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1.  Cell-type specific analysis of translating RNAs in developing flowers reveals new levels of control 
Combining translating ribosome affinity purification with RNA-seq for cell-specific profiling of translating RNAs in developing flowers.Cell type comparisons of cell type-specific hormone responses, promoter motifs, coexpressed cognate binding factor candidates, and splicing isoforms.Widespread post-transcriptional regulation at both the intron splicing and translational stages.A new class of noncoding RNAs associated with polysomes.
What constitutes a differentiated cell type? How much do cell types differ in their transcription of genes? The development and functions of tissues rely on constant interactions among distinct and nonequivalent cell types. Answering these questions will require quantitative information on transcriptomes, proteomes, protein–protein interactions, protein–nucleic acid interactions, and metabolomes at cellular resolution. The systems approaches emerging in biology promise to explain properties of biological systems based on genome-wide measurements of expression, interaction, regulation, and metabolism. To facilitate a systems approach, it is essential first to capture such components in a global manner, ideally at cellular resolution.
Recently, microarray analysis of transcriptomes has been extended to a cellular level of resolution by using laser microdissection or fluorescence-activated sorting (for review, see Nelson et al, 2008). These methods have been limited by stresses associated with cellular separation and isolation procedures, and biases associated with mandatory RNA amplification steps. A newly developed method, translating ribosome affinity purification (TRAP; Zanetti et al, 2005; Heiman et al, 2008; Mustroph et al, 2009), circumvents these problems by epitopetagging a ribosomal protein in specific cellular domains to selectively purify polysomes. We combined TRAP with deep sequencing, which we term TRAP-seq, to provide cell-level spatiotemporal maps for Arabidopsis early floral development at single-base resolution.
Flower development in Arabidopsis has been studied extensively and is one of the best understood aspects of plant development (for review, see Krizek and Fletcher, 2005). Genetic analysis of homeotic mutants established the ABC model, in which three classes of regulatory genes, A, B and C, work in a combinatorial manner to confer organ identities of four whorls (Coen and Meyerowitz, 1991). Each class of regulatory gene is expressed in a specific and evolutionarily conserved domain, and the action of the class A, B and C genes is necessary for specification of organ identity (Figure 1A).
Using TRAP-seq, we purified cell-specific translating mRNA populations, which we and others call the translatome, from the A, B and C domains of early developing flowers, in which floral patterning and the specification of floral organs is established. To achieve temporal specificity, we used a floral induction system to facilitate collection of early stage flowers (Wellmer et al, 2006). The combination of TRAP-seq with domain-specific promoters and this floral induction system enabled fine spatiotemporal isolation of translating mRNA in specific cellular domains, and at specific developmental stages.
Multiple lines of evidence confirmed the specificity of this approach, including detecting the expression in expected domains but not in other domains for well-studied flower marker genes and known physiological functions (Figures 1B–D and 2A–C). Furthermore, we provide numerous examples from flower development in which a spatiotemporal map of rigorously comparable cell-specific translatomes makes possible new views of the properties of cell domains not evident in data obtained from whole organs or tissues, including patterns of transcription and cis-regulation, new physiological differences among cell domains and between flower stages, putative hormone-active centers, and splicing events specific for flower domains (Figure 2A–D). Such findings may provide new targets for reverse genetics studies and may aid in the formulation and validation of interaction and pathway networks.
Beside cellular heterogeneity, the transcriptome is regulated at several steps through the life of mRNA molecules, which are not directly available through traditional transcriptome profiling of total mRNA abundance. By comparing the translatome and transcriptome, we integratively profiled two key posttranscriptional control points, intron splicing and translation state. From our translatome-wide profiling, we (i) confirmed that both posttranscriptional regulation control points were used by a large portion of the transcriptome; (ii) identified a number of cis-acting features within the coding or noncoding sequences that correlate with splicing or translation state; and (iii) revealed correlation between each regulation mechanism and gene function. Our transcriptome-wide surveys have highlighted target genes transcripts of which are probably under extensive posttranscriptional regulation during flower development.
Finally, we reported the finding of a large number of polysome-associated ncRNAs. About one-third of all annotated ncRNA in the Arabidopsis genome were observed co-purified with polysomes. Coding capacity analysis confirmed that most of them are real ncRNA without conserved ORFs. The group of polysome-associated ncRNA reported in this study is a potential new addition to the expanding riboregulator catalog; they could have roles in translational regulation during early flower development.
Determining both the expression levels of mRNA and the regulation of its translation is important in understanding specialized cell functions. In this study, we describe both the expression profiles of cells within spatiotemporal domains of the Arabidopsis thaliana flower and the post-transcriptional regulation of these mRNAs, at nucleotide resolution. We express a tagged ribosomal protein under the promoters of three master regulators of flower development. By precipitating tagged polysomes, we isolated cell type-specific mRNAs that are probably translating, and quantified those mRNAs through deep sequencing. Cell type comparisons identified known cell-specific transcripts and uncovered many new ones, from which we inferred cell type-specific hormone responses, promoter motifs and coexpressed cognate binding factor candidates, and splicing isoforms. By comparing translating mRNAs with steady-state overall transcripts, we found evidence for widespread post-transcriptional regulation at both the intron splicing and translational stages. Sequence analyses identified structural features associated with each step. Finally, we identified a new class of noncoding RNAs associated with polysomes. Findings from our profiling lead to new hypotheses in the understanding of flower development.
PMCID: PMC2990639  PMID: 20924354
Arabidopsis; flower; intron; transcriptome; translation
2.  Stress-Dependent Coordination of Transcriptome and Translatome in Yeast 
PLoS Biology  2009;7(5):e1000105.
Cells rapidly alter gene expression in response to environmental stimuli such as nutrients, hormones, and drugs. During the imposed “remodeling” of gene expression, changes in the levels of particular mRNAs do not necessarily correlate with those of the encoded proteins, which could in part rely on the differential recruitment of mRNAs to translating ribosomes. To systematically address this issue, we have established an approach to rapidly access the translational status of each mRNA in the yeast Saccharomyces cerevisiae by affinity purification of endogenously formed ribosomes and the analysis of associated mRNAs with DNA microarrays. Using this method, we compared changes in total mRNA levels (transcriptome) with ribosome associations (translatome) after the application of different conditions of cellular stress. Severe stresses, induced by amino acid depletion or osmotic shock, stimulated highly correlated responses affecting about 15% of both total RNA levels and translatome. Many of the regulated messages code for functionally related proteins, thus reflecting logical responses to the particular stress. In contrast, mild stress provoked by addition of Calcofluor-white and menadione altered the translatome of approximately 1% of messages with only marginal effects on total mRNA, suggesting largely uncorrelated responses of transcriptome and translatome. Among these putative translationally regulated messages were most components of the mitochondrial ATPase. Increased polysome associations of corresponding messages and higher mitochondrial ATPase activities upon treatment confirmed the relevance for regulation of this macromolecular complex. Our results suggest the presence of highly sensitive translational regulatory networks that coordinate functionally related messages. These networks are preferentially activated for rapid adaptation of cells to minor environmental perturbations.
Author Summary
Organisms respond to environmental or physiological changes by altering the amounts and activities of specific proteins that are necessary for their adaptation and survival. Importantly, protein levels can be modulated by changing either the rate of synthesis or the stability of the messenger RNA (mRNA or transcript), or the synthesis or stability of the protein itself. Scientists often measure global mRNA levels upon changing conditions to identify transcripts that are differentially regulated, and often the assumption is made that changes in transcript levels lead to corresponding changes in protein levels. Here, we systematically compared global transcript levels (transcriptome) with global alterations in the levels of ribosome association of transcripts (translatome) when yeast cells are exposed to different stresses to determine how significant the discrepancy between transcript and protein levels can be. We found that changes in the transcriptome correlate well with those in the translatome after application of harsh stresses that arrest cell growth. However, this correlation is generally lost under more mild stresses that do not affect cell growth. In this case, remodeling of gene expression is mainly executed at the translational level by modulating mRNA association with ribosomes. As one example, we show that expression for many components of the mitochondrial ATPase, the major energy production machinery in cells, is translationally but not transcriptionally activated under a specific mild stress condition. Our results therefore show that alteration of protein synthesis can be the dominant mediator of changes of gene expression during adaptation to minor changes in cellular needs.
During cellular adaptation to changing growth conditions, the extent of correlation between changes in transcriptional and translational regulation varies with the severity of the stress.
PMCID: PMC2675909  PMID: 19419242
3.  The HOG Pathway Dictates the Short-Term Translational Response after Hyperosmotic Shock 
Molecular Biology of the Cell  2010;21(17):3080-3092.
In the global osmoshock translational response in yeast, some gene products were translationally mobilized without transcriptional up-regulation. Conversely, other transcriptionally up-regulated mRNAs were translationally inhibited. Analogous changes occurred on the protein level. These translational responses were strongly dependent on Hog1 and Rck2.
Cellular responses to environmental changes occur on different levels. We investigated the translational response of yeast cells after mild hyperosmotic shock by isolating mRNA associated with multiple ribosomes (polysomes) followed by array analysis. Globally, recruitment of preexisting mRNAs to ribosomes (translational response) is faster than the transcriptional response. Specific functional groups of mRNAs are recruited to ribosomes without any corresponding increase in total mRNA. Among mRNAs under strong translational up-regulation upon shock, transcripts encoding membrane-bound proteins including hexose transporters were enriched. Similarly, numerous mRNAs encoding cytoplasmic ribosomal proteins run counter to the overall trend of down-regulation and are instead translationally mobilized late in the response. Surprisingly, certain transcriptionally induced mRNAs were excluded from ribosomal association after shock. Importantly, we verify, using constructs with intact 5′ and 3′ untranslated regions, that the observed changes in polysomal mRNA are reflected in protein levels, including cases with only translational up-regulation. Interestingly, the translational regulation of the most highly osmostress-regulated mRNAs was more strongly dependent on the stress-activated protein kinases Hog1 and Rck2 than the transcriptional regulation. Our results show the importance of translational control for fine tuning of the adaptive responses.
PMCID: PMC2930000  PMID: 20587780
4.  Translational Regulation of Specific mRNAs Controls Feedback Inhibition and Survival during Macrophage Activation 
PLoS Genetics  2014;10(6):e1004368.
For a rapid induction and efficient resolution of the inflammatory response, gene expression in cells of the immune system is tightly regulated at the transcriptional and post-transcriptional level. The control of mRNA translation has emerged as an important determinant of protein levels, yet its role in macrophage activation is not well understood. We systematically analyzed the contribution of translational regulation to the early phase of the macrophage response by polysome fractionation from mouse macrophages stimulated with lipopolysaccharide (LPS). Individual mRNAs whose translation is specifically regulated during macrophage activation were identified by microarray analysis. Stimulation with LPS for 1 h caused translational activation of many feedback inhibitors of the inflammatory response including NF-κB inhibitors (Nfkbid, Nfkbiz, Nr4a1, Ier3), a p38 MAPK antagonist (Dusp1) and post-transcriptional suppressors of cytokine expression (Zfp36 and Zc3h12a). Our analysis showed that their translation is repressed in resting and de-repressed in activated macrophages. Quantification of mRNA levels at a high temporal resolution by RNASeq allowed us to define groups with different expression patterns. Thereby, we were able to distinguish mRNAs whose translation is actively regulated from mRNAs whose polysomal shifts are due to changes in mRNA levels. Active up-regulation of translation was associated with a higher content in AU-rich elements (AREs). For one example, Ier3 mRNA, we show that repression in resting cells as well as de-repression after stimulation depends on the ARE. Bone-marrow derived macrophages from Ier3 knockout mice showed reduced survival upon activation, indicating that IER3 induction protects macrophages from LPS-induced cell death. Taken together, our analysis reveals that translational control during macrophage activation is important for cellular survival as well as the expression of anti-inflammatory feedback inhibitors that promote the resolution of inflammation.
Author Summary
When macrophages encounter pathogens, they initiate inflammation by secreting pro-inflammatory factors such as the cytokine TNF. Because a prolonged or overshooting release of these factors is harmful for the organism, their production needs to be tightly controlled and shut off in due time. To ensure a rapid but transient inflammatory response, gene expression is regulated at multiple levels, including transcription, stability and translation of mRNAs. While control of transcription and mRNA stability has been studied extensively, little is known about translational regulation in macrophages. In this study, we measured the translation of all mRNAs expressed in mouse macrophages. Upon activation of macrophages with the bacterial cell wall component lipopolysaccharide, we found that many feedback inhibitors, which are important for dampening the inflammatory response, are translationally up-regulated. Translation of these mRNAs is repressed in resting cells and de-repressed after stimulation. In contrast to feedback inhibitors, most cytokines are primarily regulated by changes in mRNA abundance. Furthermore, we could show that one of the feedback inhibitors, IER3, protects macrophages from cell death during activation. Therefore, regulation at the level of translation is important for the induction of negative feedback loops and cellular survival.
PMCID: PMC4063670  PMID: 24945926
5.  Pichia pastoris regulates its gene-specific response to different carbon sources at the transcriptional, rather than the translational, level 
BMC Genomics  2015;16(1):167.
The methylotrophic, Crabtree-negative yeast Pichia pastoris is widely used as a heterologous protein production host. Strong inducible promoters derived from methanol utilization genes or constitutive glycolytic promoters are typically used to drive gene expression. Notably, genes involved in methanol utilization are not only repressed by the presence of glucose, but also by glycerol. This unusual regulatory behavior prompted us to study the regulation of carbon substrate utilization in different bioprocess conditions on a genome wide scale.
We performed microarray analysis on the total mRNA population as well as mRNA that had been fractionated according to ribosome occupancy. Translationally quiescent mRNAs were defined as being associated with single ribosomes (monosomes) and highly-translated mRNAs with multiple ribosomes (polysomes). We found that despite their lower growth rates, global translation was most active in methanol-grown P. pastoris cells, followed by excess glycerol- or glucose-grown cells. Transcript-specific translational responses were found to be minimal, while extensive transcriptional regulation was observed for cells grown on different carbon sources. Due to their respiratory metabolism, cells grown in excess glucose or glycerol had very similar expression profiles. Genes subject to glucose repression were mainly involved in the metabolism of alternative carbon sources including the control of glycerol uptake and metabolism. Peroxisomal and methanol utilization genes were confirmed to be subject to carbon substrate repression in excess glucose or glycerol, but were found to be strongly de-repressed in limiting glucose-conditions (as are often applied in fed batch cultivations) in addition to induction by methanol.
P. pastoris cells grown in excess glycerol or glucose have similar transcript profiles in contrast to S. cerevisiae cells, in which the transcriptional response to these carbon sources is very different. The main response to different growth conditions in P. pastoris is transcriptional; translational regulation was not transcript-specific. The high proportion of mRNAs associated with polysomes in methanol-grown cells is a major finding of this study; it reveals that high productivity during methanol induction is directly linked to the growth condition and not only to promoter strength.
Electronic supplementary material
The online version of this article (doi:10.1186/s12864-015-1393-8) contains supplementary material, which is available to authorized users.
PMCID: PMC4408588  PMID: 25887254
Pichia pastoris; Methylotrophic yeast; Crabtree-negative yeast; Polysome profiling; Microarray analysis; Transcriptome; Glucose repression; Carbon substrate repression; Methanol induction
6.  Polysome profiling reveals broad translatome remodeling during endoplasmic reticulum (ER) stress in the pathogenic fungus Aspergillus fumigatus 
BMC Genomics  2014;15:159.
The unfolded protein response (UPR) is a network of intracellular signaling pathways that supports the ability of the secretory pathway to maintain a balance between the load of proteins entering the endoplasmic reticulum (ER) and the protein folding capacity of the ER lumen. Current evidence indicates that several pathogenic fungi rely heavily on this pathway for virulence, but there is limited understanding of the mechanisms involved. The best known functional output of the UPR is transcriptional upregulation of mRNAs involved in ER homeostasis. However, this does not take into account mechanisms of translational regulation that involve differential loading of ribosomes onto mRNAs. In this study, a global analysis of transcript-specific translational regulation was performed in the pathogenic mold Aspergillus fumigatus to determine the nature and scope of the translational response to ER stress.
ER stress was induced by treating the fungus with dithiothreitol, tunicamycin, or a thermal up-shift. The mRNAs were then fractionated on the basis of ribosome occupancy into an under-translated pool (U) and a well-translated pool (W). The mRNAs were used to interrogate microarrays and the ratio of the hybridization signal (W/U) was used as an indicator of the relative translational efficiency of a mRNA under each condition. The largest category of translationally upregulated mRNAs during ER stress encoded proteins involved in translation. Components of the ergosterol and GPI anchor biosynthetic pathways also showed increased polysome association, suggesting an important role for translational regulation in membrane and cell wall homeostasis. ER stress induced limited remodeling of the secretory pathway translatome. However, a select group of transcription factors was translationally upregulated, providing a link to subsequent modification of the transcriptome. Finally, we provide evidence that one component of the ER stress translatome is a novel mRNA isoform from the yvc1 gene that is induced by ER stress in a UPR-dependent manner.
Together, these findings define a core set of mRNAs subject to translational control during the adaptive response to acute ER stress in A. fumigatus and reveal a remarkable breadth of functions that are needed to resolve ER stress in this organism.
PMCID: PMC3943501  PMID: 24568630
Aspergillus fumigatus; UPR; Unfolded protein response; ER stress; Translational regulation; Polysome profiling; Yvc1
7.  Regulation of the cardiomyocyte transcriptome vs translatome by endothelin-1 and insulin: translational regulation of 5' terminal oligopyrimidine tract (TOP) mRNAs by insulin 
BMC Genomics  2010;11:343.
Changes in cellular phenotype result from underlying changes in mRNA transcription and translation. Endothelin-1 stimulates cardiomyocyte hypertrophy with associated changes in mRNA/protein expression and an increase in the rate of protein synthesis. Insulin also increases the rate of translation but does not promote overt cardiomyocyte hypertrophy. One mechanism of translational regulation is through 5' terminal oligopyrimidine tracts (TOPs) that, in response to growth stimuli, promote mRNA recruitment to polysomes for increased translation. TOP mRNAs include those encoding ribosomal proteins, but the full panoply remains to be established. Here, we used microarrays to compare the effects of endothelin-1 and insulin on the global transcriptome of neonatal rat cardiomyocytes, and on mRNA recruitment to polysomes (i.e. the translatome).
Globally, endothelin-1 and insulin (1 h) promoted >1.5-fold significant (false discovery rate < 0.05) changes in expression of 341 and 38 RNAs, respectively. For these transcripts with this level of change there was little evidence of translational regulation. However, 1336 and 712 RNAs had >1.25-fold significant changes in expression in total and/or polysomal RNA induced by endothelin-1 or insulin, respectively, of which ~35% of endothelin-1-responsive and ~56% of insulin-responsive transcripts were translationally regulated. Of mRNAs for established proteins recruited to polysomes in response to insulin, 49 were known TOP mRNAs with a further 15 probable/possible TOP mRNAs, but 49 had no identifiable TOP sequences or other consistent features in the 5' untranslated region.
Endothelin-1, rather than insulin, substantially affects global transcript expression to promote cardiomyocyte hypertrophy. Effects on RNA recruitment to polysomes are subtle, with differential effects of endothelin-1 and insulin on specific transcripts. Furthermore, although insulin promotes recruitment of TOP mRNAs to cardiomyocyte polysomes, not all recruited mRNAs are TOP mRNAs.
PMCID: PMC2900265  PMID: 20509958
8.  An approach to analyse the specific impact of rapamycin on mRNA-ribosome association 
BMC Medical Genomics  2008;1:33.
Recent work, using both cell culture model systems and tumour derived cell lines, suggests that the differential recruitment into polysomes of mRNA populations may be sufficient to initiate and maintain tumour formation. Consequently, a major effort is underway to use high density microarray profiles to establish molecular fingerprints for cells exposed to defined drug regimes. The aim of these pharmacogenomic approaches is to provide new information on how drugs can impact on the translational read-out within a defined cellular background.
We describe an approach that permits the analysis of de-novo mRNA-ribosome association in-vivo during short drug exposures. It combines hypertonic shock, polysome fractionation and high-throughput analysis to provide a molecular phenotype of translationally responsive transcripts. Compared to previous translational profiling studies, the procedure offers increased specificity due to the elimination of the drugs secondary effects (e.g. on the transcriptional read-out). For this pilot "proof-of-principle" assay we selected the drug rapamycin because of its extensively studied impact on translation initiation.
High throughput analysis on both the light and heavy polysomal fractions has identified mRNAs whose re-recruitment onto free ribosomes responded to short exposure to the drug rapamycin. The results of the microarray have been confirmed using real-time RT-PCR. The selective down-regulation of TOP transcripts is also consistent with previous translational profiling studies using this drug.
The technical advance outlined in this manuscript offers the possibility of new insights into mRNA features that impact on translation initiation and provides a molecular fingerprint for transcript-ribosome association in any cell type and in the presence of a range of drugs of interest. Such molecular phenotypes defined pre-clinically may ultimately impact on the evaluation of a particular drug in a living cell.
PMCID: PMC2533349  PMID: 18673536
9.  Concordant Regulation of Translation and mRNA Abundance for Hundreds of Targets of a Human microRNA 
PLoS Biology  2009;7(11):e1000238.
A specific microRNA reduces the synthesis of hundreds of proteins via concordant effects on the abundance and translation of the mRNAs that encode them.
MicroRNAs (miRNAs) regulate gene expression posttranscriptionally by interfering with a target mRNA's translation, stability, or both. We sought to dissect the respective contributions of translational inhibition and mRNA decay to microRNA regulation. We identified direct targets of a specific miRNA, miR-124, by virtue of their association with Argonaute proteins, core components of miRNA effector complexes, in response to miR-124 transfection in human tissue culture cells. In parallel, we assessed mRNA levels and obtained translation profiles using a novel global approach to analyze polysomes separated on sucrose gradients. Analysis of translation profiles for ∼8,000 genes in these proliferative human cells revealed that basic features of translation are similar to those previously observed in rapidly growing Saccharomyces cerevisiae. For ∼600 mRNAs specifically recruited to Argonaute proteins by miR-124, we found reductions in both the mRNA abundance and inferred translation rate spanning a large dynamic range. The changes in mRNA levels of these miR-124 targets were larger than the changes in translation, with average decreases of 35% and 12%, respectively. Further, there was no identifiable subgroup of mRNA targets for which the translational response was dominant. Both ribosome occupancy (the fraction of a given gene's transcripts associated with ribosomes) and ribosome density (the average number of ribosomes bound per unit length of coding sequence) were selectively reduced for hundreds of miR-124 targets by the presence of miR-124. Changes in protein abundance inferred from the observed changes in mRNA abundance and translation profiles closely matched changes directly determined by Western analysis for 11 of 12 proteins, suggesting that our assays captured most of miR-124–mediated regulation. These results suggest that miRNAs inhibit translation initiation or stimulate ribosome drop-off preferentially near the start site and are not consistent with inhibition of polypeptide elongation, or nascent polypeptide degradation contributing significantly to miRNA-mediated regulation in proliferating HEK293T cells. The observation of concordant changes in mRNA abundance and translational rate for hundreds of miR-124 targets is consistent with a functional link between these two regulatory outcomes of miRNA targeting, and the well-documented interrelationship between translation and mRNA decay.
Author Summary
The human genome contains directions to regulate the timing and magnitude of expression of its thousands of genes. MicroRNAs are important regulatory RNAs that tune the expression levels of tens to hundreds of specific genes by pairing to complimentary stretches in the messenger RNAs from these genes, thereby reducing their stability and their translation into protein. Although the importance of microRNAs is appreciated, little is known about the relative contributions of degradation or repression of translation of the cognate mRNAs to the overall effects on protein synthesis, or the links between these two regulatory mechanisms. We devised a simple, economical method to systematically measure mRNA translation profiles, then applied this method, in combination with gene expression analysis, to measure the effects of the human microRNA miR-124 on the abundance and apparent translation rate of its mRNA targets. We found that for the ∼600 mRNA targets of miR-124 that were identified by their association with microRNA effector complexes, around three quarters of the reduction in estimated protein synthesis was explained by changes in mRNA abundance. Although the apparent changes in translation efficiencies of the targeted mRNAs were smaller in magnitude, they were highly correlated with changes in the abundance of those RNAs, suggesting a functional link between microRNA-mediated repression of translation and mRNA decay.
PMCID: PMC2766070  PMID: 19901979
10.  Temporally Regulated Traffic of HuR and Its Associated ARE-Containing mRNAs from the Chromatoid Body to Polysomes during Mouse Spermatogenesis 
PLoS ONE  2009;4(3):e4900.
In mammals, a temporal disconnection between mRNA transcription and protein synthesis occurs during late steps of germ cell differentiation, in contrast to most somatic tissues where transcription and translation are closely linked. Indeed, during late stages of spermatogenesis, protein synthesis relies on the appropriate storage of translationally inactive mRNAs in transcriptionally silent spermatids. The factors and cellular compartments regulating mRNA storage and the timing of their translation are still poorly understood. The chromatoid body (CB), that shares components with the P. bodies found in somatic cells, has recently been proposed to be a site of mRNA processing. Here, we describe a new component of the CB, the RNA binding protein HuR, known in somatic cells to control the stability/translation of AU-rich containing mRNAs (ARE-mRNAs).
Methodology/Principal Findings
Using a combination of cell imagery and sucrose gradient fractionation, we show that HuR localization is highly dynamic during spermatid differentiation. First, in early round spermatids, HuR colocalizes with the Mouse Vasa Homolog, MVH, a marker of the CB. As spermatids differentiate, HuR exits the CB and concomitantly associates with polysomes. Using computational analyses, we identified two testis ARE-containing mRNAs, Brd2 and GCNF that are bound by HuR and MVH. We show that these target ARE-mRNAs follow HuR trafficking, accumulating successively in the CB, where they are translationally silent, and in polysomes during spermatid differentiation.
Our results reveal a temporal regulation of HuR trafficking together with its target mRNAs from the CB to polysomes as spermatids differentiate. They strongly suggest that through the transport of ARE-mRNAs from the CB to polysomes, HuR controls the appropriate timing of ARE-mRNA translation. HuR might represent a major post-transcriptional regulator, by promoting mRNA storage and then translation, during male germ cell differentiation.
PMCID: PMC2659425  PMID: 19333380
11.  Increased sucrose levels mediate selective mRNA translation in Arabidopsis 
BMC Plant Biology  2014;14:306.
Protein synthesis is a highly energy demanding process and is regulated according to cellular energy levels. Light and sugar availability affect mRNA translation in plant cells but the specific roles of these factors remain unclear. In this study, sucrose was applied to Arabidopsis seedlings kept in the light or in the dark, in order to distinguish sucrose and light effects on transcription and translation. These were studied using microarray analysis of steady-state mRNA and mRNA bound to translating ribosomes.
Steady-state mRNA levels were affected differently by sucrose in the light and in the dark but general translation increased to a similar extent in both conditions. For a majority of the transcripts changes of the transcript levels were followed by changes in polysomal mRNA levels. However, for 243 mRNAs, a change in polysomal occupancy (defined as polysomal levels related to steady-state levels of the mRNA) was observed after sucrose treatment in the light, but not in the dark condition. Many of these mRNAs are annotated as encoding ribosomal proteins, supporting specific translational regulation of this group of transcripts. Unexpectedly, the numbers of ribosomes bound to each mRNA decreased for mRNAs with increased polysomal occupancy.
Our results suggest that sucrose regulate translation of these 243 mRNAs specifically in the light, through a novel regulatory mechanism. Our data shows that increased polysomal occupancy is not necessarily leading to more ribosomes per transcript, suggesting a mechanism of translational induction not solely dependent on increased translation initiation rates.
Electronic supplementary material
The online version of this article (doi:10.1186/s12870-014-0306-3) contains supplementary material, which is available to authorized users.
PMCID: PMC4252027  PMID: 25403240
Arabidopsis; Protein synthesis; Sugar signaling; Translational regulation
12.  Distinct glucose-dependent stress responses revealed by translational profiling in pancreatic β-cells 
The Journal of endocrinology  2007;192(1):179-187.
In pancreatic β-cells, following an acute (within 1 h) increase in glucose concentration, there are rapid changes in the expression of a large subset of proteins. The change in the expression of many of these proteins is mediated by a post-transcriptional mechanism through either increases or decreases in the rate of translation from pre-existing transcripts. These proteins, whose synthesis is rapidly up- or down-regulated in response to glucose, are likely important in mounting the correct response to changes in plasma glucose concentrations. However, the vast majority of these proteins remain unidentified. Therefore, in order to identify these proteins, we analysed changes in the levels of mRNAs associated with polysomes (i.e. actively translating mRNAs) isolated from mouse insulinoma 6 cells incubated at either 0·5 or 20 mM glucose for 1 h. Changes in the levels of polysomal mRNAs in response to glucose were analysed using affymetrix oligonucleotide microarrays (translational profiling). This work revealed that, in response to a change in glucose concentration, the abundance of 313 transcripts associated with polysomes changed by more than 1·5-fold, of which the abundance of 37 changed by more than twofold. The majority of these transcripts encoded proteins associated with metabolism or gene expression. More detailed analysis showed that a number of mRNAs encoding proteins associated with the induction of oxidative stress, including thioredoxin-2 and thioredoxin-interacting protein were rapidly redistributed onto heavier polysomes at high glucose concentration, indicating an increase in their expression. At low glucose concentration, when the general rate of protein synthesis is low, a number of mRNAs encoding integrated stress response proteins, including ATF4 and CHOP10, associate with heavier polysomes, indicating that their expression is up-regulated. In conclusion, translational profiling has revealed that, at either low or at high glucose concentration, β-cells rapidly increase the synthesis of a specific subset of proteins that are likely important in maintaining β-cell integrity and survival during conditions of nutritional stress.
PMCID: PMC1831533  PMID: 17210755
13.  Distinct glucose-dependent stress responses revealed by translational profiling in pancreatic β-cells 
The Journal of Endocrinology  2007;192(1):179-187.
In pancreatic β-cells, following an acute (within 1 h) increase in glucose concentration, there are rapid changes in the expression of a large subset of proteins. The change in the expression of many of these proteins is mediated by a post-transcriptional mechanism through either increases or decreases in the rate of translation from pre-existing transcripts. These proteins, whose synthesis is rapidly up- or down-regulated in response to glucose, are likely important in mounting the correct response to changes in plasma glucose concentrations. However, the vast majority of these proteins remain unidentified. Therefore, in order to identify these proteins, we analysed changes in the levels of mRNAs associated with polysomes (i.e. actively translating mRNAs) isolated from mouse insulinoma 6 cells incubated at either 0·5 or 20 mM glucose for 1 h. Changes in the levels of polysomal mRNAs in response to glucose were analysed using affymetrix oligonucleotide microarrays (translational profiling). This work revealed that, in response to a change in glucose concentration, the abundance of 313 transcripts associated with polysomes changed by more than 1·5-fold, of which the abundance of 37 changed by more than twofold. The majority of these transcripts encoded proteins associated with metabolism or gene expression. More detailed analysis showed that a number of mRNAs encoding proteins associated with the induction of oxidative stress, including thioredoxin-2 and thioredoxin-interacting protein were rapidly redistributed onto heavier polysomes at high glucose concentration, indicating an increase in their expression. At low glucose concentration, when the general rate of protein synthesis is low, a number of mRNAs encoding integrated stress response proteins, including ATF4 and CHOP10, associate with heavier polysomes, indicating that their expression is up-regulated. In conclusion, translational profiling has revealed that, at either low or at high glucose concentration, β-cells rapidly increase the synthesis of a specific subset of proteins that are likely important in maintaining β-cell integrity and survival during conditions of nutritional stress.
PMCID: PMC1831533  PMID: 17210755
14.  Tunable protein synthesis by transcript isoforms in human cells 
eLife  null;5:e10921.
Eukaryotic genes generate multiple RNA transcript isoforms though alternative transcription, splicing, and polyadenylation. However, the relationship between human transcript diversity and protein production is complex as each isoform can be translated differently. We fractionated a polysome profile and reconstructed transcript isoforms from each fraction, which we term Transcript Isoforms in Polysomes sequencing (TrIP-seq). Analysis of these data revealed regulatory features that control ribosome occupancy and translational output of each transcript isoform. We extracted a panel of 5′ and 3′ untranslated regions that control protein production from an unrelated gene in cells over a 100-fold range. Select 5′ untranslated regions exert robust translational control between cell lines, while 3′ untranslated regions can confer cell type-specific expression. These results expose the large dynamic range of transcript-isoform-specific translational control, identify isoform-specific sequences that control protein output in human cells, and demonstrate that transcript isoform diversity must be considered when relating RNA and protein levels.
eLife digest
To produce a protein, a gene’s DNA is first copied to make molecules of messenger RNA (mRNA). The mRNAs pass through a molecular machine known as the ribosome, which translates the genetic code to make a protein. Not all of an mRNA is translated to make a protein; the “untranslated” regions play crucial roles in regulating how much of the protein is produced.
In animals, plants and other eukaryotes, many mRNAs are made up of small pieces that are “spliced” together. During this process, proteins are deposited on the mRNA to mark the splice junctions, which are then cleared when the mRNA is translated. Many different mRNAs can be produced from the same gene by splicing different combinations of RNA pieces. Each of these mRNA “isoforms” can, in principle, contain a unique set of features that control its translation. Hence each mRNA isoform can be translated differently so that different amounts of the corresponding protein product are produced. However, the relationship between the variety of isoforms and the control of translation is complex and not well understood.
To address these questions, Floor and Doudna measured the translation of over 60,000 mRNA isoforms made from almost 14,000 human genes. The experiments show that untranslated regions at the end of the mRNA (known as the 3′ end) strongly influence translation, even if the protein coding regions remain the same. Furthermore, the data showed that mRNAs with more splice junctions are translated better, implying an mRNA has some sort of memory of how many junctions it had even after the protein markers have been cleared.
Next, Floor and Doudna inserted regulatory sequences from differently translated isoforms into an unrelated “reporter” gene. This dramatically changed the amount of protein produced from the reporter gene, in a manner predicted by the earlier experiments. Untranslated regions at the beginning of the mRNAs (known as the 5′ end) controlled the amount of protein produced from the reporter consistently across different types of cells from the body. On the other hand, the 3′ regions can tune the level of protein production in particular types of cells.
Floor and Doudna’s findings demonstrate that differences between mRNA isoforms of a gene can have a big effect on the level of protein production. Changes in the types of mRNA made from a gene are often associated with human diseases, and these findings suggest one reason why. Additionally, the ability to engineer translation of an mRNA using the data is likely to aid the development of mRNA-based therapies.
PMCID: PMC4764583  PMID: 26735365
transcript isoforms; translational control; deep sequencing; engineered translation; RNA processing; RNA-seq; Human
15.  Regulation of mRNA translation during mitosis 
eLife  null;4:e07957.
Passage through mitosis is driven by precisely-timed changes in transcriptional regulation and protein degradation. However, the importance of translational regulation during mitosis remains poorly understood. Here, using ribosome profiling, we find both a global translational repression and identified ∼200 mRNAs that undergo specific translational regulation at mitotic entry. In contrast, few changes in mRNA abundance are observed, indicating that regulation of translation is the primary mechanism of modulating protein expression during mitosis. Interestingly, 91% of the mRNAs that undergo gene-specific regulation in mitosis are translationally repressed, rather than activated. One of the most pronounced translationally-repressed genes is Emi1, an inhibitor of the anaphase promoting complex (APC) which is degraded during mitosis. We show that full APC activation requires translational repression of Emi1 in addition to its degradation. These results identify gene-specific translational repression as a means of controlling the mitotic proteome, which may complement post-translational mechanisms for inactivating protein function.
eLife digest
The human body contains billions of cells, most of which formed via a process called mitosis in which a single cell divides to produce two new daughter cells. Actively dividing cells pass through a series of events (or phases) that are collectively known as the cell cycle. These phases allow the cell to grow in size, copy its genetic material, and then make preparations for cell division before taking the final decision to divide.
Many proteins are involved in regulating the cell cycle and each protein has a particular role in specific phases. The levels of these proteins in cells may change during the cycle, which is often crucial to allow the cell to progress to the next phase. For example, cells need a group of proteins called the anaphase-promoting complex (or APC for short) to destroy other specific proteins at the end of mitosis.
Another way in which the amount of protein in a cell can be adjusted is by controlling how much new protein is made during a process known as translation. During this process, a molecule called a messenger RNA (mRNA)—which contains information copied from a particular gene—is used as a template to assemble a new protein. However, it is not clear whether regulation of translation is involved in control of the cell division.
Tanenbaum et al. now address this question using a technique called ribosome profiling to measure the translation of individual mRNA molecules. The experiments analysed the changes in protein production before, during and after mitosis. The overall level of translation of all the mRNAs was about 35% lower during mitosis. However, some mRNAs in particular experienced a very large reduction in the level of translation (between three- and ten-fold less than the levels before mitosis).
One example of an mRNA whose translation is turned off in mitosis is the mRNA that makes a protein called Emi1. It is known from previous work that Emi1 inhibits the activity of the APC. Therefore, Emi1 needs to be inactivated in mitosis so that the APC can become active and promote progression to the next phase of the cell cycle. It was previously shown that Emi1 is destroyed during mitosis to allow the APC to operate. Tanenbaum et al. found that translation of the Emi1 mRNA must also be suppressed during mitosis in order to keep Emi1 protein levels very low and allow the APC to become fully active. These findings uncover a new role for the control of protein production in regulating the cell cycle. The next challenge will be to find out whether suppression of translation is also used in other biological systems where proteins need to be rapidly inactivated.
PMCID: PMC4548207  PMID: 26305499
mitosis; APC; Emi1; translation; ribosone profiling; mRNA; human
16.  Genome-wide Analysis of Transcript Abundance and Translation in Arabidopsis Seedlings Subjected to Oxygen Deprivation 
Annals of Botany  2005;96(4):647-660.
• Background and Aims DNA microarrays allow comprehensive estimation of total cellular mRNA levels but are also amenable to studies of other mRNA populations, such as mRNAs in translation complexes (polysomes). The aim of this study was to evaluate the role of translational regulation in response to oxygen deprivation (hypoxia).
• Methods Alterations in total cellular and large polysome (≥ five ribosomes per mRNA) mRNA levels were monitored in response to 12 h of hypoxia stress in seedlings of Arabidopsis thaliana with a full-genome oligonucleotide microarray.
• Key Results Comparison of two mRNA populations revealed considerable modulation of mRNA accumulation and diversity in translation in response to hypoxia. Consistent with the global decrease in protein synthesis, hypoxia reduced the average proportion of individual mRNA species in large polysome complexes from 56·1 % to 32·1 %. A significant decrease in the association with translational complexes was observed for 77 % of the mRNAs, including a subset of known hypoxia-induced gene transcripts. The examination of mRNA levels of nine genes in polysomes fractionated through sucrose density gradients corroborated the microarray data. Gene cluster analysis was used to identify mRNAs that displayed co-ordinated regulation. Fewer than half of the highly induced mRNAs circumvented the global depression of translation. Moreover, a large number of mRNAs displayed a significant decrease in polysome association without a concomitant decrease in steady-state accumulation. The abundant mRNAs that encode the ribosomal proteins behaved in this manner. By contrast, a small group of abiotic and biotic stress-induced mRNAs showed a significant increase in polysome association, without a change in abundance. Evaluation of quantitative features of mRNA sequences demonstrated that a low GC nucleotide content of the 5′-untranslated region provides a selective advantage for translation under hypoxia.
• Conclusions Alterations in transcript abundance and translation contribute to the differential regulation of gene expression in response to oxygen deprivation.
PMCID: PMC4247032  PMID: 16081496
Hypoxia; DNA microarray; polysome; translational control; mRNA sequence features; Arabidopsis thaliana
17.  Translational control analysis by translationally active RNA capture/microarray analysis (TrIP–Chip) 
Nucleic Acids Research  2010;38(9):e104.
We have developed a new approach to systematically study post-transcriptional regulation in a small number of cells. Actively translating mRNAs are associated with polysomes and the newly synthesized peptide chains are closely associated with molecular chaperones such as hsp70s, which assist in the proper folding of nascent polypeptides into higher ordered structures. These chaperones provide an anchor with which to separate actively translating mRNAs associated with polysomes from free mRNAs. Affinity capture beads were developed to capture hsp70 chaperones associated with the polysome complexes. The isolated actively translating mRNAs were used for high-throughput expression profiling analysis. Feasibility was demonstrated using an in vitro translation system with known translationally regulated mRNA transcript thymidylate synthase (TS). We further developed the approach using HCT-116 colon cancer cells with both TS and p53 as positive controls. The steady-state levels of TS and p53 mRNAs were unaltered after 5-fluorouracil treatment as assessed by real-time qRT-PCR analysis. In contrast, the protein expression and polysome-associated mRNA levels of both genes were increased. These differences in translational rate were revealed with our new approach from 500 cells. This technology has the potential to make investigation of translational control feasible with limited quantities of clinical specimens.
PMCID: PMC2875024  PMID: 20123731
18.  Coordinate translational regulation in the syntheses of elongation factor 1 alpha and ribosomal proteins in Xenopus laevis. 
Nucleic Acids Research  1993;21(20):4721-4725.
The regulation of the synthesis of elongation factor 1 alpha (EF-1 alpha) in Xenopus laevis has been analyzed from the point of view of translational control. The 5' end of EF-1 alpha mRNA, examined by primer extension, revealed the presence of a terminal pyrimidine tract that is characteristic of ribosomal protein mRNAs (rp-mRNAs). We have then compared the translation pattern of EF-1 alpha and rp-mRNAs during Xenopus embryogenesis and in Xenopus cultured cells during growth rate changes. In Xenopus embryos EF-1 alpha transcripts, that appear after midblastula transition, are initially mostly localized on mRNP and translationally inactive. Only later in embryogenesis, together with rp-mRNAs, they are gradually recruited on polysomes. Also in Xenopus cells B 3.2, EF-1 alpha mRNA shows a distribution change similar to an rp-mRNA: part of it moves from polysomes to mRNP during serum deprivation and goes back on polysomes after restitution of serum to the culture. Moreover EF-1 alpha mRNA, similarly to rp-mRNAs, is always localized on mRNP or fully loaded on polysomes but never on small polysomes. Therefore EF-1 alpha mRNA for structural features and translation behavior can be included in the 'regulatory' group of rp-mRNAs.
PMCID: PMC331496  PMID: 8233819
19.  Tuberous Sclerosis Complex Proteins 1 and 2 Control Serum-Dependent Translation in a TOP-Dependent and -Independent Manner▿ †  
Molecular and Cellular Biology  2007;27(16):5746-5764.
The tuberous sclerosis complex (TSC) proteins TSC1 and TSC2 regulate protein translation by inhibiting the serine/threonine kinase mTORC1 (for mammalian target of rapamycin complex 1). However, how TSC1 and TSC2 control overall protein synthesis and the translation of specific mRNAs in response to different mitogenic and nutritional stimuli is largely unknown. We show here that serum withdrawal inhibits mTORC1 signaling, causes disassembly of translation initiation complexes, and causes mRNA redistribution from polysomes to subpolysomes in wild-type mouse embryo fibroblasts (MEFs). In contrast, these responses are defective in Tsc1−/− or Tsc2−/− MEFs. Microarray analysis of polysome- and subpolysome-associated mRNAs uncovered specific mRNAs that are translationally regulated by serum, 90% of which are TSC1 and TSC2 dependent. Surprisingly, the mTORC1 inhibitor, rapamycin, abolished mTORC1 activity but only affected ∼40% of the serum-regulated mRNAs. Serum-dependent signaling through mTORC1 and polysome redistribution of global and individual mRNAs were restored upon re-expression of TSC1 and TSC2. Serum-responsive mRNAs that are sensitive to inhibition by rapamycin are highly enriched for terminal oligopyrimidine and for very short 5′ and 3′ untranslated regions. These data demonstrate that the TSC1/TSC2 complex regulates protein translation through mainly mTORC1-dependent mechanisms and implicates a discrete profile of deregulated mRNA translation in tuberous sclerosis pathology.
PMCID: PMC1952130  PMID: 17562867
20.  Global regulation of mRNA translation and stability in the early Drosophila embryo by the Smaug RNA-binding protein 
Genome Biology  2014;15(1):R4.
Smaug is an RNA-binding protein that induces the degradation and represses the translation of mRNAs in the early Drosophila embryo. Smaug has two identified direct target mRNAs that it differentially regulates: nanos and Hsp83. Smaug represses the translation of nanos mRNA but has only a modest effect on its stability, whereas it destabilizes Hsp83 mRNA but has no detectable effect on Hsp83 translation. Smaug is required to destabilize more than one thousand mRNAs in the early embryo, but whether these transcripts represent direct targets of Smaug is unclear and the extent of Smaug-mediated translational repression is unknown.
To gain a panoramic view of Smaug function in the early embryo, we identified mRNAs that are bound to Smaug using RNA co-immunoprecipitation followed by hybridization to DNA microarrays. We also identified mRNAs that are translationally repressed by Smaug using polysome gradients and microarrays. Comparison of the bound mRNAs to those that are translationally repressed by Smaug and those that require Smaug for their degradation suggests that a large fraction of Smaug’s target mRNAs are both translationally repressed and degraded by Smaug. Smaug directly regulates components of the TRiC/CCT chaperonin, the proteasome regulatory particle and lipid droplets, as well as many metabolic enzymes, including several glycolytic enzymes.
Smaug plays a direct and global role in regulating the translation and stability of a large fraction of the mRNAs in the early Drosophila embryo, and has unanticipated functions in control of protein folding and degradation, lipid droplet function and metabolism.
PMCID: PMC4053848  PMID: 24393533
21.  On the functions of the h subunit of eukaryotic initiation factor 3 in late stages of translation initiation 
Genome Biology  2007;8(4):R60.
Reporter transgene assays and comparative polysome-microarray analysis reveal that the intact h subunit of Arabidopsis eIF3 contributes to efficient translation initiation on mRNA leader sequences harbouring multiple uORFs.
The eukaryotic translation initiation factor 3 (eIF3) has multiple roles during the initiation of translation of cytoplasmic mRNAs. How individual subunits of eIF3 contribute to the translation of specific mRNAs remains poorly understood, however. This is true in particular for those subunits that are not conserved in budding yeast, such as eIF3h.
Working with stable reporter transgenes in Arabidopsis thaliana mutants, it was demonstrated that the h subunit of eIF3 contributes to the efficient translation initiation of mRNAs harboring upstream open reading frames (uORFs) in their 5' leader sequence. uORFs, which can function as devices for translational regulation, are present in over 30% of Arabidopsis mRNAs, and are enriched among mRNAs for transcriptional regulators and protein modifying enzymes. Microarray comparisons of polysome loading in wild-type and eif3h mutant seedlings revealed that eIF3h generally helps to maintain efficient polysome loading of mRNAs harboring multiple uORFs. In addition, however, eIF3h also boosted the polysome loading of mRNAs with long leaders or coding sequences. Moreover, the relative polysome loading of certain functional groups of mRNAs, including ribosomal proteins, was actually increased in the eif3h mutant, suggesting that regulons of translational control can be revealed by mutations in generic translation initiation factors.
The intact eIF3h protein contributes to efficient translation initiation on 5' leader sequences harboring multiple uORFs, although mRNA features independent of uORFs are also implicated.
PMCID: PMC1896003  PMID: 17439654
22.  Extensive translation of small Open Reading Frames revealed by Poly-Ribo-Seq 
eLife  null;3:e03528.
Thousands of small Open Reading Frames (smORFs) with the potential to encode small peptides of fewer than 100 amino acids exist in our genomes. However, the number of smORFs actually translated, and their molecular and functional roles are still unclear. In this study, we present a genome-wide assessment of smORF translation by ribosomal profiling of polysomal fractions in Drosophila. We detect two types of smORFs bound by multiple ribosomes and thus undergoing productive translation. The ‘longer’ smORFs of around 80 amino acids resemble canonical proteins in translational metrics and conservation, and display a propensity to contain transmembrane motifs. The ‘dwarf’ smORFs are in general shorter (around 20 amino-acid long), are mostly found in 5′-UTRs and non-coding RNAs, are less well conserved, and have no bioinformatic indicators of peptide function. Our findings indicate that thousands of smORFs are translated in metazoan genomes, reinforcing the idea that smORFs are an abundant and fundamental genome component.
eLife digest
To produce a protein, a stretch of DNA must first be transcribed to produce a molecule of messenger RNA (mRNA). The genetic information copied from the DNA is then read three letters at a time, in groups called codons. Each codon either encodes a particular amino acid to be added into a protein or provides further instructions: ‘start codons’ mark the beginning of a protein; ‘stop codons’ mark its end. The DNA between these two points is called an open reading frame (or ORF)—however, not all ORFs produce proteins.
Most proteins are made of several hundred amino acids, but the genomes of animals contain thousands of ORFs that would generate much smaller proteins made of fewer than 100 amino acids, if they were translated. It is, however, unclear how many of these small ORFs are converted into mRNA molecules and functional proteins.
Ribosomes are large molecular machines that translate the code in mRNA molecules and join together the appropriate amino acids in the right order to make a protein. Ribosome profiling is a technique that identifies which mRNA molecules are translated into proteins by determining the sequences of all the mRNA molecules bound to ribosomes at a particular moment. The mRNA sequences can then be compared with the sequence of the whole genome to work out which ORFs they correspond to. Ribosome profiling has been used to detect translated small ORFs, but the method yields a relatively high false positive rate as some mRNAs can bind to ribosomes without being translated.
To better detect small protein-producing ORFs, Aspden et al. developed a technique based on ribosome profiling called Poly-Ribo-Seq. The method takes advantage of the fact that during active translation, clusters of multiple ribosomes, called polysomes, bind mRNAs. Poly-Ribo-Seq isolates these polysomes and determines the sequence bound by each of the ribosomes, thereby reducing the number of false positives.
Applying Poly-Ribo-Seq to cells from the fruit fly Drosophila allowed Aspden et al. to identify two types of short ORF. The first type codes for proteins that are around 80 amino acids long and are translated with the same efficiency as larger ORFs. The sequences of these ORFs are found in other species, match at least in part sequences of known functional ORFs, and the proteins produced are found in specific locations inside cells. These small proteins may contribute to membrane integrity or function. Together, these properties suggest that these mRNAs create functional small proteins.
The second pool consists of very small ORFs (‘dwarf smORFs’) that code for around 20 amino acids, which are not translated so often and do not show many similarities with other species.
As the findings of Aspden et al. suggest that a large fraction of Drosophila small ORFs are translated into proteins, the next challenge will be to determine the roles of these small proteins in cells.
PMCID: PMC4359375  PMID: 25144939
small open reading Frames; non-coding RNAs; transmembrane peptides; D. melanogaster
23.  Different Mechanisms Preserve Translation of Programmed Cell Death 8 (PDCD8) and JunB in Virus-Infected Endothelial Cells 
Translation initiation of eukaryotic mRNAs typically occurs by cap-dependent ribosome scanning mechanism. However, certain mRNAs are translated by ribosome assembly at internal ribosome entry sites (IRES). Whether IRES-mediated translation occurs in stressed primary human endothelial cells (EC) is unknown.
Methods and Results
We performed microarray analysis of polyribosomal mRNA from EC to identify IRES-containing mRNAs. Cap-dependent translation was disabled by poliovirus (PV) infection and confirmed by loss of polysome peaks, detection of eIF4G cleavage, and decreased protein synthesis. 87.4% of mRNAs were dissociated from polysomes in virus-infected EC. 12% of mRNAs remained associated with polysomes and 0.6% were enriched ≥2-fold in polysome fractions from infected EC. Quantitative RT-PCR confirmed the microarray findings for 31 selected mRNAs. We found that enriched polysome associations of PDCD8 and JunB mRNA resulted in increased protein expression in PV-infected EC. The presence of IRES in the 5’UTR of PDCD8 mRNA, but not of JunB mRNA, was confirmed by dicistronic analysis.
We show that microarray profiling of polyribosomal mRNA transcripts from PV-infected EC successfully identifies mRNAs whose translation is preserved in the face of stress-induced, near complete cessation of cap-dependent initiation. Nevertheless, internal ribosome entry is not the only mechanism responsible for this privileged translation.
PMCID: PMC3310396  PMID: 22328780
IRES; microarray; poliovirus; PDCD8; JunB
24.  Thalamic WNT3 Secretion Spatiotemporally Regulates the Neocortical Ribosome Signature and mRNA Translation to Specify Neocortical Cell Subtypes 
The Journal of Neuroscience  2015;35(31):10911-10926.
Neocortical development requires tightly controlled spatiotemporal gene expression. However, the mechanisms regulating ribosomal complexes and the timed specificity of neocortical mRNA translation are poorly understood. We show that active mRNA translation complexes (polysomes) contain ribosomal protein subsets that undergo dynamic spatiotemporal rearrangements during mouse neocortical development. Ribosomal protein specificity within polysome complexes is regulated by the arrival of in-growing thalamic axons, which secrete the morphogen Wingless-related MMTV (mouse mammary tumor virus) integration site 3 (WNT3). Thalamic WNT3 release during midneurogenesis promotes a change in the levels of Ribosomal protein L7 in polysomes, thereby regulating neocortical translation machinery specificity. Furthermore, we present an RNA sequencing dataset analyzing mRNAs that dynamically associate with polysome complexes as neocortical development progresses, and thus may be regulated spatiotemporally at the level of translation. Thalamic WNT3 regulates neocortical translation of two such mRNAs, Foxp2 and Apc, to promote FOXP2 expression while inhibiting APC expression, thereby driving neocortical neuronal differentiation and suppressing oligodendrocyte maturation, respectively. This mechanism may enable targeted and rapid spatiotemporal control of ribosome composition and selective mRNA translation in complex developing systems like the neocortex.
SIGNIFICANCE STATEMENT The neocortex is a highly complex circuit generating the most evolutionarily advanced complex cognitive and sensorimotor functions. An intricate progression of molecular and cellular steps during neocortical development determines its structure and function. Our goal is to study the steps regulating spatiotemporal specificity of mRNA translation that govern neocortical development. In this work, we show that the timed secretion of Wingless-related MMTV (mouse mammary tumor virus) integration site 3 (WNT3) by ingrowing axons from the thalamus regulates the combinatorial composition of ribosomal proteins in developing neocortex, which we term the “neocortical ribosome signature.” Thalamic WNT3 further regulates the specificity of mRNA translation and development of neurons and oligodendrocytes in the neocortex. This study advances our overall understanding of WNT signaling and the spatiotemporal regulation of mRNA translation in highly complex developing systems.
PMCID: PMC4524969  PMID: 26245956
mRNA translation; neocortex; ribosome; thalamocortical; Wnt
25.  The Transacting Factor CBF-A/Hnrnpab Binds to the A2RE/RTS Element of Protamine 2 mRNA and Contributes to Its Translational Regulation during Mouse Spermatogenesis 
PLoS Genetics  2013;9(10):e1003858.
During spermatogenesis, mRNA localization and translation are believed to be regulated in a stage-specific manner. We report here that the Protamine2 (Prm2) mRNA transits through chromatoid bodies of round spermatids and localizes to cytosol of elongating spermatids for translation. The transacting factor CBF-A, also termed Hnrnpab, contributes to temporal regulation of Prm2 translation. We found that CBF-A co-localizes with the Prm2 mRNA during spermatogenesis, directly binding to the A2RE/RTS element in the 3′ UTR. Although both p37 and p42 CBF-A isoforms interacted with RTS, they associated with translationally repressed and de-repressed Prm2 mRNA, respectively. Only p42 was found to interact with the 5′cap complex, and to co-sediment with the Prm2 mRNA in polysomes. In CBF-A knockout mice, expression of protamine 2 (PRM2) was reduced and the Prm2 mRNA was prematurely translated in a subset of elongating spermatids. Moreover, a high percentage of sperm from the CBF-A knockout mouse showed abnormal DNA morphology. We suggest that CBF-A plays an important role in spermatogenesis by regulating stage-specific translation of testicular mRNAs.
Author Summary
During eukaryotic gene expression, a fraction of newly exported mRNA molecules is transported to the cellular periphery for translation. The underlying mechanisms are not fully understood even though they likely affect specialized functions in many cell types including oligodendrocyets, neurons and germ cells. We discovered that the heterogeneous nuclear ribonucleoprotein CBF-A, interacts with a conserved sequence, the RNA trafficking sequence (RTS), located in the untranslated region of transported mRNAs. This interaction facilitates transport of myelin basic protein mRNA and dendritic mRNAs in oligodendrocytes and neurons, respectively. Here we investigated whether RTS-recognition by CBF-A coordinates transport and localized translation of the Protamine 2 mRNA in spermatogenic cells. During spermatogenesis the Protamine 2 mRNAs is synthesized and kept in a silent form to be translated at later stages. We show that by interacting with the RTS of the Protamine 2 mRNA both CBF-A isoforms contribute to regulate the transcript at the translational level. In a CBF-A knockout mouse model, we demonstrate that the interplay between the CBF-A isoforms in translation regulation of the Protamine 2 mRNA and other testicular transcripts has an impact on spermatogenesis.
PMCID: PMC3798277  PMID: 24146628

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