Relationships between translation of pro α1(I) and pro α2(I) mRNAs during synthesis of the type I procollagen heterotrimer

Final assembly of the procollagen I heterotrimeric molecule is initiated by interactions between the carboxyl propeptide domains of completed, or nearly completed nascent pro α chains. These interactions register the chains for triple helix folding. Prior to these events, however, the appropriate nascent chains must be brought within the same compartments of the endoplasmic reticulum (ER). We hypothesize that the co-localization of the synthesis of the nascent pro α1(I) and pro α2(I) chains results from an interaction between their translational complexes during chain synthesis. This has been investigated by studying the polyribosomal loading of the pro α-chain messages during in vitro translation in the presence and absence of microsomal membranes, and in cells which have the ability to synthesize the pro α1 homotrimer or the normal heterotrimer. Recombinant human pro α1(I) and pro α2(I) C DNAs were inserted into plasmids and then transcribed in vitro. The resulting RNAs were translated separately and in mixture in a cell-free rabbit reticulocyte lysate ± canine pancreatic microsomes. Cycloheximide (100 μg/ml) was added and the polysomes were collected and fractionated on a 15–50% sucrose gradient. The RNA was extracted from each fraction and the level of each chain message was determined by RT-PCR. Polysomes from K16 (heterotrimer-producing), W8 (pro α1(I) homotrimer), and A2′ (heterotrimer + homotrimer) cells were similarly analyzed. Translations of the pro α1(I) and pro α2(I) messages proceeded independently in the cell-free, membrane-free systems, but were coordinately altered in the presence of membrane. The cell-free + membrane translation systems mimicked the behavior of the comparable cell polysome mRNA loading distributions. These data all suggest that there is an interaction between the pro α chain translational complexes at the ER membrane surface which temporally and spatially localize the nascent chains for efficient heteromeric selection and folding. © 1995 Wiley-Liss, Inc.     

Liver albumin synthesis increases in free ribosomes during the acute-phase reaction

Albumin concentration in the blood, and its synthesis by the liver, decrease in the rat during the acute-phase response to inflammation. In this paper we show that 24 hours after turpentine treatment free ribosomes from rat liver double their albumin synthesis and release preproalbumin in the cytosol. albumin mRNA from free polysomes, tested in reconstructed systems in vitro, directs the synthesis of preproalbumin which is correctly processed in the presence of microsomal membranes. Albumin mRNA in the free ribosomal fraction decreases in amount, but it is mainly associated with the heavier polysomal fraction. These data favor the hypothesis of a more, efficient utilization of the reduced amount of albumin mRNA, concurrent with failure of translational arrest of the nascent chain and with the release of unprocessed product in the cytosol.     

Transfer of proteins across membranes. I. Presence of proteolytically processed and unprocessed nascent immunoglobulin light chains on membrane-bound ribosomes of murine myeloma

Fractionation of MOPC 41 DL-1 tumors revealed that the mRNA for the light chain of immunoglobulin is localized exclusively in membrane- bound ribosomes. It was shown that the translation product of isolated light chain mRNA in a heterologous protein-synthesizing system in vitro is larger than the authentic secreted light chain; this confirms similar results from several laboratories. The synthesis in vitro of a precursor protein of the light chain is not an artifact of translation in a heterologous system, because it was shown that detached polysomes, isolated from detergent-treated rough microsomes, not only contain nascent light chains which have already been proteolytically processed in vivo but also contain unprocessed nascent light chains. In vitro completion of these nascent light chains thus resulted in the synthesis of some chains having the same mol wt as the authentic secreted light chains, because of completion of in vivo proteolytically processed chains and of other chains which, due to the completion of unprocessed chains, have the same mol wt as the precursor of the light chain. In contrast, completion of the nascent light chains contained in rough microsomes resulted in the synthesis of only processed light chains. Taken together, these results indicate that the processing activity is present in isolated rough microsomes, that it is localized in the membrane moiety of rough microsomes, and, therefore, that it was most likely solubilized during detergent treatment used for the isolation of detached polysomes. Furthermore, these results established that processing in vivo takes place before completion of the nascent chain. The data also indicate that in vitro processing of nascent chains by rough microsomes is dependent on ribosome binding to the membrane. If the latter process is interfered with by aurintricarboxylic acid, rough microsomes also synthesize some unprocessed chains. The data presented in this paper have been interpreted in the light of a recently proposed hypothesis. This hypothesis, referred to as the signal hypothesis, is described in greater detail in the Discussion section.     

Localization-dependent oskar protein accumulation; control after the initiation of translation

The appearance of Oskar protein occurs coincident with localization of oskar mRNA to the posterior pole of the Drosophila oocyte, and earlier accumulation of the protein is prevented by translational repression. We find that the nascent polypeptide-associated complex (NAC) is required for correct localization of oskar mRNA. The timing of the defects suggests that, if NAC acts directly via an interaction with nascent Oskar protein, oskar mRNA should be undergoing translation prior to its localization. Polysome analysis confirms that oskar mRNA is associated with polysomes even in the absence of localization of the mRNA or accumulation of Oskar protein. Thus, the mechanisms that prevent accumulation of Oskar protein until it can be secured at the posterior pole of the oocyte include regulated degradation or inhibition of translational elongation.     

Possible interaction sites of mRNA, tRNA, translation factors and the nascent peptide in 5S, 5.8S and 28S rRNA in in vivo assembled eukaryotic ribosomal complexes.

We have investigated possible interaction sites for mRNA, tRNA, translation factors and the nascent peptide on 5S, 5.8S and 28S rRNA in in vivo assembled translational active mouse ribosomes by comparing the chemical footprinting patterns derived from native polysomes, salt-washed polysomes (mainly lacking translational factors) and salt-washed runoff ribosomes (lacking mRNA, tRNA and translational factors). Several ligand-induced footprints were observed in 28S rRNA while no reactivity changes were seen in 5S and 5.8S rRNA. Footprints derived from mRNA, tRNA and/or the nascent peptide chain were observed in domain I of 28S rRNA (hairpin 23), in domain II (helix 37/38 and helices 42 and 43 and in the eukaryotic expansion segment 15), in domain IV (helices 67 and 74) and in domain V (helices 94 and 96 and in the peptidyl transferase ring). Some of the protected sites were homologous to sites previously suggested to be involved in mRNA, tRNA and/or peptide binding in in vitro assembled prokaryotic complexes. Additional footprints were located in regions that have not previously been found involved in ligand binding. Part of these sites could derive from the nascent peptide in the exit channel of the ribosome.     

The translational requirement for complete La Crosse virus mRNA synthesis is cell-type dependent.

The translational requirement to prevent premature termination during La Crosse virus S mRNA synthesis was found to be cell-type dependent. This requirement was present in the BHK, HEL, and Vero cell lines we examined, but not in C6/36 mosquito cells. The cell-dependent translational requirement could be reproduced in vitro by using either cell extracts or purified virions of BHK and C6/36 cells. In the BHK reactions, the polymerase terminated predominantly at nucleotide 175 in the absence of concurrent translation and required translation to read through this position. In the C6/36 reactions, however, the polymerase reads through nucleotide 175 efficiently independent of translation. Reconstitution studies suggested that the translational requirement was due to a factor(s) present in BHK, but not in C6/36, cells.     

Spatial regulation of nanos is required for its function in dendrite morphogenesis

Spatial control of mRNA translation can generate cellular asymmetries and functional specialization of polarized cells like neurons. A requirement for the translational repressor Nanos (Nos) in the Drosophila larval peripheral nervous system (PNS) implicates translational control in dendrite morphogenesis [1]. Nos was first identified by its requirement in the posterior of the early embryo for abdomen formation [2]. Nos synthesis is targeted to the posterior pole of the oocyte and early embryo through translational repression of unlocalized nos mRNA coupled with translational activation of nos mRNA localized at the posterior pole [3, 4]. Abolishment of nos localization prevents abdominal development, whereas translational derepression of unlocalized nos mRNA suppresses head/thorax development, emphasizing the importance of spatial regulation of nos mRNA [3, 5]. Loss and overexpression of Nos affect dendrite branching complexity in class IV dendritic arborization (da) neurons, suggesting that nos also might be regulated in these larval sensory neurons [1]. Here, we show that localization and translational control of nos mRNA are essential for da neuron morphogenesis. RNA-protein interactions that regulate nos translation in the oocyte and early embryo also regulate nos in the PNS. Live imaging of nos mRNA shows that the cis-acting signal responsible for posterior localization in the oocyte/embryo mediates localization to the processes of class IV da neurons but suggests a different transport mechanism. Targeting of nos mRNA to the processes of da neurons may reflect a local requirement for Nos protein in dendritic translational control.     

Nascent polypeptide sequences that influence ribosome function

Ribosomes catalyze protein synthesis using transfer RNAs and auxiliary proteins. Historically, ribosomes have been considered nonspecific translational machines, having no regulatory functions. However, a new class of regulatory mechanisms has been discovered that is based on interactions occurring within the ribosomal peptide exit tunnel that result in ribosome stalling during translation of an appropriate mRNA segment. These discoveries reveal an unexpectedly dynamic role ribosomes play in regulating their own activity. By using nascent leader peptides in combination with bound specific amino acids or antibiotics, ribosome functions can be altered significantly resulting in regulated expression of downstream coding regions. This review summarizes relevant findings in recent articles and outlines our current understanding of nascent peptide-induced ribosome stalling in regulating gene expression.     

Translational and pretranslational control of ornithine aminotransferase synthesis in rat liver

The mitochondrial matrix enzyme, ornithine aminotransferase, is induced in rat liver by the administration of a diet high in protein and by glucagon. The rate of synthesis of the enzyme is increased 100-fold in the livers of rats maintained on a 60% relative to a 0% protein diet, whereas the levels of functional and hybridizable mRNA measured by in vitro translation and through the use of a cloned cDNA probe increased by only 2- to 6-fold and 2- to 3-fold, respectively. Under conditions of glucagon induction that resulted in a 10- to 12-fold increase in the rate of enzyme synthesis, the relative level of functional ornithine aminotransferase mRNA increased by only 2-fold, and the level of hybridizable mRNA actually decreased. The rate of polypeptide chain elongation and the relative number of ornithine aminotransferase nascent chains on polysomes were 2-fold and 23-fold greater, respectively, in hepatocytes derived from 60% relative to 0% protein-fed rats. Using these data, a 23-fold increase in the translational efficiency of the mRNA was calculated. This increase, along with a 2-fold increase in the mRNA level, completely account for the 40-fold increase in the rate of ornithine aminotransferase synthesis observed in hepatocytes derived from 60% protein-fed rats. We conclude that ornithine aminotransferase synthesis is regulated at both a translational and a pretranslational level in rat liver.     

The study of spermidine-stimulated polypeptide synthesis in cell-free translation of mRNA from lactating mouse mammary gland

The stimulatory effect of spermidine on the translation of poly(A)⁺ mRNA from lactating mouse mammary glands in a wheat germ system was studied. Spermidine stimulated total polypeptide synthesis about 2.5-fold relative to that occurring in the presence of an optimal concentration of Mg²⁺ alone. The size and the number of polysomes were about 1.6-times larger in the presence of spermidine than in its absence. A similar magnitude of increase in peptide chain initiation, 1.4-fold, was found when the extent of peptide chain initiation was measured by determining the residual polypeptide synthesis subsequent to the addition of inhibitor(s) of peptide chain initiation to the in vitro translation system with or without spermidine at various times of the incubation. Time-course study of the release of polypeptide from polysomes showed that spermidine stimulated this process to a much greater extent than peptide chain initiation, indicating that the polyamine also increases the rate of peptide chain elongation. The extent of stimulation of peptide chain elongation by spermidine was estimated to be about 1.5-fold when the disappearance of isotope-labeled nascent peptides from polysomes was measured by pulse-chase experiments. These results indicate that spermidine stimulates the cell-free translation of mammary mRNA by increasing the rates of both initiation and elongation of polypeptide synthesis to almost the same extent. The polyamine also reduced the relative amount of incomplete polypeptides, thereby increasing the yield of full-length translational products.     

Evidence of the carboxymethylation of nascent peptide chains on ribosomes

Protein carboxymethylase from bovine anterior pituitary is found to be capable of carboxymethylating proteins in an in vitro protein synthesizing system which includes S-adenosyl-L-methionine-[¹⁴C methyl], wheat germ ribosomes and oviduct mRNA. Optimal carboxymethylation is inhibited by puromycin indicating the requirement for de novo protein synthesis. Ultracentrifugal profiles show that carboxymethylated proteins are associated with ribosomal absorption peaks. This is consistent with the carboxymethylation of proteins occurring on nascent peptide chains.     

Glycosylation causes an apparent block in translation of immunoglobulin heavy chain

Analysis of nascent heavy chains isolated from MPC11 (gamma 2b heavy chains) and MOPC 21 (gamma 1 heavy chains) mouse myeloma cells demonstrates an accumulation of nascent heavy chains which are slightly smaller in mass (approximately 35,000 daltons) than nascent heavy chains which have just been glycosylated (approximately 38,000 daltons). The accumulation of 35,000-dalton nascent heavy chain appears to be a consequence of the glycosylation process since tunicamycin, an inhibitor of glycosylation, abolishes the apparent translational block manifested by the accumulation of 35,000-dalton nascent chains. Tunicamycin also causes a 15 to 25% increase n the relative rate of synthesis of heavy chain compared to the corresponding rate of synthesis of the nonglycosylated light chain synthesized by the same cell. These results suggest that the translation block, caused by the glycosylation process, of heavy chain synthesis contributes to the imbalance of heavy chain and light chain biosynthesis observed in malignant and normal lymphoid cells.     

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the message^{for review see 1}. However, it''s now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates¹. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others². Codon bias is organism as well as tissue specific^2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs⁴. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes^5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein folding^{for review see 9}. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding^1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems^6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.     

Ribosomes specifically bind to mammalian mitochondria via protease-sensitive proteins on the outer membrane

The interaction of ribosomes with specific components of membranes is one of the central themes to the co-translational targeting and import of proteins. To examine ribosome binding to mammalian mitochondria, we used ribosome-nascent chain complexes (RNCs) to follow the in vitro binding of ribosomes that correspond to the initial targeting stage of proteins. Mitochondria were found to contain a limited number of RNC binding sites on the outer membrane. It required more than twice the amount of non-translating ribosomes to inhibit RNC binding by one-half, indicating that RNCs have a competitive binding advantage. In addition, we found that RNCs bind mainly through the ribosomal component and not the nascent chain. RNCs bind via protease-sensitive proteins on the outer membrane, as well as by protease-insensitive components suggesting that two classes of receptors exist. We also show that binding is sensitive to cation conditions. Nearly all of the binding was inhibited in 0.5 m KCl, indicating that they interact with the membrane primarily through electrostatic interactions. In addition, disruption of RNC structure by removing magnesium causes the complete inhibition of binding under normal binding conditions indicating that it is the intact ribosome that is crucial for binding and not the nascent chain. These findings support the hypothesis that the outer mitochondrial membrane contains receptors specific for ribosomes, which would support the conditions necessary for co-translational import.