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PubMed1996

Cap-binding protein (eukaryotic initiation factor 4E) and 4E-inactivating protein BP-1 independently regulate cap-dependent translation.

Cap-dependent protein synthesis in animal cells is inhibited by heat shock, serum deprivation, metaphase arrest, and infection with certain viruses such as adenovirus (Ad). At a mechanistic level, translation of capped mRNAs is inhibited by dephosphorylation of eukaryotic initiation factor 4E (eIF-4E) (cap-binding protein) and its physical sequestration with the translation repressor protein BP-1 (PHAS-I). Dephosphorylation of BP-I blocks cap-dependent translation by promoting sequestration of eIF-4E. Here we show that heat shock inhibits translation of capped mRNAs by simultaneously inducing dephosphorylation of eIF-4E and BP-1, suggesting that cells might coordinately regulate translation of capped mRNAs by impairing both the activity and the availability of eIF-4E. Like heat shock, late Ad infection is shown to induce dephosphorylation of eIF-4E. However, in contrast to heat shock, Ad also induces phosphorylation of BP-1 and release of eIF-4E. BP-1 and eIF-4E can therefore act on cap-dependent translation in either a mutually antagonistic or cooperative manner. Three sets of experiments further underscore this point: (i) rapamycin is shown to block phosphorylation of BP-1 without inhibiting dephosphorylation of eIF-4E induced by heat shock or Ad infection, (ii) eIF-4E is efficiently dephosphorylated during heat shock or Ad infection regardless of whether it is in a complex with BP-1, and (iii) BP-1 is associated with eIF-4E in vivo regardless of the state of eIF-4E phosphorylation. These and other studies establish that inhibition of cap-dependent translation does not obligatorily involve sequestration of eIF-4E by BP-1. Rather, translation is independently regulated by the phosphorylation states of eIF-4E and the 4E-binding protein, BP-1. In addition, these results demonstrate that BP-1 and eIF-4E can act either in concert or in opposition to independently regulate cap-dependent translation. We suggest that independent regulation of eIF-4E and BP-1 might finely regulate the efficiency of translation initiation or possibly control cap-dependent translation for fundamentally different purposes.

Adaptor Proteins, Signal Transducing↗

PubMed2000

Transcription-coupled translation control of AML1/RUNX1 is mediated by cap- and internal ribosome entry site-dependent mechanisms.

AML1/RUNX1 belongs to the runt domain transcription factors that are important regulators of hematopoiesis and osteogenesis. Expression of AML1 is regulated at the level of transcription by two promoters, distal (D) and proximal (P), that give rise to mRNAs bearing two distinct 5' untranslated regions (5'UTRs) (D-UTR and P-UTR). Here we show that these 5'UTRs act as translation regulators in vivo. AML1 mRNAs bearing the uncommonly long (1,631-bp) P-UTR are poorly translated, whereas those with the shorter (452-bp) D-UTR are readily translated. The low translational efficiency of the P-UTR is attributed to its length and the cis-acting elements along it. Transfections and in vitro assays with bicistronic constructs demonstrate that the D-UTR mediates cap-dependent translation whereas the P-UTR mediates cap-independent translation and contains a functional internal ribosome entry site (IRES). The IRES-containing bicistronic constructs are more active in hematopoietic cell lines that normally express the P-UTR-containing mRNAs. Furthermore, we show that the IRES-dependent translation increases during megakaryocytic differentiation but not during erythroid differentiation, of K562 cells. These results strongly suggest that the function of the P-UTR IRES-dependent translation in vivo is to tightly regulate the translation of AML1 mRNAs. The data show that AML1 expression is regulated through usage of alternative promoters coupled with IRES-mediated translation control. This IRES-mediated translation regulation adds an important new dimension to the fine-tuned control of AML1 expression.

5' Untranslated Regions↗

PubMed1999

Complex spike activity of Purkinje cells in the ventral uvula and nodulus of pigeons in response to translational optic flow.

The complex spike (CS) activity of Purkinje cells in the ventral uvula and nodulus of the vestibulocerebellum was recorded from anesthetized pigeons in response to translational optic flow. Translational optic flow was produced using a "translator" projector: a mechanical device that projected a translational optic flowfield onto the walls, ceiling, and floor of the room and encompassed the entire binocular visual field. CS activity was broadly tuned but maximally modulated in response to translational optic flow along a "best" axis. Each neuron was assigned a vector representing the direction in which the animal would need to translate to produce the optic flowfield that resulted in maximal excitation. The vector is described with reference to a standard right-handed coordinate system, where the vectors, +x, +y, and +z represent, rightward, upward, and forward translation of the animal, respectively. Neurons could be grouped into four response types based on the vector of maximal excitation. +y neurons were modulated maximally in response to a translational optic flowfield that results from self-motion upward along the vertical (y) axis. -y neurons also responded best to translational optic flow along the vertical axis but showed the opposite direction preference. The two remaining groups responded best to translational optic flow along horizontal axes: -x + z neurons and -x-z neurons. In summary, our results suggest that the olivocerebellar system dedicated to the analysis of translational optic flow is organized according to a reference frame consisting of three approximately orthogonal axes: the vertical axis, and two horizontal axes oriented 45 degrees to either side the midline. Previous research has shown that the rotational optic flow system, the eye muscles, the vestibular semicircular canals and the postural control system all share a similar spatial frame of reference.

Animals↗

PubMed2007

Phylogenetic distribution of translational GTPases in bacteria.

BACKGROUND: Translational GTPases are a family of proteins in which GTPase activity is stimulated by the large ribosomal subunit. Conserved sequence features allow members of this family to be identified. RESULTS: To achieve accurate protein identification and grouping we have developed a method combining searches with Hidden Markov Model profiles and tree based grouping. We found all the genes for translational GTPases in 191 fully sequenced bacterial genomes. The protein sequences were grouped into nine subfamilies. Analysis of the results shows that three translational GTPases, the translation factors EF-Tu, EF-G and IF2, are present in all organisms examined. In addition, several copies of the genes encoding EF-Tu and EF-G are present in some genomes. In the case of multiple genes for EF-Tu, the gene copies are nearly identical; in the case of multiple EF-G genes, the gene copies have been considerably diverged. The fourth translational GTPase, LepA, the function of which is currently unknown, is also nearly universally conserved in bacteria, being absent from only one organism out of the 191 analyzed. The translation regulator, TypA, is also present in most of the organisms examined, being absent only from bacteria with small genomes.Surprisingly, some of the well studied translational GTPases are present only in a very small number of bacteria. The translation termination factor RF3 is absent from many groups of bacteria with both small and large genomes. The specialized translation factor for selenocysteine incorporation--SelB--was found in only 39 organisms. Similarly, the tetracycline resistance proteins (Tet) are present only in a small number of species. Proteins of the CysN/NodQ subfamily have acquired functions in sulfur metabolism and production of signaling molecules. The genes coding for CysN/NodQ proteins were found in 74 genomes. This protein subfamily is not confined to Proteobacteria, as suggested previously but present also in many other groups of bacteria. CONCLUSION: Four of the translational GTPase subfamilies (IF2, EF-Tu, EF-G and LepA) are represented by at least one member in each bacterium studied, with one exception in LepA. This defines the set of translational GTPases essential for basic cell functions.

Amino Acid Sequence↗

PubMed2000

Control of oskar mRNA translation by Bruno in a novel cell-free system from Drosophila ovaries.

The coupled regulation of oskar mRNA localization and translation in time and space is critical for correct anteroposterior patterning of the Drosophila embryo. Localization-dependent translation of oskar mRNA, a mechanism whereby oskar RNA localized at the posterior of the oocyte is selectively translated and the unlocalized RNA remains in a translationally repressed state, ensures that Oskar activity is present exclusively at the posterior pole. Genetic experiments indicate that translational repression involves the binding of Bruno protein to multiple sites, the Bruno Response Elements (BRE), in the 3' untranslated region (UTR) of oskar mRNA. We have established a cell-free translation system derived from Drosophila ovaries, which faithfully reproduces critical features of mRNA translation in vivo, namely cap structure and poly(A) tail dependence. We show that this ovary extract, containing endogenous Bruno, is able to recapitulate oskar mRNA regulation in a BRE-dependent way. Thus, the assembly of a ribonucleoprotein (RNP) complex leading to the translationally repressed state occurs in vitro. Moreover, we show that a Drosophila embryo extract lacking Bruno efficiently translates oskar mRNA. Addition of recombinant Bruno to this extract establishes the repressed state in a BRE-dependent manner, providing a direct biochemical demonstration of the critical role of Bruno in oskar mRNA translation. The approach that we describe opens new avenues to investigate translational regulation in Drosophila oogenesis at a biochemical level.

Animals↗

PubMed2006

Control of mammalian translation by mRNA structure near caps.

The scanning model of RNA translation proposes that highly stable secondary structures within mRNAs can inhibit translation, while structures of lower thermal stability also affect translation if close enough to the 5' methyl G cap. However, only fragmentary information is available about the dependence of translation efficiency in live mammalian cells on the thermodynamic stability, location, and GC content of RNA structures in the 5'-untranslated region. We devised a two-color fluorescence assay for translation efficiency in single live cells and compared a wide range of hairpins with predicted thermal stabilities ranging from -10 to -50 kcal/mol and 5' G cap-to-hairpin distances of 1-46 bases. Translation efficiency decreased abruptly as hairpin stabilities increased from deltaG = -25 to -35 kcal/mol. Shifting a hairpin as little as nine bases relative to the 5' cap could modulate translation more than 50-fold. Increasing GC content diminished translation efficiency when predicted thermal stability and cap-to-hairpin distances were held constant. We additionally found naturally occurring 5'-untranslated regions affected translation differently in live cells compared with translation in in vitro lysates. Our study will assist scientists in designing experiments that deliberately modulate mammalian translation with designed 5' UTRs.

Animals↗

PubMed1999

Translation to Spanish, reproducibility, and cross-cultural adaptation of the Miller-Rahe Recent Life Change Questionnaire in Venezuela.

OBJECTIVE: To translate into Spanish a version of the Miller-Rahe Recent Life Change Questionnaire and to adapt it to Venezuelan cultural values. METHODS: The Spanish version and cross-cultural adaptation of the Miller-Rahe Recent Life Change Questionnaire was done following recently proposed guidelines to preserve semantic, idiomatic, and conceptual equivalence in translations of health assessment instruments. We performed one or more translations into the new language, as well as back-translation, test-retest reliability, and weighting score for the translated instrument. RESULTS: A Spanish version of the Recent Life Change Questionnaire was obtained. Validity of translations was demonstrated with a significant agreement for the nonliteral translations (kappa value = 0.97, P < 0.05). The conceptual equivalence was demonstrated by significant agreement in the back-translations (kappa value = 0.84, P < 0.05). The translated instrument met acceptable levels of reliability, as assessed by Spearman's rank correlation coefficients (> 0.60 for all categories of the questionnaire). The cross-cultural adaptation of the translated instrument required addition and exclusion of items as well as changes of the ranking and scaling of life units in the original questionnaire. CONCLUSION: A valid and reliable Spanish version of the Miller-Rahe Recent Life Change Questionnaire was produced and adapted to Venezuelan cultural values.

Adult↗

PubMed1996

Translational regulation of parathyroid hormone gene expression and RNA: protein interactions.

The aim of this study was to investigate the mechanism by which translation of parathyroid hormone (PTH) mRNA is regulated with regard to the subcellular distribution of PTH mRNA and RNA:protein interactions. Sucrose density ultracentrifugation of RNA from bovine parathyroid cells indicated that there was no evidence for a pool of nonribosomal PTH mRNA, and the extracellular calcium concentration had no effect on polysome size. UV cross-linking studies revealed two proteins in parathyroid cell cytosol which bound specifically to the 5'-untranslated region (UTR) of PTH mRNA with molecular masses of 66 and 68 kD while proteins with apparent molecular masses of 48 and 70 kD bound to the 3'-UTR. In vitro translation assays indicated that parathyroid cell cytosol contains factors that inhibit translation of PTH mRNA. Fractionation of cytosol revealed that this effect was associated with proteins within the molecular mass range 30-90 kD. To determine which sequences in PTH mRNA mediate translational regulation, RNA was synthesized from luciferase gene constructs containing the 5'- and/or 3'-UTR of PTH mRNA, and translated in vitro. Addition of parathyroid cell cytosol reduced the translation of RNA containing the 5'- and 3'-UTR of PTH mRNA by 44 +/- 7% but had no effect on the translation of RNA containing only the luciferase coding region. Translation of RNA containing only the 5'-UTR of PTH mRNA was unchanged; however, cytosol reduced the translation of RNA containing the 3'-UTR by 31 +/- 9%. These data demonstrate a role for RNA:protein interactions in the regulation of PTH synthesis and that translational control is mediated primarily through interactions with the 3'-UTR of PTH mRNA.

Animals↗

PubMed2006

Reading for repetition and reading for translation: do they involve the same processes?

UNLABELLED: Theories of translation differ in the role assigned to the reformulation process. One view, the "horizontal" approach, considers that translation involves on-line searches for matches between linguistic entries in the two languages involved [Gerver, D. (1976). Empirical studies of simultaneous interpretation: A review and a model. In R. W. Brislin (Ed.), Translation: Applications and research (pp. 165-207). New York: Gardiner]. The second view, the "vertical" approach, assumes that on-line reformulation does not take place while reading: translation involves giving lexical expression to the meaning extracted after comprehension [Seleskovitch, D. (1976). INTERPRETATION: A psychological approach to translating. In R.W. Brislin (Ed.), Translation: Applications and research (pp. 92-116). New York: Gardner]. In four experiments, translators or bilinguals read sentences for repetition or for translation. When participants read for translation, on-line and global comprehension was affected by lexical ambiguity and memory load (Experiment 1a and 1b). Furthermore, cognate words located at the final portion of the sentences facilitated performance (Experiment 2a and 2b). However, when participants were asked to understand and repeat the sentences, lexical ambiguity and the cognate status of the words did not have any effect. This pattern of results provides support for horizontal theories of translation.

Analysis of Variance↗

PubMed2026

Acetic acid-induced translational repression involves eIF2B body formation and Ded1 sequestration into stress granules in yeast.

Elucidating the physiological impact of acetic acid stress and the corresponding yeast responses is essential for advancing fundamental biology and improving industrial alcoholic fermentation. Despite numerous genome-wide studies, information on the effects of acetic acid stress on yeast translational regulation remains limited. We found that a sublethal concentration of acetic acid (35 mM, 0.2% v/v) causes translational repression, accompanied by the formation of eIF2B bodies and the phosphorylation of eIF2α, both of which are involved in the regulation of translation initiation. Acetic acid also caused the sequestration of Ded1, a DEAD-box RNA helicase crucial for translation initiation, into stress granules. Removal of acetic acid restored translational activity and the proper localization of eIF2B and Ded1, indicating the reversibility of acetic acid-induced translational repression. Furthermore, when yeast cells were pretreated with 0.05% acetic acid, translational repression under subsequent 0.2% acetic acid stress was attenuated in wild-type cells but not in hrk1Δ cells. This indicates that Hrk1, a Pma1 activator, is required to sufficiently enhance tolerance to acetic acid-induced translational repression. These findings provide novel insights into the physiological effects of acetic acid stress on translational activity and translation-related factors in yeast cells.

Saccharomyces cerevisiae↗

PubMed1999

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.

5' Untranslated Regions↗

PubMed1998

Light regulates the rate of translation elongation of chloroplast reaction center protein D1.

Intact and lysed chloroplasts isolated from the day or night phase of seedling growth exhibit a higher rate of [35S]Met incorporation into the D1 protein in the light than in darkness. In the presence of the translation initiation inhibitor lincomycin, radiolabel incorporation remains unaffected for 7.5-10 min of the in vitro translation reaction, indicating that radiolabel incorporation is regulated by translation elongation. The rate of [35S]Met incorporation into D1-protein can be increased by addition of exogenous ATP to the in vitro translation reactions; however, ATP cannot replace light, and at physiological concentrations of stromal ATP (40 microM), the rate is at least 25-fold higher in the light than in darkness. This indicates that translation elongation is arrested in darkness. Separation of translation-elongation reactions into polysome-bound and membrane-integrated D1 proteins demonstrates that the rate of translation elongation is higher in the presence of light. In the light, less time is required to transiently radiolabel a D1 translation intermediate of about 17 kDa and to chase the translation intermediate into mature D1 protein. We propose that light regulates the enzymatic activity of the translation-elongation process in chloroplasts.

Chloroplasts↗

PubMed1999

Reliability and validity of French, German, Italian, Dutch, and UK English translations of the Medical Outcomes Study HIV Health Survey.

OBJECTIVES: Test the reliability and validity of 5 translations of the 34-item version of the MOS HIV for use in multinational clinical trials. RESEARCH DESIGN: Investigators in five countries followed a standardized protocol and recruited HIV+ patients stratified by disease stage: asymptomatic; symptomatic; and AIDS. During routine clinic visits, patients completed the MOS HIV and a checklist of HIV-related symptoms. Clinicians reported patients' demographics, most recent CD4+ count and disease stage. SUBJECTS: Three hundred and sixty three HIV+ outpatients attending AIDS clinics in The Netherlands, France, Germany, Italy, and England. MEASURES: Dutch, French, German, Italian, and UK English translations of the MOS HIV CD4+ cell count and the SCL-57. RESULTS: All translations recruited roughly equal proportions of each disease stage, although the number of patients recruited differed by translation (n: German = 92, French = 86; Italian = 88; UK English = 72; and Dutch = 25). Internal consistency reliability was similar across translations and adequate (alpha >.70) for all scales except for Mental Health in the French sample. Multi-trait analyses supported structural validity of the MOS HIV scales in each translation. Principal component analysis of scale scores identified 2 dimensions for all translations except German. For all translations, scores were significantly correlated with symptom severity scores but were uncorrelated with CD4+ cell counts. CONCLUSIONS: In general, the 5 translations of the MOS HIV had similar psychometric properties to those reported in the validation study for the original US English version of the MOS HIV. With some revision, these translations promise to provide useful quality of life data from HIV+ subjects in clinical trials.

Activities of Daily Living↗

PubMed2003

Patterns, mechanisms, and functions of translation regulation in mammalian spermatogenic cells.

Translational regulation is a fundamental aspect of the atypical patterns of gene expression in mammalian meiotic and haploid spermatogenic cells. Every mRNA is at least partially translationally repressed in meiotic and haploid spermatogenic cells, but the extent of repression of individual mRNA species is regulated individually and varies greatly. Many mRNA species, such as protamine mRNAs, are stored in translationally repressed free-mRNPs in early haploid cells and translated actively in late haploid cells. However, translation does not regulate developmental expression of all mRNAs. Some mRNAs appear to be partially repressed for the entire period that the mRNA is expressed in meiotic and haploid cells, while other mRNAs, some of which are expressed at high levels, are almost totally inactivated in free-mRNPs and/or generate little or no protein. This distinctive phenomenon can be explained by the hypothesis that translational repression is used to prevent the potentionally deleterious effects of overproduction of proteins encoded by overexpressed mRNAs. Translational regulation also appears to be frequently altered by the widespread usage of alternative transcription start sites in spermatogenic cells. Many ubiquitously expressed genes generate novel transcripts in somatic spermatogenic cells containing elements, uORFs and secondary structure that are inhibitory to mRNA translation, while the ribosomal proten L32 mRNA lacks a repressive element that is present in somatic cells. Very little is known about the mechanisms that regulate mRNA translation in spermatogenic cells, largely because few labs have utilized in vivo genetic approaches, although there have been important insights into the repression and activation of protamine 1 mRNA, and the role of Y-box proteins and poly(A) lengthening in mRNA-specific translational activation mediated by the cytoplasmic poly(A) element binding protein and a testis-specific isoform of poly(A) polymerase. A very large literature by evolutionary biologists suggests that the atypical patterns of gene expression in spermatogenic cells are the consequence of the powerful and unusual selective pressures on male reproductive success.

3' Untranslated Regions↗

PubMedDate not supplied

Instrument translation and evaluation of equivalence and psychometric properties: the Chinese Sense of Coherence Scale.

Translating well-established English instruments into target languages other than English and testing cross-cultural validity to prove that the same attributes are being measured in each cultural group or country are required before a multicultural or international study can be conducted and cross-cultural comparisons of study results can be applied. However, rigorous and systematic cross-cultural efforts to test the effectiveness of specific translation methods are rare. This article presents a model of translation processes and empirical validation of the translated instrument through description of the translation of a selected instrument--the Sense of Coherence (SOC) Scale--from English into Chinese. A cyclic process of forward translations, back translations, and expert evaluation of equivalence by bilingual and English speaking experts was conducted to achieve conceptual equivalence between the original and translated instruments. Empirical validation of the Chinese SOC scale data from a group of bilingual Chinese people demonstrated non-identical, but comparable, item and scale means and variances, internal consistency, and relationships to an external criterion. Advantages and difficulties of using multi-rounds, multi-experts, and multi-methods to establish translation equivalence and to validate the translated Chinese SOC scale are discussed.

Adaptation, Psychological↗

PubMed1995

Translation initiation and assembly of peripherin in cultured cells.

The peripherin gene has three potential ATG translation initiation sites at positions 38, 56, and 290. The second ATG has been proposed to be the initiation codon used for translation of the protein, but there is no experimental evidence for this conjecture. We have isolated a full-length peripherin cDNA (designated as p61-11) from a rat brain cDNA library. Upon sequencing, we found that this cDNA contains a point mutation at the second potential translation initiation codon, which changes this ATG to ACG. When expressed in SW13 cl.2 vim- cells, a cell line without any detectable cytoplasmic intermediate filaments, the protein product of p61-11 cannot form a filamentous network and the major product is 45 kDa in size, which is most likely initiated from the third ATG. The protein product from the first ATG (57 kDa in size) of p61-11 is also detected albeit in smaller amounts. We introduced a frame-shift mutation upstream of the third ATG in p61-11 to create p61-11FS and showed that the third ATG is able to initiate translation efficiently even in the presence of the first ATG, and the 45 kDa protein leads to a diffuse nonfilamentous staining pattern in vim- cells confirming that the first ATG may not be the preferred translation initiation codon, since it cannot suppress a downstream ATG. We increased the translation efficiency from the first ATG of p61-11 by mutating the three nucleotides preceding this first ATG and thereby placing it in a better Kozak consensus sequence for translation initiation. The resulting 57 kDa protein is able to form a filamentous network in vim- cells. We corrected the mutation in the original p61-11 by polymerase chain reaction and generated two peripherin constructs: perM1M2 (which contains all three translation initiation codons) and per delta 1M2 (the first ATG is deleted, but the other two are present). When transfected, their protein products, about 57 kDa in size, form filamentous networks in the absence of other cytoplasmic intermediate filaments. Since there is no 45 kDa protein detected for these latter two constructs, it is reasonable to conclude that in the presence of the second ATG, little or no translation is initiated from the third ATG. Taken together, these results strongly suggest that the second ATG is the preferred translation initiation codon for the peripherin gene.

Adenocarcinoma↗

PubMed2005

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.

DNA-Binding Proteins↗

PubMed2025

Effects of light on chloroplast translation in Marchantia polymorpha are similar to those in angiosperms and are not influenced by light-independent chlorophyll synthesis.

Translation of the chloroplast psbA mRNA in angiosperms is activated by photodamage of its gene product, the D1 subunit of photosystem II (PSII), providing nascent D1 for PSII repair. The involvement of chlorophyll in the regulatory mechanism has been suggested due to the regulatory roles of proteins proposed to mediate chlorophyll/D1 transactions and the fact that chlorophyll is synthesized only in the light in angiosperms. We used ribosome profiling and RNA-seq to address whether the effects of light on chloroplast translation are conserved in the liverwort Marchantia (Marchantia polymorpha), which synthesizes chlorophyll in both the dark and the light. As in angiosperms, ribosome occupancy on psbA mRNA decreased rapidly upon shifting plants to the dark and was rapidly restored upon a transfer back to the light, whereas ribosome occupancy on other chloroplast mRNAs changed very little. The results were similar in a Marchantia mutant unable to synthesize chlorophyll in the dark. Those results, in conjunction with pulse-labeling data, suggest that light elicits a plastome-wide activation of translation elongation and a specific increase in psbA translation initiation in Marchantia, as in angiosperms. These findings show that light regulates chloroplast translation similarly in vascular and non-vascular plants, and that constitutive chlorophyll synthesis does not affect light-regulated psbA translation initiation. Additionally, the translational outputs of chloroplast genes are similar in Marchantia and angiosperms but result from differing contributions of mRNA abundance and translational efficiencies. This adds to the evidence that chloroplast mRNA abundance and translational efficiencies co-evolve under selection to maintain protein outputs.

Chloroplasts↗