Bridging IRES elements in mRNAs to the eukaryotic translation apparatus

IRES elements are highly structured RNA sequences that function to recruit ribosomes for the initiation of translation. In contrast to the canonical cap-binding, ribosome-scanning model, the mechanism of IRES-mediated translation initiation is not well understood. IRES elements, first discovered in viral RNA genomes, were subsequently found in a subset of cellular RNAs as well. Interestingly, these cellular IRES-containing mRNAs appear to play important roles during conditions of cellular stress, development, and disease (e.g., cancer). It has been shown for viral IRESes that some require specific IRES trans-acting factors (ITAFs), while others require few if any additional proteins and can bind ribosomes directly. Current studies are aimed at elucidating the mechanism of IRES-mediated translation initiation and features that may be common or differ greatly among cellular and viral IRESes. This review will explore IRES elements as important RNA structures that function in both cellular and viral RNA translation and the significance of these structures in providing an alternative mechanism of eukaryotic translation initiation.     

Attachment of ribosomes to endoplasmic membranes in mouse pancreas. Degranulation in vivo caused by the inducers of autophagocytosis neutral red, vinblastine, puromycin, and cadmium ions, and prevention by cycloheximide.

The present investigation was undertaken to discover whether in vivo inducers of autophagocytosis such as neutral red (0.40 mg/g body weight), cadmium chloride (0.15 mg/animal), vinblastine sulfate (7.5 mg/kg b.w.), and puromycin dihydrochloride (0.20 mg/g b.w.) are able to produce degranulation of rough surfaced endoplasmic reticulum (ER) of pancreatic acinar cells as we suggested earlier. Using a modified method of Blobel and Potter about 30% of the total ribosomes of untreated control pancreas were recovered in the free form, and 70% in the membrane-bound form. Cycloheximide (0.20 mg/g body weight) had no effect on this distribution of ribosomes, while neutral red, cadmium ions, vinblastine, and puromycin led to the presence of more free ribosomes: thus up to 70% of the total cytoplasmic ribosomes were recovered in the free form 30 to 60 min after treatments with the autophagic inducers. Pretreatments with cycloheximide prevented this shift and the distribution of ribosomes remained normal. On the basis of these and previous results we conclude that in pancreas an initial degranulation of the ER caused by these agents is a precondition of membrane transformation in autophagocytosis which is preventable by cycloheximide. Confirming the ultrastructural data of Longnecker et al. on rat pancreas, puromycin was shown to cause different forms of cellular injury including autophagocytosis. All forms of cellular injury were also prevented by pretreatment with cycloheximide in mouse pancreas.     

Dynamics of cellular growth.

The assumption that prompted the studies reported in this paper was that the unsatisfactory state of our knowledge on the regulation of cellular growth might derive from the reductionistic approach used to investigate it. Thus an analysis of cellular growth which applied concepts derived from systems dynamics was undertaken. First of all a dynamic model of cellular growth has been constructed. It has the following features: the levels of DNA, ribosomes and proteins are the defining levels; cellular growth is expressed by a close loop in which the level of ribosomes per genome and, indirectly, the level of proteins per genome are stabilized around goal values by the action of negative feed backs. The validity of the model has been tested by its ability to predict the growth kinetics of a real system (exponentially growing Neurospora cells). The simulated growth has been found to reproduce with great accuracy that of Neurospora cells. A slightly modified model, which takes into consideration also the degradation of ribosomes and of proteins, is shown to predict with accuracy the dynamics of growth of both growing and resting fibroblasts. These latter results suggest that the rates of macromolecular turnovers play a central role in the control of proliferation of mammalian cells: the condition of zero growth seems to be achieved when the rate of synthesis and the rate of degradation of proteins are the same. The possibility is discussed that the model indicates a unifying hypothesis of the mode of action of growth controlling conditions (hormones, growth factors, contact inhibition).     

A cytoskeletal structure with associated polyribosomes obtained from HeLa cells

A method is described by which HeLa cells can be fractionated to reveal a skeletal-like structure in the cytoplasm. This cytoskeleton has many of the cell's ultrastructural features, such as 100Å filaments, microfilaments, centrioles, and microspikes, although most of the cellular protein, membranes, and microtubules have been extracted. Associated with the cytoskeleton are most of the polysomal, but not the monomeric, ribosomes. These polysomes are distributed throughout the cytoskeleton except in the region of the 100Å filaments, which resembles the distribution in intact cells. Degradation of mRNA with low levels of ribonuclease releases most ribosomes from the cytoskeleton. Prior disaggregation of polyribosomes in vivo releases ribosomes but not mRNA. Cytochalasin B administered in vivo releases the mRNA from the cytoskeleton. These results suggest an attachment of polyribosomes to the cytoskeleton via mRNA.     

Ribosome degradation in growing bacteria

Ribosomes are large ribozymes that synthesize all cellular proteins. As protein synthesis is rate-limiting for bacterial growth and ribosomes can comprise a large portion of the cellular mass, elucidation of ribosomal turnover is important to the understanding of cellular physiology. Although ribosomes are widely believed to be stable in growing cells, this has never been rigorously tested, owing to the lack of a suitable experimental system in commonly used bacterial model organisms. Here, we develop an experimental system to directly measure ribosomal stability in Escherichia coli. We show that (i) ribosomes are stable when cells are grown at a constant rate in the exponential phase; (ii) more than half of the ribosomes made during exponential growth are degraded during slowing of culture growth preceding the entry into stationary phase; and (iii) ribosomes are stable for many hours in the stationary phase. Ribosome degradation occurs in growing cultures that contain almost no dead cells and coincides with a reduction of comparable magnitude in the cellular RNA concentration.     

Translation of poliovirus mRNA

Ribosome binding to cellular eukaryotic mRNAs is proposed to occur by initial attachment at or near the mRNA 5' cap structure (m7 GppN, where N is any nucleotide) followed by scanning till the appropriate initiator AUG is encountered. A pivotal aspect of this model is the obligatory entry of the ribosomes at the 5' end of the mRNA (regardless if it contains a cap structure). Recent experiments, however, demonstrated that ribosomes can access certain mRNAs by internal binding to the 5'-untranslated region. This was most clearly demonstrated for members of the picornavirus family such as poliovirus and encephalomyocarditis virus. Further experiments suggest that other viral mRNAs and even cellular mRNAs may use similar mechanisms of ribosome binding. Here we describe some features of the poliovirus 5'-untranslated region and possible trans-acting factors that are involved in this mechanism of translation.     

Fish liver protein synthesis during cold acclimatization: seasonal changes of the ultrastructure of the carp hepatocyte

A cell-free system, active in protein synthesis, was constructed using ribosomes and supernatant factors isolated from the livers of winter-acclimatized carps. Upon seasonal adaptation, the hepatocytes of these fishes exhibited distinctive morphological features that were not sex dependent. The cytoarchitecture of the liver cell from summer carps is consistent with the cellular arrangements found when the rate of gene expression is high. During winter, several morphological features reveal that macromolecular synthetic activity may be clearly diminished.     

Electron microscopic examination of ribosome preparations from germinated spores of Aspergillus fumigatus

Ribosomes were isolated from germinated spores (germlings) of Aspergillus fumigatus and electron microscopy was used to determine qualitatively the extent of cellular contamination. After differential centrifugation, the initial crude preparation contained 80S ribosomes and numerous membrane contaminants in the form of membrane sheets, palisade aggregates, and vesicles 0.10–0.18 m in size. Gel filtration chromatography of crude ribosomes in Sepharose CL-4B did not remove all of the membrane contamination. Homogenous ribosome suspensions, morphologically free of other cellular component contamination, were then obtained after gel filtration fractions were centrifuged through a 35% sucrose solution. The total ribosome yield from a germling preparation was approximately four times the yield of ribosomes from a comparable spore preparation. Gel diffusion precipitin patterns of germling ribosomes were identical to those of spore ribosomes with both germling and spore ribosomes antisera.     

Protein synthesis factors (RF1, RF2, RF3, RRF, and tmRNA) and peptidyl-tRNA hydrolase rescue stalled ribosomes at sense codons

During translation, ribosomes stall on mRNA when the aminoacyl-tRNA to be read is not readily available. The stalled ribosomes are deleterious to the cell and should be rescued to maintain its viability. To investigate the contribution of some of the cellular translation factors on ribosome rescuing, we provoked stalling at AGA codons in mutants that affected the factors and then analyzed the accumulation of oligopeptidyl (peptides of up to 6 amino acid residues, oligopep-)-tRNA or polypeptidyl (peptides of more than 300 amino acids in length, polypep-)-tRNA associated with ribosomes. Stalling was achieved by starvation for aminoacyl-tRNA(Arg4) upon induced expression of engineered lacZ (β-galactosidase) reporter gene harboring contiguous AGA codons close to the initiation codon or at internal codon positions together with minigene ATGAGATAA accompanied by reduced peptidyl-tRNA hydrolase (Pth). Our results showed accumulations of peptidyl-tRNA associated with ribosomes in mutants for release factors (RF1, RF2, and RF3), ribosome recycling factor (RRF), Pth, and transfer-messenger RNA (tmRNA), implying that each of these factors cooperate in rescuing stalled ribosomes. The role of these factors in ribosome releasing from the stalled complex may vary depending on the length of the peptide in the peptidyl-tRNA. RF3 and RRF rescue stalled ribosomes by "drop-off" of peptidyl-tRNA, while RF1, RF2 (in the absence of termination codon), or Pth may rescue by hydrolyzing the associated peptidyl-tRNA. This is followed by the disassembly of the ribosomal complex of tRNA and mRNA by RRF and elongation factor G.     

Stm1p, a ribosome-associated protein, is important for protein synthesis in Saccharomyces cerevisiae under nutritional stress conditions

Stm1p is a Saccharomyces cerevisiae protein that has been implicated in several biological processes, ranging from apoptosis to telomere biosynthesis. Likewise, Stm1p has been identified as a protein associated with supramolecular structures, including ribosomes and nuclear telomere cap complexes. Using a variety of biochemical methods, we found that the vast majority of cellular Stm1p is associated with free cytosolic 80S ribosomes and polysomes. In its association with ribosomes, Stm1p interacts in an equimolar complex with both ribosomal subunits and is not associated with mRNA. Functionally, targeted disruption of the STM1 gene results in rapamycin hypersensitivity and a defect in recovery following nitrogen starvation and replenishment. These effects coincide with severe polysome depletion and reduced total protein synthesis. Taken together, our data indicate that Stm1p plays a critical role in facilitating translation under nutrient stress conditions and suggest that Stm1p acts in concert with the target of rapamycin (TOR) signaling pathway.     

Translational regulation of the expression of ribosomal protein genes in Xenopus laevis

The mRNAs coding for ribosomal proteins (rp-mRNA) are subjected to translational control during Xenopus oogenesis and embryogenesis, and also during nutritional changes in Xenopus cultured cells. This regulation, which appears to respond to the cellular need for new ribosomes, operates by changing the fraction of rp-mRNA engaged on polysomes, each translated rp-mRNA molecule always remaining fully loaded with ribosomes. All rp-mRNAs analyzed up to now show this translational behavior, and also share some structural features in their untranslated portions. In particular they all have rather short 5' untranslated regions, similar to each other, and always start at the very 5' end with a stretch of several pyrimidines. Fusion to a reporter-coding sequence of the 5' untranslated region of r-protein S19 has shown that this is involved in the translational regulation.     

Structural and Functional Topography of Human Ribosomes Deduced from Crosslinking with mRNA Analogs,Oligoribonucleotide Derivatives

Reviewed are data on the position of template codons with respect to 18S rRNA and certain proteins on human ribosome obtained using a set of mRNA analogs, oligoribonucleotide derivatives carrying alkylating or photoactivatable groups at different positions. A comparison of data on the template position on the human and Escherichia coliribosomes has revealed both the similarity in the structure of the mRNA-binding site of bacterial and mammalian ribosomes and the peculiarities of the functioning of mammalian (in particular, human) ribosomes. The similarity manifests itself in that the template codons at the A-, P-, and E-sites of bacterial and human ribosomes are surrounded by similar nucleotides (occupying similar positions in the conserved regions of secondary structure) of small subunit rRNA. The template forms a loop whose foot is in proximity to the 530 stem–loop conserved region of rRNA. The specific features of mammalian ribosomes appear to be associated with their lower conformational mobility as compared with bacterial ribosomes, owing to which their interaction with the template involves a lesser number of molecular contacts.     

Cellular content of ribonucleic acid and protein in Saccharomyces cerevisiae as a function of exponential growth rate: calculation of the apparent peptide chain elongation rate.

The average cellular content of ribonucleic acid and protein was determined in cultures of Saccharomyces cerevisiae growing exponentially at different rates in a variety of media. Estimations of the proportion of total cellular ribonucleic acid that is made up of ribosomal ribonucleic acid were used to calculate the average number of ribosomes per cell at the different growth rates. The fraction of ribosomes actively engaged in translation was estimated by sucrose gradient centrifugation of ribosomes and polysomes. These data were used in a calculation of the apparent time taken for the addition of an amino acid to the growing polypeptide chain; this value was found to vary linearly with growth rate over a fivefold range of doubling times.     

Expression of fibronectin,laminin and ribosomes in normal and nocodazole-treated neonatal heart cells in culture: a study by laser scanning confocal microscopy and immunocytochemistry

Polyclonal and monoclonal antibodies were used to examine the effects of the synthetic microtubule disruptive drug nocodazole on the subcellular expression of fibronectin, laminin, and ribosomes in primary cultures of neonatal cardiac ventricular cells. Non-invasive serial optical sectioning was carried out by immunolaser scanning confocal microscopy. In addition, fibronectin and laminin were immunolabelled with peroxidase or gold conjugates for electron-microscopic examination. Immunolabelling for the large 60S ribosome subunit in fibroblast-like non-myocytes showed that punctate ribosome structures with a multi-subunit composition were present in perinuclear region. Double immunostaining with antibodies directed against ribosomes and cellular fibronectin indicated that the punctate structures were cisternae of the rough endoplasmic reticulum. No clear effects of nocodazole treatment were detected on the distribution of cytoskeleton-bound ribosomes. Following immunolabelling for both glycoproteins and double immunolabelling for cellular fibronectin and the 60 S ribosome subunit, fibronectin and laminin were found in the perinuclear cisternae of the rough endoplasmic reticulum and in pleomorphic secretory vesicles. The cisternal stacks of the Golgi complex appeared either unstained or were only weakly labelled. When these cells were exposed to nocodazole, fibronectin and laminin accumulated in peripheral parts of the cytoplasm, including cellular processes. These peripheral accumulations of immunostaining for fibronectin and laminin did not reflect Golgi staining, as shown by double labelling experiments versus wheat-germ-agglutinin staining, and, by exposing cultures to a high dose of brefeldin A.     

Changes in ribosome-polyribosome balances in chick muscle cells during tissue dissociation, development in culture, and exposure to simplified culture-medium

The populations of polyribosomes, monomeric ribosomes, and ribosomal subunits are described from the time of tissue explantation to the time of complete muscle differentiation in primary cultures of chick muscle cells. There is extensive degradation of polyribosomes, and a net loss of ribosomes recovered, as cells of embryonic muscle are dissociated with proteolytic enzymes. The cells rapidly restore a high polysome: monomeric ribosome ratio. This recovery of the polyribosome population occurs before there is any detectable net increase in ribosome number. Ribosome production begins after a lag of approximately 15 hours in culture. Number of ribosomes/cell triples by 60 hours, at which time cell fusion (myotube formation) is complete. Unlike developing muscle in vivo, cultured cells have a very reduced pool of monomeric ribosomes. Medium simplification experiments done with fully differentiated cultures show, however, that monomers accumulate during starvation. These monomers reassociate to form polyribosomes during medium replenishment. Subunit complements are maintained at a constant level regardless of nutritional conditions. These features of cultured muscle are discussed as possible tools for further study of muscle development.     

The primitive ribosome model

A new model is proposed on the origin of crystallizable ribosomes and the kinetics of ribosome crystallization. The model assumes that ribosome crystallizability is a property the ribosomes have only in a well-defined period of their life cycle and implies a close relationship between cellular differentiation and structural rearrangements at the ribosome level. The model is used to interpret a variety of cases in which ribosome crystallization occurs, such as chick embryo tissues during development, tissue cultures treated with different antibiotics, chick adult tissues infected by viruses, lizard oocytes, degenerating cells and dedifferentiated cellular systems.