Cartilage-specific 5' end of chick alpha 2(I) collagen mRNAs

Chondrocytes grown in suspension contain both type I and type II collagen mRNAs, yet synthesize only type II collagen. The inability of chondrocytes to synthesize the alpha 2 subunit of type I collagen, alpha 2(I), results from a severely reduced translation elongation rate (Bennett, V.D., and Adams, S.L. (1987) J. Biol. Chem. 262, 14806-14814). Furthermore, the alpha 2(I) collagen mRNAs from chondrocytes are translated inefficiently in vitro and appear slightly smaller than those from other cells (Focht, R.J., and Adams, S.L. (1984) Mol. Cell. Biol. 4, 1843-1852). These observations suggest that the reduced translation elongation rate may be due to an intrinsic property of the mRNAs. In this report we demonstrate that the alpha 2(I) collagen mRNAs from suspended chondrocytes are 120 bases shorter than those from other cells, and that the first 94 bases of the chondrocyte mRNAs differ from the corresponding region of the calvaria mRNAs. The unique 5' end of the chondrocyte alpha 2(I) collagen mRNAs accounts for their smaller size and may be responsible for the translation elongation defect. Interestingly, the alpha 2(I) collagen mRNAs from chondrocytes grown in monolayer, rather than in suspension, no longer display the cartilage-specific 5' end, suggesting that cell shape and/or adhesion may modulate the structure of the 5' end of the chondrocyte alpha 2(I) collagen mRNAs.     

RNA metabolism in cultures of corneal stromal cells from patients with keratoconus

Total cellular RNA was extracted from cultured keratoconus and normal human corneal stromal cells. The translational activity of these RNAs was examined in a cell-free translation system derived from reticulocyte lysate. Results indicated that keratoconus cells can be separated into two groups, as has been shown previously. Group I keratoconus cells contained the same amount of total RNA as normal cells. RNA activity and the rate of mRNA synthesis in this group of keratoconus cells were also normal. By these criteria it seems that the protein synthesizing system is functioning properly, and group I keratoconus cells should have a normal rate of protein synthesis. These results correlate well with previous findings. Group II keratoconus cells, in contrast, contained more RNA than normal cells. The translational efficiency of RNA was so markedly reduced that the elevation in RNA content did not compensate for the decrease in translational efficiency. It is likely that the reduced protein and collagen synthesis in this group of cells is related to the reduction in the RNA activity. An inhibitory component was present in the keratoconus RNA which affected synthesis of all proteins and suppressed translation of normal RNA.     

Type I collagen messenger RNA levels in experimental granulation tissue and silicosis in rats

A complementary DNA (cDNA) clone was constructed for chick pro alpha 2(I) collagen mRNA. This and previously constructed cDNA clones for chick and human pro alpha 1(I) collagen mRNAs were used to measure levels of type I procollagen messenger RNAs in two experimental models: viscose cellulose sponge-induced experimental granulation tissue and silica-induced experimental lung fibrosis in rats. Both Northern RNA blot and RNA dot hybridizations were used to quantitate procollagen mRNAs during formation of granulation tissue. The period of rapid collagen synthesis was characterized by high levels of procollagen mRNAs, which were reduced when collagen production returned to a low basal level. The rate of collagen synthesis and the levels of procollagen mRNAs during the period of rapid reduction in collagen production did not, however, parallel with each other. This suggests that translational control mechanisms are important during this time in preventing overproduction of collagen. In silicotic lungs, the early stages of fibroblast activation follow a similar path but appear faster. At a later stage, however, the RNA levels increase again and permit collagen synthesis to continue at a high rate, resulting in massive collagen accumulation.     

Scp160p is required for translational efficiency of codon-optimized mRNAs in yeast

The budding yeast multi-K homology domain RNA-binding protein Scp160p binds to >1000 messenger RNAs (mRNAs) and polyribosomes, and its mammalian homolog vigilin binds transfer RNAs (tRNAs) and translation elongation factor EF1alpha. Despite its implication in translation, studies on Scp160p''s molecular function are lacking to date. We applied translational profiling approaches and demonstrate that the association of a specific subset of mRNAs with ribosomes or heavy polysomes depends on Scp160p. Interaction of Scp160p with these mRNAs requires the conserved K homology domains 13 and 14. Transfer RNA pairing index analysis of Scp160p target mRNAs indicates a high degree of consecutive use of iso-decoding codons. As shown for one target mRNA encoding the glycoprotein Pry3p, Scp160p depletion results in translational downregulation but increased association with polysomes, suggesting that it is required for efficient translation elongation. Depletion of Scp160p also decreased the relative abundance of ribosome-associated tRNAs whose codons show low potential for autocorrelation on mRNAs. Conversely, tRNAs with highly autocorrelated codons in mRNAs are less impaired. Our data indicate that Scp160p might increase the efficiency of tRNA recharge, or prevent diffusion of discharged tRNAs, both of which were also proposed to be the likely basis for the translational fitness effect of tRNA pairing.     

Differential utilization of poly(rC) binding protein 2 in translation directed by picornavirus IRES elements

The translation of picornavirus genomic RNAs occurs by a cap-independent mechanism that requires the formation of specific ribonucleoprotein complexes involving host cell factors and highly structured regions of picornavirus 5' noncoding regions known as internal ribosome entry sites (IRES). Although a number of cellular proteins have been shown to be involved in picornavirus RNA translation, the precise role of these factors in picornavirus internal ribosome entry is not understood. In this report, we provide evidence for the existence of distinct mechanisms for the internal initiation of translation between type I and type II picornavirus IRES elements. In vitro translation reactions were conducted in HeLa cell cytoplasmic translation extracts that were depleted of the cellular protein, poly(rC) binding protein 2 (PCBP2). Upon depletion of PCBP2, these extracts possessed a significantly diminished capacity to translate reporter RNAs containing the type I IRES elements of poliovirus, coxsackievirus, or human rhinovirus linked to luciferase; however, the addition of recombinant PCBP2 could reconstitute translation. Furthermore, RNA electrophoretic mobility-shift analysis demonstrated specific interactions between PCBP2 and both type I and type II picornavirus IRES elements; however, the translation of reporter RNAs containing the type II IRES elements of encephalomyocarditis virus and foot-and-mouth disease virus was not PCBP2 dependent. These data demonstrate that PCBP2 is essential for the internal initiation of translation on picornavirus type I IRES elements but is dispensable for translation directed by the structurally distinct type II elements.     

Ribosomal stress activates eEF2K–eEF2 pathway causing translation elongation inhibition and recruitment of Terminal Oligopyrimidine (TOP) mRNAs on polysomes

The synthesis of adequate amounts of ribosomes is an essential task for the cell. It is therefore not surprising that regulatory circuits exist to organize the synthesis of ribosomal components. It has been shown that defect in ribosome biogenesis (ribosomal stress) induces apoptosis or cell cycle arrest through activation of the tumor suppressor p53. This mechanism is thought to be implicated in the pathophysiology of a group of genetic diseases such as Diamond Blackfan Anemia which are called ribosomopathies. We have identified an additional response to ribosomal stress that includes the activation of eukaryotic translation elongation factor 2 kinase with a consequent inhibition of translation elongation. This leads to a translational reprogramming in the cell that involves the structurally defined group of messengers called terminal oligopyrimidine (TOP) mRNAs which encode ribosomal proteins and translation factors. In fact, while general protein synthesis is decreased by the impairment of elongation, TOP mRNAs are recruited on polysomes causing a relative increase in the synthesis of TOP mRNA-encoded proteins compared to other proteins. Therefore, in response to ribosomal stress, there is a change in the translation pattern of the cell which may help restore a sufficient level of ribosomes.     

Hypothesis: Emergence of Translation as a Result of RNA Helicase Evolution

The origin of translation and the genetic code is one of the major mysteries of evolution. The advantage of templated protein synthesis could have been achieved only when the translation apparatus had already become very complex. This means that the translation machinery, as we know it today, must have evolved towards some different essential function that subsequently sub-functionalised into templated protein synthesis. The hypothesis presented here proposes that translation originated as the result of evolution of a primordial RNA helicase, which has been essential for preventing dying out of the RNA organism in sterile double-stranded form. This hypothesis emerges because modern ribosome possesses RNA helicase activity that likely dates back to the RNA world. I hypothesise that codon-anticodon interactions of tRNAs with mRNA evolved as a mechanism used by RNA helicase, the predecessor of ribosomes, to melt RNA duplexes. In this scenario, peptide bond formation emerged to drive unidirectional movement of the helicase via a molecular ratchet mechanism powered by Brownian motion. I propose that protein synthesis appeared as a side product of helicase activity. The first templates for protein synthesis were functional RNAs (ribozymes) that were unwound by the helicase, and the first synthesised proteins were of random or non-sense sequence. I further suggest that genetic code emerged to avoid this randomness. The initial genetic code thus emerged as an assignment of amino acids to codons according to the sequences of the pre-existing RNAs to take advantage of the side products of RNA helicase function.     

Short RNAs repress translation after initiation in mammalian cells

MicroRNAs (miRNAs) are predicted to regulate 30% of mammalian protein-encoding genes by interactions with their 3' untranslated regions (UTRs). We use partially complementary siRNAs to investigate the mechanism by which miRNAs mediate translational repression in human cells. Repressed mRNAs are associated with polyribosomes that are engaged in translation elongation, as shown by puromycin sensitivity. The inhibition appears to be postinitiation because translation driven by the cap-independent processes of HCV IRES and CrPV IRES is repressed by short RNAs. Further, metabolic labeling suggests that silencing occurs before completion of the nascent polypeptide chain. In addition, silencing by short RNAs causes a decrease in translational readthrough at a stop codon, and ribosomes on repressed mRNAs dissociate more rapidly after a block of initiation of translation than those on control mRNAs. These results suggest that repression by short RNAs, and thus probably miRNAs, is primarily due to ribosome drop off during elongation of translation.     

Early and late functions in a bipartite RNA virus: evidence for translational control by competition between viral mRNAs.

It has been shown previously that Drosophila cells infected with black beetle virus synthesize an early viral protein, protein A, a putative element of the viral RNA polymerase. Synthesis of protein A declines sharply by 6 h postinfection, whereas synthesis of viral coat protein alpha continues for at least 14 h. The early shutoff in protein A synthesis occurred despite the presence of equimolar proportions of the mRNAs for proteins A and alpha, RNAs 1 and 2, respectively. We have now been able to mimic this translational discrimination in a cell-free protein-synthesizing system prepared from infected or uninfected Drosophila cells, thus allowing further analysis of the mechanism by which translation of RNA 1 is selectively turned off. The results revealed no evidence for control by virus-encoded proteins or by virus-induced modification of mRNAs by the cell-free system. Rather, with increasing RNA concentration, viral RNA 1 was outcompeted by its genomic partner, RNA 2. This suggests that the early shutoff in intracellular synthesis of protein A is due to decreasing ability of RNA 1 to compete for a rate-controlling translational factor(s) as the concentration of viral RNAs accumulates within the infected cell.     

Formation of a type I collagen RNA dimer by intermolecular base-pairing of a conserved sequence around the translation initiation site.

A symmetrical sequence around the translation initiation site of several collagen genes is highly conserved. Deletions in this sequence increase translational efficiency of an alpha 2(I) collagen - CAT chimeric gene after DNA transfection of NIH 3T3 fibroblasts (Schmidt, Rossi and de Crombrugghe, submitted). The secondary structure, predicted by the sequence of this segment, was shown to exist in solution in 200 mM NaCl at 37 degrees C. Cell-free translation of the corresponding RNA using a reticulocyte lysate is inhibited 2 to 4-fold by preincubation with a 0.5 M NaCl extract of an NIH 3T3 ribosomal eluate. Cell-free translation of two mutant RNAs, with partial deletions of the conserved sequence, is not inhibited by such preincubation. This inhibition is not due to degradation of the RNA and requires a protein component of the ribosomal eluate, which, however, is not required after the preincubation step. Preincubation of the RNA with the ribosomal eluate from NIH 3T3 fibroblasts causes the reversible formation of an intermolecular dimer in which the conserved symmetrical sequences hybridize to each other. This results in an increase in the degree of secondary structure of the conserved segment around the translation initiation site. We speculate that translational efficiency could be modulated by influencing the equilibrium between monomer and dimer.     

Translation elongation by a hybrid ribosome in which proteins at the GTPase center of the Escherichia coli ribosome are replaced with rat counterparts.

Ribosomal L10-L7/L12 protein complex and L11 bind to a highly conserved RNA region around position 1070 in domain II of 23 S rRNA and constitute a part of the GTPase-associated center in Escherichia coli ribosomes. We replaced these ribosomal proteins in vitro with the rat counterparts P0-P1/P2 complex and RL12, and tested them for ribosomal activities. The core 50 S subunit lacking the proteins on the 1070 RNA domain was prepared under gentle conditions from a mutant deficient in ribosomal protein L11. The rat proteins bound to the core 50 S subunit through their interactions with the 1070 RNA domain. The resultant hybrid ribosome was insensitive to thiostrepton and showed poly(U)-programmed polyphenylalanine synthesis dependent on the actions of both eukaryotic elongation factors 1alpha (eEF-1alpha) and 2 (eEF-2) but not of the prokaryotic equivalent factors EF-Tu and EF-G. The results from replacement of either the L10-L7/L12 complex or L11 with rat protein showed that the P0-P1/P2 complex, and not RL12, was responsible for the specificity of the eukaryotic ribosomes to eukaryotic elongation factors and for the accompanying GTPase activity. The presence of either E. coli L11 or rat RL12 considerably stimulated the polyphenylalanine synthesis by the hybrid ribosome, suggesting that L11/RL12 proteins play an important role in post-GTPase events of translation elongation.     

Yeast Lsm1p-7p/Pat1p deadenylation-dependent mRNA-decapping factors are required for brome mosaic virus genomic RNA translation

Previously, we used the ability of the higher eukaryotic positive-strand RNA virus brome mosaic virus (BMV) to replicate in yeast to show that the yeast LSM1 gene is required for recruiting BMV RNA from translation to replication. Here we extend this observation to show that Lsm1p and other components of the Lsm1p-Lsm7p/Pat1p deadenylation-dependent mRNA decapping complex were also required for translating BMV RNAs. Inhibition of BMV RNA translation was selective, with no effect on general cellular translation. We show that viral genomic RNAs suitable for RNA replication were already distinguished from nonreplication templates at translation, well before RNA recruitment to replication. Among mRNA turnover pathways, only factors specific for deadenylated mRNA decapping were required for BMV RNA translation. Dependence on these factors was not only a consequence of the nonpolyadenylated nature of BMV RNAs but also involved the combined effects of the viral 5' and 3' noncoding regions and 2a polymerase open reading frame. High-resolution sucrose density gradient analysis showed that, while mutating factors in the Lsm1p-7p/Pat1p complex completely inhibited viral RNA translation, the levels of viral RNA associated with ribosomes were only slightly reduced in mutant yeast. This polysome association was further verified by using a conditional allele of essential translation initiation factor PRT1, which markedly decreased polysome association of viral genomic RNA in the presence or absence of an LSM7 mutation. Together, these results show that a defective Lsm1p-7p/Pat1p complex inhibits BMV RNA translation primarily by stalling or slowing the elongation of ribosomes along the viral open reading frame. Thus, factors in the Lsm1p-7p/Pat1p complex function not only in mRNA decapping but also in translation, and both translation and recruitment of BMV RNAs to viral RNA replication are regulated by a cell pathway that transfers mRNAs from translation to degradation.