Translation affects YoeB and MazF messenger RNA interferase activities by different mechanisms

Prokaryotic toxin–antitoxin loci encode mRNA cleaving enzymes that inhibit translation. Two types are known: those that cleave mRNA codons at the ribosomal A site and those that cleave any RNA site specifically. RelE of Escherichia coli cleaves mRNA at the ribosomal A site in vivo and in vitro but does not cleave pure RNA in vitro. RelE exhibits an incomplete RNase fold that may explain why RelE requires its substrate mRNA to presented by the ribosome. In contrast, RelE homologue YoeB has a complete RNase fold and cleaves RNA independently of ribosomes in vitro. Here, we show that YoeB cleavage of mRNA is strictly dependent on translation of the mRNA in vivo. Non-translated model mRNAs were not cleaved whereas the corresponding wild-type mRNAs were cleaved efficiently. Model mRNAs carrying frameshift mutations exhibited a YoeB-mediated cleavage pattern consistent with the reading frameshift thus giving strong evidence that YoeB cleavage specificity was determined by the translational reading frame. In contrast, site-specific mRNA cleavage by MazF occurred independently of translation. In one case, translation seriously influenced MazF cleavage efficiency, thus solving a previous apparent paradox. We propose that translation enhances MazF-mediated cleavage of mRNA by destabilization of the mRNA secondary structure.     

In vivo effect of inactivation of ribosome recycling factor - fate of ribosomes after unscheduled translation downstream of open reading frame

The post-termination ribosomal complex is disassembled by ribosome recycling factor (RRF) and elongation factor G. Without RRF, the ribosome is not released from mRNA at the termination codon and reinitiates translation downstream. This is called unscheduled translation. Here, we show that at the non-permissive temperature of a temperature-sensitive RRF strain, RRF is lost quickly, and some ribosomes reach the 3' end of mRNA. However, instead of accumulating at the 3' end of mRNA, ribosomes are released as monosomes. Some ribosomes are transferred to transfer-messenger RNA from the 3' end of mRNA. The monosomes thus produced are able to translate synthetic homopolymer but not natural mRNA with leader and canonical initiation signal. The pellet containing ribosomes appears to be responsible for rapid but reversible inhibition of most but not all of protein synthesis in vivo closely followed by decrease of cellular RNA and DNA synthesis.     

Ribosomes inhibit an RNase E cleavage which induces the decay of the rpsO mRNA of Escherichia coli.

The hypothesis generally proposed to explain the stabilizing effect of translation on many bacterial mRNAs is that ribosomes mask endoribonuclease sites which control the mRNA decay rate. We present the first demonstration that ribosomes interfere with a particular RNase E processing event responsible for mRNA decay. These experiments used an rpsO mRNA deleted of the translational operator where ribosomal protein S15 autoregulates its synthesis. We demonstrate that ribosomes inhibit the RNase E cleavage, 10 nucleotides downstream of the rpsO coding sequence, responsible for triggering the exonucleolytic decay of the message mediated by polynucleotide phosphorylase. Early termination codons and insertions which increase the length of ribosome-free mRNA between the UAA termination codon and this RNase E site destabilize the translated mRNA and facilitate RNase E cleavage, suggesting that ribosomes sterically inhibit RNase E access to the processing site. Accordingly, a mutation which reduces the distance between these two sites stabilizes the mRNA. Moreover, an experiment showing that a 10 nucleotide insertion which destabilizes the untranslated mRNA does not affect mRNA stability when it is inserted in the coding sequence of a translated mRNA demonstrates that ribosomes can mask an RNA feature, 10-20 nucleotides upstream of the processing site, which contributes to the RNase E cleavage efficiency.     

Neuronal RNA granules: a link between RNA localization and stimulation-dependent translation.

RNA granules are a macromolecular structure observed in neurons, where they serve as motile units that translocate mRNAs. Isolated RNA granules are highly enriched in Staufen protein and ultrastructurally contain densely packed clusters of ribosomes. With depolarization, many mRNAs, including those involved in plasticity, rapidly shift from the RNA granule fraction to polysomes. Depolarization reorganizes granules and induces a less compact organization of their ribosomes. RNA granules are not translationally competent, as indicated by the failure to incorporate radioactive amino acids and the absence of eIF4E, 4G, and tRNAs. We concluded that RNA granules are a local storage compartment for mRNAs under translational arrest but are poised for release to actively translated pools. Local release of mRNAs and ribosomes from granules may serve as a macromolecular mechanism linking RNA localization to translation and synaptic plasticity.     

Autogenous regulation of RNA translation and packaging by Rous sarcoma virus Pr76gag.

Unspliced cytoplasmic retroviral RNA in chronically infected cells either is encapsidated by Gag proteins in the manufacture of virus or is used to direct synthesis of Gag proteins. Several models have been suggested to explain the sorting of viral RNA for these two purposes. Here we present evidence supporting a simple biochemical mechanism that accounts for the routing of retroviral RNA. Our results indicate that ribosomes compete with the Gag proteins to determine the fate of nascent retroviral RNA. Although the integrity of the entire Rous sarcoma virus leader sequence is important for retroviral packaging and translation, the RNA structure around the third small open reading frame, which neighbors the psi site required for packaging of the RNA, is particularly critical for maintenance of the balance between translation and packaging. These results support the hypothesis that Gag proteins autogenously regulate their synthesis and encapsidation of retroviral RNA and that an equilibrium exists between RNA destined for translation and packaging that is based on the intracellular levels of Gag proteins and ribosomes. To test the model, mRNAs with natural or mutated 5' leader sequences from Rous sarcoma virus were expressed in avian cells in the presence and absence of Pr76gag. We demonstrate that Pr76gag acts as a translational repressor of these mRNAs in a dose-dependent manner, supporting the hypothesis that Pr76gag can sort retroviral RNA for translation and encapsidation.     

On the accessibility and selection of the initiator site of mRNA in protein synthesis.

The specificity of the cell-free system of Escherichia coli for mRNA was examined, and the "accessibility" of some natural and synthetic RNAs to the ribosomes was determined by measurement of AcPhe-tRNA and fMet-tRNA binding, AcPhe-puromycin and fMet-puromycin formation, and polypeptide synthesis. The E. coli system effectively initiates the translation of various synthetic RNAs with AcPhe-tRNA or fMet-tRNA under conditions optimal for the translation of viral RNA. Poly(A,G,U) is accessible to the ribosomes according to all of the above criteria. Poly(A,C,G,U), 23 S rRNA, R17 RNA, and MS2 RNA, on the other hand, show limited accessibility when tested for initiator tRNA binding, or for AcPhe-puromycin and fMet-puromycin formation. MS2 and R17 RNA, but not poly(A,C,G,U) and 23 S rRNA, show accessibility when measured by polypeptide synthesis. The results suggest that, except at initiator sites of natural mRNA, an RNA containing about equal amounts of all four bases is inaccessible to E. coli ribosomes for polypeptide synthesis. Rate constants obtained for fMet-tRNA binding with MS2 RNA, poly(A,G,U), and poly(C,G,U) indicate that the ribosomes do not have any special affinity for the viral RNA. Thus, the selection of the initiator site in protein synthesis may be critically determined more by the accessibility of the initiator codon than by ribosomal recognition of the site.     

Characterization of the translational control mechanism preventing synthesis of alpha 2(I) collagen in chicken vertebral chondroblasts

Chondrogenesis, the differentiation of mesenchyme into cartilage, involves a transition from synthesis of type I to type II collagen. Chicken vertebral chondroblasts contain both type II and alpha 2 type I collagen RNAs but synthesize only type II collagen, suggesting the existence of translational discrimination between these RNAs. The experiments outlined in this report examine the translational control mechanism preventing the synthesis of alpha 2(I) collagen in chondroblasts. Specifically, the alpha 2(I) collagen RNA in the cytoplasm of mature chondroblasts does not appear to be sequestered in ribonucleoprotein particles that could prevent its translation in these cells. Instead, the RNA associates with an average of only three ribosomes; each of these ribosomes appears to be capable of forming at least one peptide bond. However, treatment of chondroblasts with low concentrations of cycloheximide, an elongation inhibitor, suggests movement of the ribosomes on the alpha 2(I) collagen RNA may be partially blocked, resulting in a severe reduction in the translation elongation rate. This translational mechanism may constitute an important regulatory function mediating the cessation of type I collagen synthesis during chondrogenesis.     

Prolyl-tRNA(Pro) in the A-site of SecM-arrested ribosomes inhibits the recruitment of transfer-messenger RNA

Translational pausing can lead to cleavage of the A-site codon and facilitate recruitment of the transfer-messenger RNA (tmRNA) (SsrA) quality control system to distressed ribosomes. We asked whether aminoacyl-tRNA binding site (A-site) mRNA cleavage occurs during regulatory translational pausing using the Escherichia coli SecM-mediated ribosome arrest as a model. We find that SecM ribosome arrest does not elicit efficient A-site cleavage, but instead allows degradation of downstream mRNA to the 3'-edge of the arrested ribosome. Characterization of SecM-arrested ribosomes shows the nascent peptide is covalently linked via glycine 165 to tRNA(3Gly) in the peptidyl-tRNA binding site, and prolyl-tRNA(2Pro) is bound to the A-site. Although A-site-cleaved mRNAs were not detected, tmRNA-mediated ssrA tagging after SecM glycine 165 was observed. This tmRNA activity results from sequestration of prolyl-tRNA(2Pro) on overexpressed SecM-arrested ribosomes, which produces a second population of stalled ribosomes with unoccupied A-sites. Indeed, compensatory overexpression of tRNA(2Pro) readily inhibits ssrA tagging after glycine 165, but has no effect on the duration of SecM ribosome arrest. We conclude that, under physiological conditions, the architecture of SecM-arrested ribosomes allows regulated translational pausing without interference from A-site cleavage or tmRNA activities. Moreover, it seems likely that A-site mRNA cleavage is generally avoided or inhibited during regulated ribosome pauses.     

Frameshifting at the internal stop codon within the mRNA for bacterial release factor-2 on eukaryotic ribosomes

A translational frameshift is necessary in the synthesis of Escherichia coli release factor 2 (RF-2) to bypass an in-frame termination codon within the coding sequence. High-efficiency frameshifting around this codon can occur on eukaryotic ribosomes as well as prokaryotic ribosomes. This was determined from the relative efficiency of translation of RF-2 RNA compared with that for the other release factor RF-1, which lacks the in-frame premature stop codon. Since the termination product is unstable an absolute measure of the efficiency of frameshifting has not been possible. A gene fusion between trpE and RF-2 was carried out to give a stable termination product as well as the frameshift product, thereby allowing a direct determination of frameshifting efficiency. The extension of RF-2 RNA near its start codon with a fragment of the trpE gene, while still allowing high efficiency frameshifting on prokaryotic ribosomes, surprisingly gives a different estimate of frameshifting on the eukaryotic ribosomes than that obtained with RF-2 RNA alone. This paradox may be explained by long distance context effects on translation rates in the frameshift region created by the trpE sequences in the gene fusion, and may reflect that pausing and translation rate are fundamental factors in determining the efficiency of frameshifting.     

Binding of wheat germ ribosomes to fragmented viral mRNA.

The specificity of binding of wheat germ ribosomes to mRNA was greatly altered by cleavage of the message. Fragmentation of reovirus mRNA allowed wheat germ ribosomes to bind and protect a variety of internal sequences which were not accessible to ribosomes in the intact message. In experiments using the polycistronic mRNA from bacteriophage R17, wheat germ ribosomes bound preferentially at the beginning of the lysis peptide and synthetase cistrons, and at a third site which may be derived from the C-terminal region of the A protein cistron. This result is similar to that reported previously in a mammalian translational system (J.F. Atkins et al., Cell 18:246-256, 1979) except that, in the present study, limited cleavage of the phage RNA was necessary to activate these sites. More extensive fragmentation of R17 RNA permitted wheat germ ribosomes to bind and protect a great many additional sites. Thus, presence of an (exposed) 5'-terminus on an RNA molecule appears to be necessary and sufficient for attachment of eucaryotic ribosomes.     

The RACK1 signal anchor protein from Trypanosoma brucei associates with eukaryotic elongation factor 1A: a role for translational control in cytokinesis

RACK1 is a WD-repeat protein that forms signal complexes at appropriate locations in the cell. RACK1 homologues are core components of ribosomes from yeast, plants and mammals. In contrast, a cryo-EM analysis of trypanosome ribosomes failed to detect RACK1, thus eliminating an important translational regulatory mechanism. Here we report that TbRACK1 from Trypanosoma brucei associates with eukaryotic translation elongation factor-1a (eEF1A) as determined by tandem MS of TAP-TbRACK1 affinity eluates, co-sedimentation in a sucrose gradient, and co-precipitation assays. Consistent with these observations, sucrose gradient purified 80S monosomes and translating polysomes each contained TbRACK1. When RNAi was used to deplete cells of TbRACK1, a shift in the polysome profile was observed, while the phosphorylation of a ribosomal protein increased. Under these conditions, cell growth became hypersensitive to the translational inhibitor anisomycin. The kinetoplasts and nuclei were misaligned in the postmitotic cells, resulting in partial cleavage furrow ingression during cytokinesis. Overall, these findings identify eEF1A as a novel TbRACK1 binding partner and establish TbRACK1 as a component of the trypanosome translational apparatus. The synergy between anisomycin and TbRACK1 RNAi suggests that continued translation is required for complete ingression of the cleavage furrow.     

Protein synthesis in Bacillus subtilis. II. Selective translation of natural mRNAs and its possible relation to the species-specific inhibition of protein synthesis by lincomycin and erythromycin.

Vacant ribosomal couples from Bacillus subtilis W168 incorporate only very small amounts of amino acids into polypeptides in response to Escherichia coli cellular RNA or bacteriophage f2 RNA, but are observed to form initiation complexes in the presence of f2 RNA. Vacant ribosomal couples from E. coli acquire pressure-resistance, but do not bind fMet-tRNA, when incubated with B. subtilis RNA in the absence of ribosomal wash fraction. The implied mRNA binding in the absence of salt wash fraction, taken with previously reported observations of salt wash-independent translation of mRNAs from Grampositive bacteria, suggests that mRNAs from Gram-positive bacteria have an active functional character which is masked or absent in mRNAs from Gram-negative sources. It is proposed that this property of B. subtilis mRNAs is required by B. subtilis ribosomes for some translational function subsequent to the formation of the 70 S initiation complex, and that f2 RNA, while it is bound by B. subtilis ribosomes in initiation complexes, is not translated because it lacks this feature.The antibiotic lincomycin has been found to inhibit translation of natural mRNAs in vitro in systems from Gram-positive bacteria at concentrations 10 to 100 times lower than those necessary to inhibit translation in systems from Gram-negative species. Lincomycin does not inhibit formation of initiation complexes by vacant couples from B. subtilis or E. coli. Taken with the published findings of other investigators, these results are interpreted as indicating that the first translocation step following assembly of the initiation complex may coincide with a transition between distinct “initiating” and “elongating” states of the ribosome, and that this transition may involve structural elements, and possibly mechanisms, which are different in Gram-positive systems than in Gram-negative systems.A comprehensive model is constructed to account for the results of these studies and for the published findings of other investigators. It is proposed that some feature of Gram-positive mRNA, perhaps a vestige of early protein synthetic systems, is required by the ribosomes of Gram-positive bacteria to facilitate the transition between initiating and elongating ribosomal states. Inhibition of protein synthesis by lincomycin and the similarly species-specific macrolide antibiotic erythromycin is interpreted as an allosteric effect on the transition between initiating and elongating ribosomal states, in which the different binding affinities of ribosomes from Gram-positive and Gram-negative bacteria for the drugs are related to the functional differences between the two types of systems at this critical step. The implications of this interpretation of interspecies translational specificity for mechanisms of translational control in the cell and for the nature of the divergence of bacterial protein synthesis systems into Gram-positive and Gram-negative types are discussed.     

Human ribosomal protein L13a is dispensable for canonical ribosome function but indispensable for efficient rRNA methylation

Previously, we demonstrated that treatment of monocytic cells with IFN-gamma causes release of ribosomal protein L13a from the 60S ribosome and subsequent translational silencing of Ceruloplasmin (Cp) mRNA. Here, evidence using cultured cells demonstrates that Cp mRNA silencing is dependent on L13a and that L13a-deficient ribosomes are competent for global translational activity. Human monocytic U937 cells were stably transfected with two different shRNA sequences for L13a and clonally selected for more than 98% abrogation of total L13a expression. Metabolic labeling of these cells showed rescue of Cp translation from the IFN-gamma mediated translational silencing activity. Depletion of L13a caused significant reduction of methylation of ribosomal RNA and of cap-independent translation mediated by Internal Ribosome Entry Site (IRES) elements derived from p27, p53, and SNAT2 mRNAs. However, no significant differences in the ribosomal RNA processing, polysome formation, global translational activity, translational fidelity, and cell proliferation were observed between L13a-deficient and wild-type control cells. These results support the notion that ribosome can serve as a depot for releasable translation-regulatory factors unrelated to its basal polypeptide synthetic function. Unlike mammalian cells, the L13a homolog in yeast is indispensable for growth. Thus, L13a may have evolved from an essential ribosomal protein in lower eukaryotes to having a role as a dispensable extra-ribosomal function in higher eukaryotes.     

Nucleotide sequence of the small double-stranded RNA segment of bacteriophage phi 6: novel mechanism of natural translational control.

The lipid-containing bacteriophage phi 6 has a genome composed of three segments of double-stranded RNA. We determined the nucleotide sequence of a cDNA copy of the smallest RNA segment. The coding sequences of the four proteins on this segment were identified. These sequences were clustered. Three of the genes had overlapping initiation-termination codons. All noncoding sequences were at the ends of the molecule. The genes of the small double-stranded RNA segment comprised two translational polarity groups. We propose that the translational coupling is the result of an inability of ribosomes to bind independently to two of the four genes. Translation of these genes occurred when ribosomes were delivered to them by translation of an upstream gene.     

Kinetics of ribosomal pausing during programmed -1 translational frameshifting

In the Saccharomyces cerevisiae double-stranded RNA virus, programmed -1 ribosomal frameshifting is responsible for translation of the second open reading frame of the essential viral RNA. A typical slippery site and downstream pseudoknot are necessary for this frameshifting event, and previous work has demonstrated that ribosomes pause over the slippery site. The translational intermediate associated with a ribosome paused at this position is detected, and, using in vitro translation and quantitative heelprinting, the rates of synthesis, the ribosomal pause time, the proportion of ribosomes paused at the slippery site, and the fraction of paused ribosomes that frameshift are estimated. About 10% of ribosomes pause at the slippery site in vitro, and some 60% of these continue in the -1 frame. Ribosomes that continue in the -1 frame pause about 10 times longer than it takes to complete a peptide bond in vitro. Altering the rate of translational initiation alters the rate of frameshifting in vivo. Our in vitro and in vivo experiments can best be interpreted to mean that there are three methods by which ribosomes pass the frameshift site, only one of which results in frameshifting.     

Dissecting eukaryotic translation and its control by ribosome density mapping

Translation of an mRNA is generally divided into three stages: initiation, elongation and termination. The relative rates of these steps determine both the number and position of ribosomes along the mRNA, but traditional velocity sedimentation assays for the translational status of mRNA determine only the number of bound ribosomes. We developed a procedure, termed Ribosome Density Mapping (RDM), that uses site-specific cleavage of polysomal mRNA followed by separation on a sucrose gradient and northern analysis, to determine the number of ribosomes associated with specified portions of a particular mRNA. This procedure allows us to test models for translation and its control, and to examine properties of individual steps of translation in vivo. We tested specific predictions from the current model for translational control of GCN4 expression in yeast and found that ribosomes were differentially associated with the uORFs elements and coding region under different growth conditions, consistent with this model. We also mapped ribosome density along the ORF of several mRNAs, to probe basic kinetic properties of translational steps in yeast. We found no detectable decline in ribosome density between the 5′ and 3′ ends of the ORFs, suggesting that the average processivity of elongation is very high. Conversely, there was no queue of ribosomes at the termination site, suggesting that termination is not very slow relative to elongation and initiation. Finally, the RDM results suggest that less frequent initiation of translation on mRNAs with longer ORFs is responsible for the inverse correlation between ORF length and ribosomal density that we observed in a global analysis of translation. These results provide new insights into eukaryotic translation in vivo.     

Identification of a ribosome-binding site for a leader peptide encoded by Rous sarcoma virus RNA.

A new method for identifying ribosome-binding sites was developed to determine whether AUG codons in the 5'-terminal RNA sequence of Rous sarcoma virus were used to initiate protein synthesis. We found that when translation is inhibited, the major ribosome-binding site on Rous sarcoma virus RNA is at the 5'-proximal AUG codon, even though the primary translational product from this RNA, Pr76gag, is encoded behind the fourth AUG codon 331 bases downstream from the observed initiation site. These results suggest that ribosomes can initiate translation on Rous sarcoma virus RNA at more than one site, thereby producing a seven-amino-acid peptide, as well as the gag gene polyprotein precursor of Mr 76,000.     

The Kissing-Loop T-Shaped Structure Translational Enhancer of Pea Enation Mosaic Virus Can Bind Simultaneously to Ribosomes and a 5′ Proximal Hairpin

The Pea Enation Mosaic Virus (PEMV) 3′ translational enhancer, known as the kissing-loop T-shaped structure (kl-TSS), binds to 40S subunits, 60S subunits, and 80S ribosomes, whereas the Turnip crinkle virus (TCV) TSS binds only to 60S subunits and 80S ribosomes. Using electrophoretic mobility gel shift assay (EMSA)-based competition assays, the kl-TSS was found to occupy a different site in the ribosome than the P-site-binding TCV TSS, suggesting that these two TSS employ different mechanisms for enhancing translation. The kl-TSS also engages in a stable, long-distance RNA-RNA kissing-loop interaction with a 12-bp 5′-coding-region hairpin that does not alter the structure of the kl-TSS as revealed by molecular dynamics simulations. Addition of the kl-TSS in trans to a luciferase reporter construct containing either wild-type or mutant 5′ and 3′ PEMV sequences suppressed translation, suggesting that the kl-TSS is required in cis to function, and both ribosome-binding and RNA interaction activities of the kl-TSS contributed to translational inhibition. Addition of the kl-TSS was more detrimental for translation than an adjacent eIF4E-binding 3′ translational enhancer known as the PTE, suggesting that the PTE may support the ribosome-binding function of the kl-TSS. Results of in-line RNA structure probing, ribosome filter binding, and high-throughput selective 2′-hydroxyl acylation analyzed by primer extension (hSHAPE) of rRNAs within bound ribosomes suggest that kl-TSS binding to ribosomes and binding to the 5′ hairpin are compatible activities. These results suggest a model whereby posttermination ribosomes/ribosomal subunits bind to the kl-TSS and are delivered to the 5′ end of the genome via the associated RNA-RNA interaction, which enhances the rate of translation reinitiation.