Was the initiation of translation in early eukaryotes IRES-driven?

The initiation of translation in eukaryotes generally involves the recognition of a 'cap' structure at the 5' end of the mRNA. However, for some viral and cellular mRNAs, a cap-independent mechanism occurs through an mRNA structure known as the internal ribosome entry site (IRES). Here, I postulate that the first eukaryotic mRNAs were translated in a cap-independent, IRES-driven manner that was then superseded in evolution by the cap-dependent mechanism, rather than vice versa. This hypothesis is supported by the following observations: (i) IRES-dependent, but not cap-dependent, translation can take place in the absence of not only a cap, but also many initiation factors; (ii) eukaryotic initiation factor 4E (eIF4E) and eIF4G, molecules absolutely required for cap-dependent translation, are among the most recently evolved translation factors; and (iii) functional similarities suggest the evolution of IRESs from spliceosomal introns. Thus, the contemporary cellular IRESs might be relics of the past.     

Head tilt–translation combinations distinguished at the level of neurons

Angular and linear accelerations of the head occur throughout everyday life, whether from external forces such as in a vehicle or from volitional head movements. The relative timing of the angular and linear components of motion differs depending on the movement. The inner ear detects the angular and linear components with its semicircular canals and otolith organs, respectively, and secondary neurons in the vestibular nuclei receive input from these vestibular organs. Many secondary neurons receive both angular and linear input. Linear information alone does not distinguish between translational linear acceleration and angular tilt, with its gravity-induced change in the linear acceleration vector. Instead, motions are thought to be distinguished by use of both angular and linear information. However, for combined motions, composed of angular tilt and linear translation, the infinite range of possible relative timing of the angular and linear components gives an infinite set of motions among which to distinguish the various types of movement. The present research focuses on motions consisting of angular tilt and horizontal translation, both sinusoidal, where the relative timing, i.e. phase, of the tilt and translation can take any value in the range −180° to 180°. The results show how hypothetical neurons receiving convergent input can distinguish tilt from translation, and that each of these neurons has a preferred combined motion, to which the neuron responds maximally. Also shown are the values of angular and linear response amplitudes and phases that can cause a neuron to be tilt-only or translation-only. Such neurons turn out to be sufficient for distinguishing between combined motions, with all of the possible relative angular–linear phases. Combinations of other neurons, as well, are shown to distinguish motions. Relative response phases and in-phase firing-rate modulation are the key to identifying specific motions from within this infinite set of combined motions.     

Differential contribution of the m7G-cap to the 5′ end-dependent translation initiation of mammalian mRNAs

Many mammalian mRNAs possess long 5′ UTRs with numerous stem-loop structures. For some of them, the presence of Internal Ribosome Entry Sites (IRESes) was suggested to explain their significant activity, especially when cap-dependent translation is compromised. To test this hypothesis, we have compared the translation initiation efficiencies of some cellular 5′ UTRs reported to have IRES-activity with those lacking IRES-elements in RNA-transfected cells and cell-free systems. Unlike viral IRESes, the tested 5′ UTRs with so-called ‘cellular IRESes’ demonstrate only background activities when placed in the intercistronic position of dicistronic RNAs. In contrast, they are very active in the monocistronic context and the cap is indispensable for their activities. Surprisingly, in cultured cells or cytoplasmic extracts both the level of stimulation with the cap and the overall translation activity do not correlate with the cumulative energy of the secondary structure of the tested 5′ UTRs. The cap positive effect is still observed under profound inhibition of translation with eIF4E-BP1 but its magnitude varies for individual 5′ UTRs irrespective of the cumulative energy of their secondary structures. Thus, it is not mandatory to invoke the IRES hypothesis, at least for some mRNAs, to explain their preferential translation when eIF4E is partially inactivated.     

Inhibition of HCV translation by disrupting the structure and interactions of the viral CRE and 3′ X-tail

A phylogenetically conserved RNA structure within the NS5B coding region of hepatitis C virus functions as a cis-replicating element (CRE). Integrity of this CRE, designated SL9266 (alternatively 5BSL3.2), is critical for genome replication. SL9266 forms the core of an extended pseudoknot, designated SL9266/PK, involving long distance RNA–RNA interactions between unpaired loops of SL9266 and distal regions of the genome. Previous studies demonstrated that SL9266/PK is dynamic, with ‘open’ and ‘closed’ conformations predicted to have distinct functions during virus replication. Using a combination of site-directed mutagenesis and locked nucleic acids (LNA) complementary to defined domains of SL9266 and its interacting regions, we have explored the influence of this structure on genome translation and replication. We demonstrate that LNAs which block formation of the closed conformation inhibit genome translation. Inhibition was at least partly independent of the initiation mechanism, whether driven by homologous or heterologous internal ribosome entry sites or from a capped message. Provision of SL9266/PK in trans relieved translational inhibition, and mutational analysis implied a mechanism in which the closed conformation recruits a cellular factor that would otherwise suppresses translation. We propose that SL9266/PK functions as a temporal switch, modulating the mutually incompatible processes of translation and replication.     

How strong is the case for regulation of the initiation step of translation by elements at the 3' end of eukaryotic mRNAs?

The belief that initiation of translation requires communication between the 5' and 3' ends of the mRNA guides--or misguides--the interpretation of many experiments. The closed-loop model for initiation creates the expectation that sequences at the 3' end of eukaryotic mRNAs should regulate translation. This review looks closely at the evidence in three prominent cases where such regulation is claimed. The mRNAs in question encode 15-lipoxygenase, ceruloplasmin, and histones. Vertebrate histone mRNAs lack a poly(A) tail, instead of which a 3' stem-loop structure is said to promote translation by binding a protein which purportedly binds initiation factors. The proffered evidence for this hypothesis has many flaws. Temporal control of 15-lipoxygenase production in reticulocytes is often cited as another well-documented example of translational regulation via the 3' untranslated region, but inspection of the evidence reveals significant gaps and contradictions. Solid evidence is lacking also for the idea that a ribosomal protein binds to and shuts off translation of ceruloplasmin mRNA. Some viral RNAs that lack a poly(A) tail have alternative 3' structures which are said to promote translation via circularization of the mRNA, but in no case has this been shown convincingly. Interpretation of many experiments is compromised by possible effects of the 3' structures on mRNA stability rather than translation. The functional-half-life assay, which is often employed to rule out effects on mRNA stability, might not be adequate to settle the question. Other issues, such as the possibility of artifacts caused by overexpression of RNA-binding proteins, can complicate studies of translational regulation. There is no doubt that elements at the 3' end of eukaryotic mRNAs can regulate gene expression in a variety of ways. It has not been shown unequivocally that one of these ways involves direct participation of the 3' untranslated region in the initiation step of translation.     

Effect of deletions 5′ to the translation initiation sequence on the expression of an mRNA in animal cells

To learn if an mRNA·18S rRNA interaction or a special secondary structure in the mRNA start region is essential for translation in eukaryotic cells, we constructed recombinant plasmids with the SV40 early promoter 5 to part of the Escherichia coli tuf B-lacZ gene. Deletion of bases potentially complementary to the 18S rRNA highly increased the transient -galactosidase expressed in transfected CHO cells. Deletion of bases that fostered formation of potential hairpins with the mRNA 5-terminus or altered the structure of the coding region reduced -galactosidase activity suggesting that these features of the mRNA secondary structure may be essential for initiation of translation. Computer aided analysis of the potential structure of 290 mRNAs suggests these are conserved features of the initiation region.     

Effect of 7-methylguanosine-5′-phosphate on the translation of rat milk-protein mRNAs in the wheat-germ cell-free system

The translation of polyadenylated and of non-poly-adenylated RNA obtained from lactating rat mammary gland was almost totally inhibited by 0,5 mM 7-methylguanosine-5-phosphate in the wheat-germ cell-free system, This inhibition was maintained during the preparation of the 9S whey-protein mRNA and of the 12S and ISS casein mRNAs, Chemical decapping of these mRNAs caused a similar reduction of their activity . Although a large fraction of milk-protein mRNAs have been reported to lack 3-polyadenylation, these results show that the mRNAs in the mammary gland do contain a 5-terminal 7-methylguanosine cap.     

Do universal codon-usage patterns minimize the effects of mutation and translation error?

Background

Do species use codons that reduce the impact of errors in translation or replication? The genetic code is arranged in a way that minimizes errors, defined as the sum of the differences in amino-acid properties caused by single-base changes from each codon to each other codon. However, the extent to which organisms optimize the genetic messages written in this code has been far less studied. We tested whether codon and amino-acid usages from 457 bacteria, 264 eukaryotes, and 33 archaea minimize errors compared to random usages, and whether changes in genome G+C content influence these error values.

Results

We tested the hypotheses that organisms choose their codon usage to minimize errors, and that the large observed variation in G+C content in coding sequences, but the low variation in G+U or G+A content, is due to differences in the effects of variation along these axes on the error value. Surprisingly, the biological distribution of error values has far lower variance than randomized error values, but error values of actual codon and amino-acid usages are actually greater than would be expected by chance.

Conclusion

These unexpected findings suggest that selection against translation error has not produced codon or amino-acid usages that minimize the effects of errors, and that even messages with very different nucleotide compositions somehow maintain a relatively constant error value. They raise the question: why do all known organisms use highly error-minimizing genetic codes, but fail to minimize the errors in the mRNA messages they encode?

     

Novel cap-independent translation with the 5′-noncoding region of L-A virus mRNA

A novel cap-independent translation has been performed where the ribosome entry is regulated by the 5-noncoding region (NCR) of L-A virus mRNA. Despite L-A virus mRNA containing neither cap structure nor a poly(A) tail, the reconstructed mRNA encoding the 5 NCR of L-A virus mRNA and a reporter gene (luciferase) was translated, in yeast lysate, 60 times more efficiently than control mRNA. The 5 NCR from L-A virus was effective in regulating the recruitment of ribosome in vitro. A possible mechanism in Saccharomyces cerevisiae is also suggested, whereby the ribosome entry is regulated by the 5 NCR of L-A virus mRNA.     

Influence of cation–π interactions to the structural stability of prokaryotic and eukaryotic translation elongation factors

We have investigated the role of cation–π interactions on translation elongation factors. In our investigation, an average of four significant cation–π interactions were found, that is, an average of one cation–π interaction per 44 residues in the ten elongation factors were observed. The analysis on the influence of short (<±4), medium (>±4 to <±20) and long (>20) range contacts showed that cation–π interactions are mainly formed by medium and long-range contacts. Arg-Tyr pair was found largest in number but energetic contribution of Arg-Trp pair was found most. Preferred secondary structural conformation analysis of the residues involved in cation–π interaction indicates that the cationic Arg prefers to be in helix and Lys having equal probability for helix and strand, whereas the aromatic Phe and Trp were found mostly in helix while Tyr in strand regions. The cation–π interaction residues involved in these proteins were found highly conserved with 48.86% residues having conservation score of ≥6. Analysis of secondary structure preference of the energetically significant cation–π residues in different solvent accessible range indicates that most of the π residues are found buried or partially buried whereas cationic residues were found mostly at the protein surface. The results presented in this study will be useful for structural stability studies in translation elongation factors.     

14 What RNA world ?? Ancestral polypeptides likely participated in the origins of translation

A widespread consensus holds that protein synthesis according to a genetic code was launched entirely by sophisticated RNA molecules that played both coding and functional roles. This belief persists, unsupported by phylogenetic evidence for ancestral ribozymes that catalyzed either amino acid activation or tRNA aminoacylation. By contrast, we have adduced strong experimental evidence that the most highly conserved portions of contemporary aminoacyl-tRNA synthetases (aaRS) accelerate both reactions well in excess of rates achieved by RNA aptomers derived from combinatorial libraries and of rates required for primordial protein synthesis. Such ancestral enzymes, or “Urzymes”, characterized for Class I (TrpRS (Pham et al., 2010, 2007) and LeuRS (Collier et al., 2013); 130 residues) and Class II (HisRS; 120–140 residues; (Li et al., 2011)) synthetases generally have promiscuous amino acid specificities, whereas ATP and cognate tRNA affinities are within an order of magnitude of those for contemporary enzymes. These characteristics match or exceed expectations for the primordial catalysts necessary to launch protein synthesis. Structural hierarchies in Class I and II aaRS also exhibit plateaus of increasing enzymatic activity, suggesting that catalysis by peptides similar to the Aleph motif identified by Trifonov (Sobolevsky et al.) may have been both necessary and sufficient to launch protein synthesis. Sense/antisense alignments of TrpRS and HisRS Urzyme coding sequences reveal unexpectedly high middle-base complementarity that increases in reconstructed ancestral nodes (Chandrasekaran et al.), consistent with the proposal of Rodin and Ohno (Rodin & Ohno, 1995). Thus, these ancestors were likely coded by opposite strands of the same gene, favoring simultaneous expression of aaRS activating both hydrophobic (core) and hydrophilic (surface) amino acids. Our results support the view that aaRS coevolved with cognate tRNAs from a much earlier stage than that envisioned under the RNA World hypothesis, and that their descendants make up appreciable portions of the proteome.     

Polyamine modulon in yeast—Stimulation of COX4 synthesis by spermidine at the level of translation

We proposed that a group of genes whose expression is enhanced by polyamines at the level of translation in Escherichia coli and mammalian cells be referred to as a “polyamine modulon”. In Saccharomyces cerevisiae, proteins whose synthesis is enhanced by polyamines at the level of translation were searched for using a polyamine-requiring mutant of S. cerevisiae deficient in ornithine decarboxylase (YPH499 Δspe1). Addition of spermidine to the medium recovered the spermidine content and enhanced cell growth of the YPH499 Δspe1 mutant by 3–5-fold. Under these conditions, synthesis of COX4, one of the subunits of cytochrome C oxidase (complex IV), was enhanced by polyamines about 2.5-fold at the level of translation. Accordingly, the COX4 gene is the first member of a polyamine modulon in yeast. Polyamines enhanced COX4 synthesis through stimulation of the ribosome shunting of the stem–loop structures (hairpin structures) during the scanning of the 5′-untranslated region (5′-UTR) of COX4 mRNA by 40S ribosomal subunit-Met-tRNA_i complex.     

Detection of an internal translation activity in the 5′ region of Bombyx mori infectious flacherie virus

The 5′ untranslated region plays an important role in positive-sense single-stranded RNA virus translation initiation, as it contains an internal ribosome entry site (IRES) that mediates cap-independent translation and is applied to simultaneously express several proteins. Infectious flacherie virus (IFV) is a positive-sense single-stranded RNA virus; however, the IRES function is still not proved. To investigate whether the sequences of IFV contain IRES activity, a series of bicistronic reporter (DsRed and enhanced green fluorescent protein) recombinant baculoviruses were constructed to infect the insect cells and silkworm using the Bombyx mori baculovirus expression system. Results showed that the upstream 311, 323, 383, 551, and 599 nt have IRES activity except for the 155-nt region in BmN cells. More importantly, the tetraloop structure containing region between 551 and 599 nt appeared to be responsible for the enhanced IRES activity in different insect cell lines and silkworm. These results indicated that the IRES activity is not species specific and tissue specific. Therefore, our findings may provide the basis for the simultaneous expression of two or various different genes under the same promoter in baculovirus expression system.     

Processing of the 5′-UTR and existence of protein factors that regulate translation of tobacco chloroplast psbN mRNA

The chloroplast psbB operon includes five genes encoding photosystem II and cytochrome b ₆ /f complex components. The psbN gene is located on the opposite strand. PsbN is localized in the thylakoid and is present even in the dark, although its level increases upon illumination and then decreases. However, the translation mechanism of the psbN mRNA remains unclear. Using an in vitro translation system from tobacco chloroplasts and a green fluorescent protein as a reporter protein, we show that translation occurs from a tobacco primary psbN 5′-UTR of 47 nucleotides (nt). Unlike many other chloroplast 5′-UTRs, the psbN 5′-UTR has two processing sites, at ?39 and ?24 upstream from the initiation site. Processing at ?39 enhanced the translation rate fivefold. In contrast, processing at ?24 did not affect the translation rate. These observations suggest that the two distinct processing events regulate, at least in part, the level of PsbN during development. The psbN 5′-UTR has no Shine–Dalgarno (SD)-like sequence. In vitro translation assays with excess amounts of the psbN 5′-UTR or with deleted psbN 5′-UTR sequences demonstrated that protein factors are required for translation and that their binding site is an 18 nt sequence in the 5′-UTR. Mobility shift assays using 10 other chloroplast 5′-UTRs suggested that common or similar proteins are involved in translation of a set of mRNAs lacking SD-like sequences.