Translational Elements Research Articles

In vivo contextual requirements for UAG translation as pyrrolysine

Pyrrolysine and selenocysteine have infiltrated natural genetic codes via the translation of canonical stop codons. UGA translation as selenocysteine is absolutely dependent on message context. Here we describe the first experimental examination of contextual requirements for UAG translation as pyrrolysine. A hexahistidine-tagged Methanosarcina barkeri mtmB1 gene, encoding monomethylamine methyltransferase MtmB1, was introduced into Methanosarcina acetivorans. Host mtmB expression was minimized by growth on methanol and recombinant mtmB1 products monitored by anti-MtmB and anti-hexahistidine immunoblotting. UAG translation was not compromised, as recombinant MtmB1 was 1% of cellular protein with only trace UAG-terminated mtmB1 product detectable. Untranslated regions flanking mtmB1 were not required for UAG translation, but loss of a downstream pyrrolysine insertion sequence (PYLIS) significantly increased the UAG-termination product of mtmB1 and decreased the UAG-translation product, which nonetheless contained pyrrolysine. An in-frame UAG within a bacterial uidA transcript was translated in the methanogen as pyrrolysine with 20% efficiency, suggesting UAG translation in the absence of evolved context. However, predominant UAG-directed termination with enhancement of UAG translation by the PYLIS appears analogous to cis-acting elements for UGA translation as selenocysteine, although different mechanisms may underlie these recoding events.

Cap-dependent and hepatitis C virus internal ribosome entry site-mediated translation are modulated by phosphorylation of eIF2α under oxidative stress

Chronic hepatitis C is often associated with oxidative stress. Hepatitis C virus (HCV) utilizes an internal ribosome entry site (IRES) element for translation, in contrast to cap-dependent translation of the majority of cellular proteins. To understand how virus translation is modulated under oxidative stress, HCV IRES-mediated translation was compared with cap-dependent translation using a bicistronic reporter construct and hydrogen peroxide (H2O2) as a stress inducer. In H2O2-sensitive HeLa cells, H2O2 repressed translation in a time- and dose-dependent manner, concomitant with the kinetics of eIF2alpha phosphorylation. A phosphomimetic of eIF2alpha, which mimics the structure of the phosphorylated eIF2alpha, was sufficient to repress translation in the absence of H2O2. In H2O2-resistant HepG2 cells, H2O2 activated both HCV IRES-mediated and cap-dependent translation, associated with an increased level of phospho-eIF2alpha. It was postulated that H2O2 might stimulate translation in HepG2 cells via an eIF2alpha-independent mechanism, whereas the simultaneous phosphorylation of eIF2alpha repressed part of the translational activities. Indeed, the translational repression was released in the presence of a non-phosphorylatable mutant, eIF2alpha-SA, resulting in further enhancement of both translational activities after exposure to H2O2. In HuH7 cells, which exhibited an intermediate level of sensitivity towards H2O2, both HCV IRES-mediated and cap-dependent translational activities were upregulated after treatment with various doses of H2O2, but the highest level of induction was achieved with a low level of H2O2, which may represent the physiological level of H2O2. At this level, the HCV IRES-mediated translation was preferentially upregulated compared with cap-dependent translation.

Structures required for poly(A) tail-independent translation overlap with, but are distinct from, cap-independent translation and RNA replication signals at the 3′ end of Tobacco necrosis virus RNA

Tobacco necrosis necrovirus (TNV) RNA lacks both a 5′ cap and a poly(A) tail but is translated efficiently, owing in part to a Barley yellow dwarf virus ( BYDV)-like cap-independent translation element (BTE) in its 3′ untranslated region (UTR). Here, we identify sequence downstream of the BTE that is necessary for poly(A) tail-independent translation in vivo by using RNA encoding a luciferase reporter gene flanked by viral UTRs. Deletions and point mutations caused loss of translation that was restored by adding a poly(A) tail, and not by adding a 5′ cap. The two 3′-proximal stem–loops in the viral genome contribute to poly(A) tail-independent translation, as well as RNA replication. For all necroviruses, we predict a conserved 3′ UTR secondary structure that includes the BTE at one end of a long helical axis and the stem–loops required for poly(A) tail-independent translation and RNA replication at the other end. This work shows that a viral genome can harbor distinct cap- and poly(A) tail-mimic sequences in the 3′ UTR.

CONVERSION OF CASSINI COORDINATES TO UTM COORDINATES ON THE EXCEL SPREADSHEET

A procedure for transforming Cassini grid coordinates (planimetric only) to UTM coordinates on the Excel spreadsheet is outlined. A general second degree polynomial is used to compute six parameters which includes a scale, a rotation two translation elements and two other unknowns. Three interconnected worksheets are used to compute the transformation. The first worksheet is used for data entry. The second worksheet computes the six transformation parameters necessary for the transformation by use of entries in the first worksheet, while the third worksheet computes the transformed coordinates in UTM by use of the entries in the first worksheet and the computed parameters in the second worksheet. Ellipsodial Cassini co-ordinates for the common points can be also computed on the excel spreadsheet, and where corresponding ellipsoidal coordinates in UTM are available, a seven parameter transformation can be obtained.

Translational control ofregA, a key gene controlling cell differentiation inVolvox carteri

The complete division of labour between the reproductive and somatic cells of the green alga Volvox carteri is controlled by three types of genes. One of these is the regA gene, which controls terminal differentiation of the somatic cells. Here, we examined translational control elements located in the 5' UTR of regA, particularly the eight upstream start codons (AUGs) that have to be bypassed by the translation machinery before regA can be translated. The results of our systematic mutational, structural and functional analysis of the 5' UTR led us to conclude that a ribosome-shunting mechanism--rather than leaky scanning, ribosomal reinitiation, or internal ribosome entry site (IRES)-mediated initiation--controls the translation of regA mRNA. This mechanism, which involves dissociation of the 40S initiation complex from the message, followed by reattachment downstream, in order to bypass a secondary structure block in the mRNA, was validated by deleting the predicted ;landing site' (which prevented regA expression) and inserting a stable 64 nucleotide hairpin just upstream of this site (which did not prevent regA expression). We believe that this is the first report suggesting that translation of an mRNA in a green eukaryote is controlled by ribosome shunting.

Phase‐Transformation‐Induced Anti‐Phase Boundary Domains in Barium Cerate Perovskite

Anti‐phase boundary (APB) domains resulting from the orthorhombic (o1, Ibmm (No. 74)) to orthorhombic (o2, Pbnm (No. 62)) phase transformation at ∼290°C in pressureless‐sintered barium cerate (BaCeO3) ceramics have been investigated by transmission electron microscopy and large‐angle convergent beam electron diffraction (LACBED). APB exhibiting symmetric π‐fringe patterns in both bright‐field and dark‐field images, lying in {011) with the fault vector R=1/2〈111〉, were determined adopting the invisibility criteria of 2πg·R=0 or 2nπ. The curved boundaries due to symmetry change can be related to the low symmetry phase by a pure translation upon phase transition. The displacement vector, R, relating the domains is the translational symmetry element from the origin to the lattice point that was lost upon transition. The formation of such domains induced by the o1→o2 phase transition is discussed in terms of change in the Bravais lattice and loss of lattice point upon the transition. The identification has also confirmed the existence of a higher symmetry orthorhombic o1 phase along the transition sequence of R3c→Ibmm→Pbnm. The increased spatial frequency of APB domains is attributed to A‐site ordering due to vacancies created by BaO loss and/or disordering of the B‐site substitutional defects generated extrinsically when doped with either a Y3+‐acceptor or a Nb5+‐donor.

Oscillating kissing stem–loop interactions mediate 5′ scanning-dependent translation by a viral 3′-cap-independent translation element

The 3'-untranslated regions (UTRs) of a group of novel uncapped viral RNAs allow efficient translation initiation at the 5'-proximal AUG. A well-characterized model is the Barley yellow dwarf virus class of cap-independent translation elements (BTE). It facilitates translation by forming kissing stem-loops between the BTE in the 3'-UTR and a BTE-complementary loop in the 5'-UTR. Here we investigate the mechanisms of the long-distance interaction and ribosome entry on the RNA. Upstream AUGs or 5'-extensions of the 5'-UTR inhibit translation, indicating that, unlike internal ribosome entry sites in many viral RNAs, the BTE relies on 5'-end-dependent ribosome scanning. Cap-independent translation occurs when the kissing sites are moved to different regions in either UTR, including outside of the BTE. The BTE can even confer cap-independent translation when fused to the 3'-UTR of a reporter RNA harboring dengue virus sequences that cause base-pairing between the 3'- and 5'-ends. Thus, the BTE serves as a functional sensor to detect sequences capable of long-distance base-pairing. We propose that the kissing interaction is repeatedly disrupted by the scanning ribosome and re-formed in an oscillating process that regulates ribosome entry on the RNA.

Xenopus ElrB, but not ElrA, binds RNA as an oligomer: Possible role of the linker

We have shown that ElrA and ElrB, Xenopus ELAV homologues, bind the Vg1 mRNA 3′UTR translation element in Xenopus oocytes and implicated ElrB in mediating translational repression during oogenesis. Here we report that, while ElrA and ElrB are 69% identical and both exhibit RNA binding in the nM range, recombinant ElrB, but not ElrA, is able to oligomerise. This oligomerisation is also seen with the endogenous protein. Both RNA binding and oligomerisation require the linker region flanked with two RNA recognition motifs. Our data demonstrate a novel and unique property of ElrB which may be important for its function as a translational regulator.

Ribosomal shunting mediated by a translational enhancer element that base pairs to 18S rRNA

In eukaryotes, 40S ribosomal subunits move from their recruitment site on the mRNA to the initiation codon by an as yet poorly understood process. One postulated mechanism involves ribosomal shunting, in which ribosomal subunits completely bypass regions of the 5' leader. For some mRNAs, shunting has been shown to require various mRNA elements, some of which are thought to base pair to 18S rRNA; however, the role of base pairing has not yet been tested directly. In earlier studies, we demonstrated that a short mRNA element in the 5' leader of the Gtx homeodomain mRNA functioned as a ribosomal recruitment site by base pairing to the 18S rRNA. Using a model system to assess translation in transfected cells, we now show that this intermolecular interaction also facilitates ribosomal shunting across two types of obstacles: an upstream AUG codon in excellent context or a stable hairpin structure. Highly efficient shunting occurred when multiple Gtx elements were present upstream of the obstacles, and a single Gtx element was present downstream. Shunting was less efficient, however, when the multiple Gtx elements were present only upstream of the obstacles. In addition, control experiments with mRNAs lacking the upstream elements showed that these results could not be attributed to recruitment by the single downstream element. Experiments in yeast in which the mRNA elements and 18S rRNA sequences were both mutated indicated that shunting required an intact complementary match. The data obtained by this model system provide direct evidence that ribosomal shunting can be mediated by mRNA-rRNA base pairing, a finding that may have general implications for mechanisms of ribosome movement.

In vitro selection of translational regulatory elements

Untranslated regions (UTRs) of mRNAs carry various kinds of translational regulatory elements; however, our knowledge of them is still limited. We created an in vitro selection system that allows us to make a systematic enrichment of the sequences that alter translation efficiency (SESTRE) in any given mRNA and translation system. This method consists of the introduction of random nucleotide sequences into the UTRs of given mRNAs, followed by translation, size fractionation of the polyribosomes, and reverse transcription and PCR amplification (RT-PCR), with repeated cycles of these steps to enrich highly or poorly translatable mRNAs. With this experimental method, we examined how and where translational enhancer motifs emerge on mRNAs using the in vitro translation systems of wheat germ extract. The results indicate that the translational enhancers differentially emerge in response to the presence or absence of the 5′ cap. Interestingly, the translational enhancers that activate cap-independent translation evolved more readily in the 3′ UTR than in the 5′ UTR in wheat germ extract. This SESTRE method should be a powerful tool with which to improve the translational efficiency of given mRNAs in given translation systems and to investigate the structure–function relationship of eukaryotic mRNAs underlying translational control.

Derepression of the Her-2 uORF is mediated by a novel post-transcriptional control mechanism in cancer cells

Transcripts harboring 5' upstream open reading frames (uORFs) are often found in genes controlling cell growth including receptors, oncogenes, or growth factors. uORFs can modulate translation or RNA stability and mediate inefficient translation of these potent proteins under normal conditions. In dysregulated cancer cells, where the gene product, for example Her-2 receptor, is overexpressed, post-transcriptional processes must exist that serve to override the inhibitory effects of the uORFs. The 5' untranslated region (UTR) of Her-2 mRNA contains a short uORF that represses translation of the downstream coding region. We demonstrate that in Her-2 overexpressing breast cancer cells, the 3' UTR of the Her-2 mRNA can override translational inhibition mediated by the Her-2 uORF. Within this 3' UTR, a translational derepression element (TDE) that binds to a 38-kDa protein was identified. These results define a novel biological mechanism in which translational control of genes harboring a 5' uORF can be modulated by elements in their 3' UTRs.

Characterization and gene expression of Babesia bovis elongation factor-1α

Elongation factor 1 alpha (EF-1α) is a constitutively expressed, abundant protein that is a key element in eukaryotic protein translation. Because of its high level of transcription, the EF-1α promoter has been utilised to drive exogenous gene expression in transfected cells. In this study, we identified and characterised the ef-1α locus of Babesia bovis, a causative agent of bovine babesiosis, and examined the transcriptional activity of the EF-1α promoter. The ef-1α locus in the T 2Bo strain of B. bovis contains two identical ef-1α genes (‘A’ and ‘B’) arranged in a head to head orientation and separated by a 1.4 kb intergenic (IG) region containing a 260 bp terminal inverted repeat. Both ef-1α genes encode identical proteins with 448 amino acids and a calculated molecular mass of 49 kDa. While the B. bovis ef-1α-IG sequence is conserved among multiple strains of B. bovis, it is not significantly related to any regulatory sequence in the DNA databases. The IG region promotes expression of both ef-1α genes. Both fragment Ig-A containing 730 bp upstream of ef-1α open reading frame A and fragment Ig-B containing 720 bp upstream of ef-1α open reading frame B were able to promote luciferase in transient transfection. In the 5′ to 3′ orientation, the Ig-B fragment resulted in the highest level of luciferase activity, 10 times higher than positive control plasmid p40-15-luc containing the rap-1 IG region, suggesting that this fragment contains a very strong promoter. Analysis of ef-1α transcripts confirms that both ef-1α genes are transcribed in merozoites. Interestingly, in contrast to other related intra-erythrocytic apicomplexans, the ef-1α locus of B. bovis contains a 160 bp intron in the 5′ untranslated region.

Glorund, a Drosophila hnRNP F/H Homolog, Is an Ovarian Repressor of nanos Translation

Patterning of the anterior-posterior body axis of the Drosophila embryo requires production of Nanos protein selectively in the posterior. Spatially restricted Nanos synthesis is accomplished by translational repression of unlocalized nanos mRNA together with translational activation of posteriorly localized nanos. Repression of unlocalized nanos mRNA is mediated by a bipartite translational control element (TCE) in its 3' untranslated region. TCE stem-loop II functions during embryogenesis, through its interaction with the Smaug repressor. Stem-loop III represses unlocalized nanos mRNA during oogenesis, but trans-acting factors that carry out this function have remained elusive. Here we identify a Drosophila hnRNP, Glorund, that interacts specifically with stem-loop III. We establish that the ability of the TCE to repress translation in vivo reflects its ability to bind Glorund in vitro. These data, together with the analysis of a glorund null mutant, reveal a specific role for an hnRNP in repression of nanos translation during oogenesis.

In Drosophila, don juan and don juan like encode proteins of the spermatid nucleus and the flagellum and both are regulated at the transcriptional level by the TAFII80 cannonball while translational repression is achieved by distinct elements

The genes don juan (dj) and don juan like (djl) encode basic proteins expressed in the male germline. Both proteins show a similar expression pattern being localized in the sperm heads during chromatin condensation and along the flagella. Prematurely expressed Don Juan-eGFP and Myc-Don Juan Like localize to the cytoplasm of spermatocytes and in mitochondrial derivatives from the nebenkern stage onward suggesting that both proteins associate with the mitochondria along the flagella in elongated spermatids. Premature expression of Myc-Don Juan Like does not impair spermatogenesis where-as Don Juan-eGFP when prematurely expressed causes male sterility as spermatids fail to individualize. In spite of the sequence identity of 72% on the nucleotide level and 42% on the protein level, the presumptive promoter regions and the untranslated regions of the mRNA are diverged. Our in vivo analysis revealed that don juan and don juan like are transcriptionally and translationally controlled by distinct short cis regulatory regions. Transcription of don juan and don juan like depends on the male germ line specific TAF(II)80, Cannonball (Can). Translational repression elements for both mRNAs are localized in the 5' UTR and are capable to form distinct secondary structures in close proximity to the translational initiation codon.

Cap-independent translation of plant viral RNAs

The RNAs of many plant viruses lack a 5′ cap and must be translated by a cap-independent mechanism. Here, we discuss the remarkably diverse cap-independent translation elements that have been identified in members of the Potyviridae, Luteoviridae, and Tombusviridae families, and genus Tobamovirus. Many other plant viruses have uncapped RNAs but their translation control elements are uncharacterized. Cap-independent translation elements of plant viruses differ strikingly from those of animal viruses: they are smaller (<200 nt), some are located in the 3′ untranslated region, some require ribosome scanning from the 5′ end of the mRNA, and the 5′ UTR elements are much less structured than those of animal viruses. We discuss how these elements may interact with host translation factors, and speculate on their mechanism of action and their roles in the virus replication cycle. Much remains to be learned about how these elements enable plant viruses to usurp the host translational machinery.

The autoregulatory translational control element of poly(A)-binding protein mRNA forms a heteromeric ribonucleoprotein complex

Repression of poly(A)-binding protein (PABP) mRNA translation involves the binding of PABP to the adenine-rich autoregulatory sequence (ARS) in the 5′-untranslated region of its own mRNA. In this report, we show that the ARS forms a complex in vitro with PABP, and two additional polypeptides of 63 and 105 kDa. The 63 and 105 kDa polypeptides were identified, as IMP1, an ortholog of chicken zip-code binding polypeptide, and UNR, a PABP binding polypeptide, respectively, by mass spectrometry of the ARS RNA affinity purified samples. Using a modified ribonucleoprotein (RNP) immunoprecipitation procedure we further show that indeed, both IMP1 and UNR bind to the ARS containing reporter RNA in vivo. Although both IMP1 and UNR could bind independently to the ARS RNA in vitro, their RNA-binding ability was stimulated by PABP. Mutational analyses of the ARS show that the presence of four of the six oligo(A) regions of the ARS was sufficient to repress translation and the length of the conserved pyrimidine spacers between the oligo(A) sequences was important for ARS function. The ability of mutant ARS RNAs to form the PABP, IMP1 and UNR containing RNP complex correlates well with the translational repressor activity of the ARS. There is also a direct relationship between the length of the poly(A) RNAs and their ability to form a trimeric complex with PABP, and to repress mRNA translation. UV crosslinking studies suggest that the ARS is less efficient than a poly(A) RNA of similar length, to bind to PABP. We show here that the ARS cannot efficiently form a trimeric complex with PABP; therefore, additional interactions with IMP1 and UNR to form a heteromeric RNP complex may be required for maximal repression of PABP mRNA translation under physiological conditions.

Dendritic BC1 RNA in translational control mechanisms

Translational control at the synapse is thought to be a key determinant of neuronal plasticity. How is such control implemented? We report that small untranslated BC1 RNA is a specific effector of translational control both in vitro and in vivo. BC1 RNA, expressed in neurons and germ cells, inhibits a rate-limiting step in the assembly of translation initiation complexes. A translational repression element is contained within the unique 3′ domain of BC1 RNA. Interactions of this domain with eukaryotic initiation factor 4A and poly(A) binding protein mediate repression, indicating that the 3′ BC1 domain targets a functional interaction between these factors. In contrast, interactions of BC1 RNA with the fragile X mental retardation protein could not be documented. Thus, BC1 RNA modulates translation-dependent processes in neurons and germs cells by directly interacting with translation initiation factors.

In Vivo Translation Studies of Plant Viral RNAs Using Reporter Genes

This unit describes the analysis of the translation-regulating properties of viral RNAs in cell-based assays using a luciferase reporter system. Electroporation and polyethylene glycol-mediated introduction of luciferase reporter RNA into cowpea protoplasts are also presented. The Commentary section discusses employing the luciferase reporter system to study translational control by the 5'- and 3'-untranslated regions of a viral mRNA. Inoculum RNA quality and half-life after delivery to the protoplasts should be monitored closely for careful investigation of translational regulation elements.

The Xenopus ELAV protein ElrB represses Vg1 mRNA translation during oogenesis.

Xenopus laevis Vg1 mRNA undergoes both localization and translational control during oogenesis. We previously characterized a 250-nucleotide AU-rich element, the Vg1 translation element (VTE), in the 3'-untranslated region (UTR) of this mRNA that is responsible for the translational repression. UV-cross-linking and immunoprecipitation experiments, described here, revealed that the known AU-rich element binding proteins, ElrA and ElrB, and TIA-1 and TIAR interact with the VTE. The levels of these proteins during oogenesis are most consistent with a possible role for ElrB in the translational control of Vg1 mRNA, and ElrB, in contrast to TIA-1 and TIAR, is present in large RNP complexes. Immunodepletion of TIA-1 and TIAR from Xenopus translation extract confirmed that these proteins are not involved in the translational repression. Mutagenesis of a potential ElrB binding site destroyed the ability of the VTE to bind ElrB and also abolished translational repression. Moreover, multiple copies of the consensus motif both bind ElrB and support translational control. Therefore, there is a direct correlation between ElrB binding and translational repression by the Vg1 3'-UTR. In agreement with the reporter data, injection of a monoclonal antibody against ElrB into Xenopus oocytes resulted in the production of Vg1 protein, arguing for a role for the ELAV proteins in the translational repression of Vg1 mRNA during early oogenesis.

Evaluation of cis-acting elements in the rubella virus subgenomic RNA that play a role in its translation

The subgenomic (SG) mRNA of rubella virus (RUB) contains the structural protein open reading frame (SP-ORF) that is translated to produce the three virion structural proteins: capsid (C) and glycoproteins E2 and E1. RUB expression vectors have been developed that express heterologous genes from the SG RNA, including replicons which replace the SP-ORF with a heterologous gene, and these expression vectors are candidate vaccine vectors. In the related alphaviruses, translational enhancing elements have been identified in both the 5' untranslated region (UTR) of the SG RNA and the N-terminal region of the C gene. To optimize expression from RUB vectors, both the 5'UTR of the SG RNA and the C gene were surveyed for translational enhancing elements using both plasmids and replicons expressing reporter genes from the SG RNA. In replicons, the entire 5'UTR was necessary for translation; interestingly, when plasmids were used the 5'UTR was dispensable for optimal translation. The RUB C gene contains a predicted long stem-loop starting 62 nts downstream from the initiation codon (SLL) that has a structure and stability similar to SL's found in the C genes of two alphaviruses, Sindbis virus (SIN) and Semliki Forest virus, that have been shown to enhance translation of the SG RNA in infected cells. However, a series of fusions of various lengths of the N-terminus of the RUB C protein with reporter genes showed that the SLL had an attenuating effect on translation that was overcome by mutagenesis that destabilized the SLL or by adding downstream sequences of the C gene to the fusion. Thus, for optimal expression efficiency from RUB expression vectors, only the 5'UTR of the SG RNA is required. Further investigation of the differing effects of the SLL on RUB and alphavirus SG RNA translation revealed that the SIN and RUB SLLs could enhance translation when expressed from a SIN cytopathic replicon, but not when expressed from a plasmid, a RUB replicon, or a SIN noncytopathic replicon. Thus, the SLL only functions in a "cytopathic environment" in which cell translation has been altered.