Translation Of Total RNA Research Articles

Abstract B19: Functional translation of total RNA packaged in microparticles shed from multidrug resistant cancer cells

Abstract Microparticles (MPs) are submicron (0.1 -1 µM) membrane vesicles released from various cell types including malignant cells. Long considered to be inert cellular debris, MPs have recently been identified to play a crucial role in cellular cross-talk. MPs represent a heterogeneous mixture of surface antigens, proteins, lipids and cytoplasmic components resembling its parental cell. Our previous studies have identified the packaging and transfer of the transcripts, regulatory nucleic acids and proteins which are associated with the acquirement of multidrug resistance (MDR) between cancer cells. In this study we verify the functionality of packaged transcripts via in vitro translation of extracted total RNA from MPs isolated from cancer cells. We confirm the translation of exogenous RNA in a rabbit reticulocyte translation system via Western Blot analysis, 2-D SDS and mass spectrometry. These findings have significance in understanding the impact of MPs on cancer cell biology and reiterate their potential as a target in clinical oncology. Citation Format: Jamie Fung Lu, Deep Pokharel, Frederick Luk, Mary Bebawy. Functional translation of total RNA packaged in microparticles shed from multidrug resistant cancer cells. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr B19.

Incorporation of arylphorins (LSP-1) and LSP-2 like protein into the integument of Ceratitis capitata during pupariation

The arylphorins and LSP-2 like polypeptide were detected by immunoblotting analysis during development in the integument of C. capitata. In vitro translation of total RNA from fat body and integument during pupariation, clearly revealed that the polypeptides under consideration were exclusively synthesized in the fat body. Furthermore, in vitro experiments demonstrated that radiolabeled arylphorins and LSP-2 like polypeptide were taken up by the integument, in an undegraded state. Immunofluorescence experiments in cross sections of wandering stage larvae and white pupae revealed that the LSP-2 like polypeptide was mainly localized in the epidermal cells, and a very weak signal was also given by the cuticle. Furthermore, the presented results indicated that a small portion of the extracted proteins exist in high molecular weight aggregate(s).

Stress-induced proteins in aortic smooth muscle cells and aorta of hypertensive rats

The current studies were performed to determine whether vascular smooth muscle cells produce stress-induced proteins when subjected to experimental hypertension. Two-dimensional gel electrophoresis was used to analyze labeled proteins in cultured cells in response to either heat shock or arsenite treatment. The major heat shock proteins (HSPs) induced had molecular masses of 70, 90, and 110 kDa. Arsenite treatment produced a similar response with the additional induction of a 30-kDa protein. In vitro translations of total RNA from heat-shocked cells, and RNA blot hybridization using HSP 70 cDNA suggested that HSP 70 induction was transcriptionally regulated. Treatment of cells with norepinephrine or angiotensin II induced cellular hypertrophy without eliciting HSPs. In vitro translation of aortic RNA from rats rendered hypertensive by administration of deoxycorticosterone and a high salt intake also did not reveal HSP induction. The absence of HSP induction using an in vivo model of hypertension suggests that those proteins may not be required to mediate the vascular response to experimental hypertension.

Molecular Changes Involved in the Ripening of Tomato Fruit

Summary In vitro translation of total RNA extracted from developing and wounded tomato fruit ( Lycopersicon esculentum Mill. cv. Rutgers) indicated that ripening-related changes to polypeptide populations are initiated in the gel and placental tissues before spreading to external tissues. Expression of ripening-related cDNA probes showed that polygalacturonase (PG) activity was first activated in placental tissue, while photosynthesis first declined in the centre of the fruit. These results support the view that ripening in tomato fruit is under genetic control, and are consistant with the theory that ripening is initiated in tomato gel tissue.

The biosynthesis and assembly of methanol dehydrogenase in bacterium W3A1.

Bacterium W3A1, a restricted facultative methylotroph, produces a periplasmic methanol dehydrogenase composed of two identical subunits of Mr = 57,300, and two noncovalently bound methoxatin prosthetic groups. A precursor form of Mr = 1,500 larger than the mature subunit was identified among the products of an in vitro translation of total RNA isolated from bacterium W3A1. The precursor form of the protein could not be detected in cells during in vivo pulse-labeling studies, suggesting that the processing of this precursor occurs entirely co-translationally. Whereas the holoenzyme was detectable only as a dimer, removal of the prosthetic group yielded an apoenzyme that could be detected as either a dimeric or monomeric species. After readdition of the purified prosthetic group to the apoenzyme, only the dimeric form of the protein, bearing the cofactor and exhibiting an absorption spectrum similar to that of the holoenzyme, was detected. Neither the mature apoprotein nor the holoenzyme demonstrated any affinity for phospholipid membranes, as assayed by their inability to bind to liposomes. Taken together, these data suggest a scheme of co-translational processing and export of the apoprotein subunits, followed by assembly of the subunits and prosthetic groups in the periplasmic space to form the mature holoenzyme. The suitability of bacterium W3A1, and other methylotrophic bacteria, for use in studies of protein biosynthesis and export, is also discussed.

Translatable mRNA for GP140 (a subunit of type VI collagen) is absent in SV40 transformed fibroblasts.

Production of GP140, a major component of the extracellular matrix of cultured fibroblasts, is markedly decreased in SV40 transformed cells as compared with normal cells (Carter, W. G., 1982, J. Biol. Chem., 257:13805-13815). To determine at what step the biosynthesis is inhibited, we compared the levels of functional mRNA for GP140 in normal and transformed fibroblasts. Translation of total RNA from W138 cells in a reticulocyte lysate, followed by immunoprecipitation with affinity-purified antibodies to GP140, yielded a single polypeptide with an Mr of 125,000. This polypeptide was identified as GP140 based on its immunoreactivity, collagenase sensitivity, and comigration on polyacrylamide gels with GP140 synthesized by cells in the presence of tunicamycin and 2,2'-bipyridyl. No cell-free synthesis of GP140 was observed with total RNA from SV40 transformed W138 cells, indicating that these cells contain very low levels of GP140-specific mRNA. The biosynthesis of GP140 might therefore be blocked at the transcriptional level.

Coordinate regulation of synthesis of ribosomal proteins during encystation of Acanthamoeba castellanii.

The synthesis of ribosomal proteins has been examined during growth and encystment of Acanthamoeba castellanii. Cells have been radiolabelled with [35S]methionine and ribosomal proteins have been extracted either from ribosomes or from total cell extracts. The results show that there is less synthesis of ribosomal proteins relative to total cell proteins soon after transfer of growing cells into non-nutrient medium, suggesting that the synthesis of all ribosomal proteins is under coordinate control. Furthermore, synthesis of ribosomal RNA and ribosomal proteins seems to be regulated coordinately, since within 1 h there is 70-80% decline in synthesis of both. Translation of total RNA in vitro indicates that the relative decrease in the synthesis of ribosomal proteins during development is not only due to a relative decrease in the content of ribosomal protein mRNA. However, a considerable (about 10-fold) and coordinate decrease in the relative content of ribosomal protein mRNA occurs during encystment. These data suggest a difference in the control of the synthesis of total proteins and ribosomal proteins as well as in the regulation of the content of total mRNA and ribosomal protein mRNA. The comparison of patterns of ribosomal proteins indicates that during development no qualitative changes occur. The content of one protein of the small ribosomal subunit is low in growing cells and increases during encystation. Since the differences in the concentration of this protein are neither due to changing levels of the corresponding mRNA nor to changing rates of synthesis, changing affinities of this protein to ribosomes in vivo are suggested.

The gcr (glycolysis regulation) mutation of Saccharomyces cerevisiae.

gcr is a mutation considerably decreasing the assayed amounts of most glycolysis enzymes in Saccharomyces cerevisiae (Clifton, D., Weinstock, S. B., and Fraenkel, D. G. (1978) Genetics 88, 1-11). We show here that although in the wild type strain the amounts of these enzymes do not greatly differ between cells from different media, in the gcr mutant strain most of the enzyme amounts are 5% or less, relative to wild type, from cells grown without sugars, but 20-50% from cells grown with sugars. Lower relative values were found for phosphoglycerate mutase and enolase. A corresponding alteration in the mutant in the intensities of several major protein bands could even be seen in stained gels after electrophoresis of crude extracts: the profiles were otherwise normal. Results of titration of phosphoglycerate kinase with antibody accorded with activity. Transfer of cells between the two types of media did not lead to a more rapid adjustment of enzyme amounts than expected from the steady state levels. gcr is not allelic to GPM (the gene for phosphoglycerate mutase) or to RNA1 (which affects transport of RNA from the nucleus). Translation of total RNA in a rabbit reticulocyte lysate gave a pattern of polypeptides similar to the in vivo one. Thus, gcr is likely to affect somehow mRNA synthesis or lifetime for a discrete number of proteins.

Frequency and diversity of RNA sequences in different cell types of the fungus Schizophyllum commune.

In the basidiomycete Schizophyllum commune an RNA complexity of 13,500 average-sized (1100 nucleotides) RNA sequences was found by means of RNA excess hybridization to complementary DNA. 600 abundant poly(A)-containing RNA sequences were present in about 100 copies per cell, whereas the rare 13,000 poly(A)-containing RNA sequences were present in about 5 copies per cell on average. Homologous and heterologous cDNA hybridizations to poly(A)-rich RNAs revealed some quantitative, but no significant qualitative differences between the monokaryotic and the dikaryotic cell type. Heterologous hybridization and subsequent hybridizations of remaining single-stranded cDNA to poly(A)-rich RNAs from both cell types showed qualitative differences amounting to 2-3% of the rare sequences in the two cell types. However, from the results of similar hybridizations with total RNAs, i.e. including non-polyadenylated RNA, it could be calculated that the RNA sequences in monokaryons and dikaryons are at least 99% the same. Identity of RNA sequences in monokaryons and dikaryons was also inferred by comparison of the patterns of polypeptides synthesized by translation of total RNA in cell-free systems.

Immune and injury responses in Cecropia pupae—RNA isolation and comparison of protein synthesis in vivo and in vitro

If pupae of Hyalophora cecropia are injected with live bacteria they respond by producing about ten immune proteins ( Boman, 1980). At least four of these proteins are bactericidal. A sham injection with a sterile salt solution gave rise to a related injury response which may be nearly similar in total protein synthesis but with only a low bactericidal activity. This difference in response was correlated to the amounts of some small basic antibacterial proteins. Inhibition of transcription by actinomycin D at different times showed that 5 hr were required for the formation of the RNA needed for the synthesis of the antibacterial proteins. A method was developed for the isolation of total RNA from immunized and injured pupae. The key step in this RNA purification was lanthanum precipitation followed by extractions with ethanol-EDTA. The RNA fraction obtained after affinity chromatography on poly-U-Sepharose was a complex mixture of high mol. wt species with no differences between the RNA obtained from immunized and injured pupae. Using a cell-free system for studying protein synthesis, total RNA from both types of pupae produced rather similar dose responses of 13–15 proteins. Translation of total RNA from an untreated pupa gave a partially different protein pattern. The regulatory implications of these results are discussed.

The majority of calf muscle cell messenger RNAs contain poly(A)

Previous studies from our laboratory have investigated messenger RNA metabolism in calf muscle cells in tissue culture. The analysis of mRNA was based on its poly(A) content. We have now examined directly the proportion of mRNA which contains poly(A) in these cells. After separation of poly(A) +- and poly(A) −-RNA on oligo(dT)-cellulose, the two fractions were translated in a reconstituted, heterologous cell-free protein-synthesizing system and the products were compared with those from the translation of total RNA. The great majority of mRNA form either prefusion or postfusion cultures was poly(A)-containing; quantitative determinations show that about 70–90% of the actin mRNA is poly(A)-containing. In order to determine if a large fraction of the calf muscle mRNA can be translated by a heterologous cell-free system, [ 3H]-poly(A) +-RNA was added to reticulocyte lysates and the formation of initiation complexes was followed. These experiments suggest that the bulk of calf muscle cell mRNA would be utilized in such a system and justify the use of cell-free systems to examine the poly(A) content of total mRNA. Thus, differential polyadenylation does not seem to be an important aspect of mRNA metabolism in cultured muscle cells. The previous study of mRNA in these cells, based on poly(A) content, is apparently a valid measure of overall mRNA metabolism.