expressed in human and mouse cells. Both proteins interact with the mRNA export factor Aly and both are able to rescue the loss of Sub2p (the yeast homolog of UAP56), indicating that both proteins have similar functions. UAP56 mRNA is more abundant than URH49 mRNA in many tissues, although in testes URH49 mRNA is much more abundant. UAP56 and URH49 mRNAs are present at similar levels in proliferating cultured cells. However, when the cells enter quiescence, the URH49 mRNA level decreases 3–6-fold while the UAP56 mRNA level remains relatively constant. The amount of URH49 mRNA increases to the level found in proliferating cells within 5 h when quiescent cells are growth-stimulated or when protein synthesis is inhibited. URH49 mRNA is relatively unstable (T_½ = 4 h) in quiescent cells, but is stabilized immediately following growth stimulation or inhibition of protein synthesis. In contrast, there is much less change in the content or stability of UAP56 mRNA following growth stimulation. Our observations suggest that in mammalian cells, two UAP56-like RNA helicases are involved in splicing and nuclear export of mRNA. Differential expression of these helicases may lead to quantitative or qualitative changes in mRNA expression.     

Electrophoretic analysis of proteins synthesized by preimplantation mouse embryos

Proteins synthesized during the preimplantation period of mouse embryogenesis were labeled with radioactive tyrosine and lysine and fractionated by electrophoresis on polyacrylamide disc gels containing sodium dodecyl sulfate. For interstage comparisons and comparisons of the incorporation of different amino acids at the same developmental stages, the embryos were incubated with either ³H- or ¹⁴C-labeled amino acids. The embryos were then combined, and the proteins were isolated and electrophoresed simultaneously. The data were analyzed with a dual isotope computer program and expressed in the form of ¹⁴C/³H ratios.Approximately 20–25 labeled protein components of apparent molecular weights between 25,000 and 115,000 can be defined, and 5 are most significant quantitatively. Of the latter, there are developmental increases in the rates of synthesis of 3 (with apparent molecular weights of 35,000 to 37,000, 37,000 to 41,000, and 66,000 to 70,000), a decrease in the rate of synthesis of another (53,000 to 57,000), and little change in the last (46,000 to 49,000). Developmental changes in the rates of synthesis of several other components are also demonstrated by the ¹⁴C/³H incorporation ratios. The relative amounts of the different proteins synthesized by day 3 (early blastocyst) embryos over an 8-hr period remain constant, as does the relative labeling by lysine and tyrosine at each developmental stage examined. Similarly, there is no change in the pattern of the radioactive proteins when day 2 (8–16 cell) embryos are labeled for 2 hr and then incubated for an additional 24 hr. The greatest change in the overall pattern of protein synthesis occurs quite early, between day 1 (2 cell) and day 2, and lesser changes occur at later stages. These findings are in contrast to the major change in the rate of protein synthesis which occurs after day 2.     

Specific protein synthesis in cellular differentiation: IV. The chorion proteins of Bombyx mori and their program of synthesis

Detailed analysis of the chorion proteins of Bombyx mori reveals more than 70 components, each distinguished by electrophoretic mobility, relative abundance, and kinetics of synthesis. Many proteins are strain specific. A protein numbering system is established, based on isoelectric point and molecular weight. As in Antheraea polyphemus, chorion proteins are produced asynchronously, individual proteins showing characteristic developmental kinetics. The synthetic program is analyzed in detail. Stages of choriogenesis are defined according to the pattern of protein synthesis, and their relative and absolute durations are determined. In a few stages, synthesis of numerous protein cohorts is coordinately initiated.     

The site of synthesis of mitochondrial proteins in Krebs II ascites-tumour cells

At 22° in Earle's medium, Krebs cells synthesize proteins. After a brief `pulse' with [<sup>14</sup>C]valine followed by a `chase' of [¹²C]valine the radioactivity appears first in microsomes and is transferred after `chase' to the cell sap. Kinetics of labelling of the mitochondrial protein are different from that of either microsomal or cell-sap protein. When Krebs cells in buffer are mixed with ribonuclease in water the nuclease penetrates the cell membrane. The ribonuclease-treated cells are still viable but have lost most of their cytoplasmic ribosomes (electron micrograph). Such cells still synthesize mitochondrial protein at near normal rate but synthesis of microsomal protein is severely inhibited. The results indicate that some mitochondrial proteins are synthesized independently of the microsome–cell-sap system.