mRNA Pool Research Articles

Секвенирование и GO аннотация транскриптома культуры клеток и тканей Rumex acetosa in vitro

The paper presents sequencing data, de novo assemblies and a functional annotation of transcriptome of the callus culture of Rumex acetosa L. The callus culture was maintained for twelve months in the presence of benzyladenine and naphthylacetic acid. The cDNA library was prepared using the adapter ligation method according to the standard protocol of the sequencer manufacturer. In the work, the “shotgun” technique was not used, since only the spectrum of the expression genes was required. In total it was obtained 74067 reads with a total 25,110,194 bp nucleotides. 10169 contigs were obtained in the resulting assembly with a medium length of 339 bp. Based on the sequence homology of total contigs in the category of biological processes for transcriptome callus, “cell metabolism”, “nitrogen metabolism”, “biosynthetic processes” and “metabolic processes of organic substances” dominated. In the category of molecular function, “transferase activity”, “heterocyclic compounds binding activity” and “binding the ions” dominated. In the category of the cellular component, the “macromolecular complex”, “organelles”, “cell”, “membrane” dominated. In comparison with the control transcriptome, the result obtained by us was distinguished by a reduced variety of GO subcategories. Variations in the expression of gene groups may proof epigenetic variation, which we have identified by GO annotations and transcriptome analysis. The mRNA pool is less diverse in callus lines, which, apparently, is associated with reduced specialization and dedifferentiation of cells. For the mRNA pool of R. acetosa cell culture, a high content of transcripts of retroelements, such as Copia and Gypsy, was noted. Similar results indicate a possible stress response of in vitro culture of cells and tissues of R. acetosa and high genome variability.

Methods for the Detection of Adenosine-to-Inosine Editing Events in Cellular RNA.

Modification of RNA is essential for properly expressing the repertoire of RNA transcripts necessary for both cell type and developmental specific functions. RNA modifications serve to dynamically re-wire and fine-tune the genetic information carried by an invariable genome. One important type of RNA modification is RNA editing and the most common and well-studied type of RNA editing is the hydrolytic deamination of adenosine to inosine. Inosine is a biological mimic of guanosine; therefore, when RNA is reverse transcribed, inosine is recognized as guanosine by the reverse transcriptase and a cytidine is incorporated into the complementary DNA (cDNA) strand. During PCR amplification, guanosines pair with the newly incorporated cytidines. As a result, the adenosine-to-inosine (A-to-I) editing events are recognized as adenosine to guanosine changes when comparing the sequences of the genomic DNA to the cDNA. This chapter describes the methods for extracting endogenous RNA for subsequent analyses of A-to-I RNA editing using reverse transcriptase-based approaches. We discuss techniques for the detection of A-to-I RNA editing events in messenger RNA (mRNA), including analyzing editing levels at specific adenosines within the total pool of mRNA versus analyzing editing patterns that occur in individual transcripts and a method for detecting editing events across the entire transcriptome. The detection of RNA editing events and editing levels can be used to better understand normal biological processes and disease states.

Functional differences between Tcf1 isoforms in early Xenopus development.

In Xenopus gastrula stage embryos, four isoforms of Tcf1 (B, C, D and E) are present with high amino acid sequence conservation compared to fish, mice and human. We studied possible functional differences between these Tcf1 isoforms during early Xenopus development. After overexpression of single Tcf1 isoforms, two distinct phenotypes were observed. Overexpression of the B or D isoforms of Tcf1, which both lack a C-clamp, enhances early canonical Wnt signaling and induces ectopic dorsal mesoderm at the expense of ventrolateral mesoderm prior to gastrulation, causing severe antero-dorzalization of embryos. Overexpression of the E-isoform, which contains a complete C-clamp, does not induce ectopic dorsal mesoderm, but rather leads to severe caudal truncation. Overexpression of the C-isoform, which contains a partial C-clamp, induces a similar phenotype. Mutation of a single amino acid in the C-clamp, known to produce a hypomorphic mutant in D. melanogaster, led to a gain of function in inducing ectopic organizer tissue, as observed after overexpression of the B or D isoforms of Tcf1. Depletion of the C-clamp exon from the zygotic mRNA pool, by injection of a morpholino oligo that targets the splice acceptor site of the exon containing the C-clamp, caused a severe shortening of the AP-axis. Furthermore, embryos showed poor development of the CNS, paraxial mesoderm and primary blood vessels. In situ hybridization analysis showed that Lef1 expression was downregulated at the mid-hindbrain boundary, in the otic vesicles and the branchial arches. The results indicate that in post-gastrula stage Xenopus embryos, the E-tail of Tcf1 is required for expression of Lef1 and for blood vessel formation.

RNA-associated protein LSM family member 14 controls oocyte meiotic maturation through regulating mRNA pools.

LSM family member 14 (LSM14) belongs to the RNA-associated protein (RAP) family that is widely expressed in different species, and whose functions include associating and storing mRNAs. In the present study, we found that LSM14b was essential for oocyte meiotic maturation. Lack of LSM14b caused oocyte meiotic arrest at metaphase, and misalignment of chromosomes, as well as abnormal spindle assembly checkpoint (SAC) and maturation promoting factor (MPF) activation. Cyclin B1 and Cdc20 mRNAs, whose contents changed with LSM14b expression, were likely direct targets of LSM14b. We conclude that LSM14b, by functioning as a container of mRNAs, controls protein expression, and thus regulates the oocyte meiotic maturation process.

Targeted Intron Retention and Excision for Rapid Gene Regulation in Response to Neuronal Activity

Activity-dependent transcription has emerged as a major source of gene products that regulate neuronal excitability, connectivity, and synaptic properties. However, the elongation rate of RNA polymerases imposes a significant temporal constraint for transcript synthesis, in particular for long genes where new synthesis requires hours. Here we reveal a novel, transcription-independent mechanism that releases transcripts within minutes of neuronal stimulation. We found that, in the mouse neocortex, polyadenylated transcripts retain select introns and are stably accumulated in the cell nucleus. A subset ofthese intron retention transcripts undergoes activity-dependent splicing, cytoplasmic export, and ribosome loading, thus acutely releasing mRNAs in response to stimulation. This process requires NMDA receptor- and calmodulin-dependent kinase pathways, and it is particularly prevalent for long transcripts. We conclude that regulated intron retention in fully transcribed RNAs represents a mechanism to rapidly mobilize a pool of mRNAs in response to neuronal activity.

Closing in on the causes of host shutoff

Influenza A virus suppresses the translation of host mRNA by selectively remodeling and dominating the pool of mRNA in infected cells.

Hypoxia and ER stress promote Staufen1 expression through an alternative translation mechanism

Under physiological stress conditions the cell protects itself through a global blockade on cap-dependent translation of mRNA. This allows cap-independent mechanisms such as internal ribosome entry site (IRES)-mediated translation to take over and initiate the translation of a specific pool of mRNAs that encode proteins involved in protecting the cell from stress. Staufen 1 (Stau1) is an RNA-binding protein that has been previously implicated in the regulation of stress granule formation and therefore could play a key role in protecting the cell against stress stimuli such as oxidative and endoplasmic reticulum (ER) stress. We hypothesized that Stau1 mRNA could, like many stress response genes, contain an IRES in its 5′UTR. Here we describe that a bona fide IRES element is present in the 5′UTR of Stau1 mRNA, which is activated under hypoxic and ER stress conditions. Further, we show that the activity of PERK kinase, a major effector of the ER stress response, is required for Stau1 IRES-mediated translation during ER stress. These results suggest that Stau1 is a stress response gene that remains efficiently translated during hypoxia and ER stress despite the substantial global inhibition of cap-dependent protein translation, promoting cell recovery following stress.

Maternal expression and early induction of histone gene transcription factor Hinfp sustains development in pre-implantation embryos

Fidelity of histone gene expression is important for normal cell growth and differentiation that is stringently controlled during development but is compromised during tumorigenesis. Efficient production of histones for packaging newly replicated DNA is particularly important for proper cell division and epigenetic control during the initial pre-implantation stages of embryonic development. Here, we addressed the unresolved question of when the machinery for histone gene transcription is activated in the developing zygote to accommodate temporal demands for histone gene expression. We examined induction of Histone Nuclear Factor P (HINFP), the only known transcription factor required for histone H4 gene expression, that binds directly to a unique H4 promoter-specific element to regulate histone H4 transcription. We show that Hinfp gene transcripts are stored in oocytes and maternally transmitted to the zygote. Transcripts from the paternal Hinfp gene, which reflect induction of zygotic gene expression, are apparent at the 4- to 8-cell stage, when most maternal mRNA pools are depleted. Loss of Hinfp expression due to gene ablation reduces cell numbers in E3.5 stage embryos and compromises implantation. Reduced cell proliferation is attributable to severe reduction in histone mRNA levels accompanied by reduced cell survival and genomic damage as measured by cleaved Caspase 3 and phospho-H2AX staining, respectively. We conclude that transmission of maternal Hinfp transcripts and zygotic activation of the Hinfp gene together are necessary to control H4 gene expression in early pre-implantation embryos in order to support normal embryonic development.

A systematic view on influenza induced host shutoff.

Host shutoff is a common strategy used by viruses to repress cellular mRNA translation and concomitantly allow the efficient translation of viral mRNAs. Here we use RNA-sequencing and ribosome profiling to explore the mechanisms that are being utilized by the Influenza A virus (IAV) to induce host shutoff. We show that viral transcripts are not preferentially translated and instead the decline in cellular protein synthesis is mediated by viral takeover on the mRNA pool. Our measurements also uncover strong variability in the levels of cellular transcripts reduction, revealing that short transcripts are less affected by IAV. Interestingly, these mRNAs that are refractory to IAV infection are enriched in cell maintenance processes such as oxidative phosphorylation. Furthermore, we show that the continuous oxidative phosphorylation activity is important for viral propagation. Our results advance our understanding of IAV-induced shutoff, and suggest a mechanism that facilitates the translation of genes with important housekeeping functions.

Neurons That UnderlieDrosophila melanogasterReproductive Behaviors: Detection of a Large Male-Bias in Gene Expression infruitless-Expressing Neurons

Male and female reproductive behaviors in Drosophila melanogaster are vastly different, but neurons that express sex-specifically spliced fruitless transcripts (fru P1) underlie these behaviors in both sexes. How this set of neurons can generate such different behaviors between the two sexes is an unresolved question. A particular challenge is that fru P1-expressing neurons comprise only 2–5% of the adult nervous system, and so studies of adult head tissue or whole brain may not reveal crucial differences. Translating Ribosome Affinity Purification (TRAP) identifies the actively translated pool of mRNAs from fru P1-expressing neurons, allowing a sensitive, cell-type-specific assay. We find four times more male-biased than female-biased genes in TRAP mRNAs from fru P1-expressing neurons. This suggests a potential mechanism to generate dimorphism in behavior. The male-biased genes may direct male behaviors by establishing cell fate in a similar context of gene expression observed in females. These results suggest a possible global mechanism for how distinct behaviors can arise from a shared set of neurons.

Applying Subtractive Hybridization Technique to Enrich and Amplify Tumor-Specific Transcripts of Esophageal Squamous Cell Carcinoma

Subtractive hybridization (SH) as an efficient and powerful approach can be applied to isolate differentially expressed transcripts as well as detect of involved mRNAs in various cellular processes, particularly diseases and malignancies. This procedure leads to the enrichment of specific low copy transcripts of tumor cells. Having developed a new approach for SH to isolate tumor specific transcripts, we facilitated discovery of uniquely expressed genes in esophageal squamous cell carcinoma (ESCC). Total RNA was extracted from the fresh tumoral and their adjacent normal tissues, and purified using the Switch Mechanism At the 5' end of Reverse Transcript (SMART) method. Following cDNA synthesis of normal mRNAs using magnetic beads, it was hybridized with tumor mRNAs. To enhance efficiency of subtraction, hybridization was repeated three rounds. Finally, amplification of subtracted tumor-specific transcripts was carried out using in vitro transcription. The subtracted tumoral mRNAs was analyzed quantitatively using real-time PCR for both tumor-specific and housekeeping genes. The subtracted mRNA was confirmed as tumor-specific mRNA pool using RT-PCR and quantitative real-time PCR assessment. The elevated level of tumor-specific transcripts such as MAGE-A4 and CD44 as well as declined copy number of housekeeping genes such as GAPDH, β actin and β2-microglobulin, were confirmed in subtracted tumoral mRNA. The presence of tumor genes was confirmed after the SH procedure. The designed SH method in combination with SMART technique can isolate and amplify high quality tumor-specific transcripts even from small amount of tumor tissues. Removal of common transcripts from the extracted tumoral mRNAs using SH, leads to the enrichment of tumor-specific transcripts. The isolated transcripts are of interest because of their probable roles in ESCC progression and development. In addition, these tumor-specific mRNAs can be applied for future vaccine cancer studies.

The ribosome in action: Tuning of translational efficiency and protein folding.

The cellular proteome is shaped by the combined activities of the gene expression and quality control machineries. While transcription plays an undoubtedly important role, in recent years also translation emerged as a key step that defines the composition and quality of the proteome and the functional activity of proteins in the cell. Among the different post-transcriptional control mechanisms, translation initiation and elongation provide multiple checkpoints that can affect translational efficiency. A multitude of specific signals in mRNAs can determine the frequency of translation initiation, choice of the open reading frame, global and local elongation velocities, and the folding of the emerging protein. In addition to specific signatures in the mRNAs, also variations in the global pools of translation components, including ribosomes, tRNAs, mRNAs, and translation factors can alter translational efficiencies. The cellular outcomes of phenomena such as mRNA codon bias are sometimes difficult to understand due to the staggering complexity of covariates that affect codon usage, translation, and protein folding. Here we summarize the experimental evidence on how the ribosome-together with the other components of the translational machinery-can alter translational efficiencies of mRNA at the initiation and elongation stages and how translation velocity affects protein folding. We seek to explain these findings in the context of mechanistic work on the ribosome. The results argue in favour of a new understanding of translation control as a hub that links mRNA homeostasis to production and quality control of proteins in the cell.

The multiple functions of RNA helicases as drivers and regulators of gene expression.

RNA helicases comprise the largest family of enzymes involved in the metabolism of mRNAs, the processing and fate of which rely on their packaging into messenger ribonucleoprotein particles (mRNPs). In this Review, we describe how the capacity of some RNA helicases to either remodel or lock the composition of mRNP complexes underlies their pleiotropic functions at different steps of the gene expression process. We illustrate the roles of RNA helicases in coordinating gene expression steps and programmes, and propose that RNA helicases function as molecular drivers and guides of the progression of their mRNA substrates from one RNA-processing factory to another, to a productive mRNA pool that leads to protein synthesis or to unproductive mRNA pools that are stored or degraded.

Seasonal immunoregulation in a naturally-occurring vertebrate.

BackgroundFishes show seasonal patterns of immunity, but such phenomena are imperfectly understood in vertebrates generally, even in humans and mice. As these seasonal patterns may link to infectious disease risk and individual condition, the nature of their control has real practical implications. Here we characterize seasonal dynamics in the expression of conserved vertebrate immunity genes in a naturally-occurring piscine model, the three-spined stickleback.ResultsWe made genome-wide measurements (RNAseq) of whole-fish mRNA pools (n = 36) at the end of summer and winter in contrasting habitats (riverine and lacustrine) and focussed on common trends to filter habitat-specific from overarching temporal responses. We corroborated this analysis with targeted year-round whole-fish gene expression (Q-PCR) studies in a different year (n = 478). We also considered seasonal tissue-specific expression (6 tissues) (n = 15) at a third contrasting (euryhaline) locality by Q-PCR, further validating the generality of the patterns seen in whole fish analyses. Extremes of season were the dominant predictor of immune expression (compared to sex, ontogeny or habitat). Signatures of adaptive immunity were elevated in late summer. In contrast, late winter was accompanied by signatures of innate immunity (including IL-1 signalling and non-classical complement activity) and modulated toll-like receptor signalling. Negative regulators of T-cell activity were prominent amongst winter-biased genes, suggesting that adaptive immunity is actively down-regulated during winter rather than passively tracking ambient temperature. Network analyses identified a small set of immune genes that might lie close to a regulatory axis. These genes acted as hubs linking summer-biased adaptive pathways, winter-biased innate pathways and other organismal processes, including growth, metabolic dynamics and responses to stress and temperature. Seasonal change was most pronounced in the gill, which contains a considerable concentration of T-cell activity in the stickleback.ConclusionsOur results suggest major and predictable seasonal re-adjustments of immunity. Further consideration should be given to the effects of such responses in seasonally-occurring disease.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2701-7) contains supplementary material, which is available to authorized users.

Human Cells Cultured under Physiological Oxygen Utilize Two Cap-binding Proteins to recruit Distinct mRNAs for Translation

Translation initiation is a focal point of translational control and requires the binding of eIF4E to the 5' cap of mRNA. Under conditions of extreme oxygen depletion (hypoxia), human cells repress eIF4E and switch to an alternative cap-dependent translation mediated by a homolog of eIF4E, eIF4E2. This homolog forms a complex with the oxygen-regulated hypoxia-inducible factor 2α and can escape translation repression. This complex mediates cap-dependent translation under cell culture conditions of 1% oxygen (to mimic tumor microenvironments), whereas eIF4E mediates cap-dependent translation at 21% oxygen (ambient air). However, emerging evidence suggests that culturing cells in ambient air, or "normoxia," is far from physiological or "normal." In fact, oxygen in human tissues ranges from 1-11% or "physioxia." Here we show that two distinct modes of cap-dependent translation initiation are active during physioxia and act on separate pools of mRNAs. The oxygen-dependent activities of eIF4E and eIF4E2 are elucidated by observing their polysome association and the status of mammalian target of rapamycin complex 1 (eIF4E-dependent) or hypoxia-inducible factor 2α expression (eIF4E2-dependent). We have identified oxygen conditions where eIF4E is the dominant cap-binding protein (21% normoxia or standard cell culture conditions), where eIF4E2 is the dominant cap-binding protein (1% hypoxia or ischemic diseases and cancerous tumors), and where both cap-binding proteins act simultaneously to initiate the translation of distinct mRNAs (1-11% physioxia or during development and stem cell differentiation). These data suggest that the physioxic proteome is generated by initiating translation of mRNAs via two distinct but complementary cap-binding proteins.

728. Tissue-Directed Transgene Engineering for AAV and Lentivector Gene Therapy Approaches

Clinical gene therapy frequently is encumbered by low transgene product biosynthesis at predictably safe vector doses. It has been hypothesized that the presence of rare codons may regulate transgene product expression through depletion of the available cognate tRNA pool. Codon optimization is the prominent strategy utilized to overcome this hypothesized limitation and involves replacing rare, presumably translation rate-limiting, codons with the most frequent ones. Typical algorithms attempt to match the codon usage frequency of the transgene coding sequence to that of target organism's total mRNA pool, which has been shown to approximate the overall available tRNA concentrations. Upon closer examination, it appears that both codon frequency and tRNA content vary between tissue types. Therefore, we hypothesize that codon-optimization can be improved by tailoring to the codon-frequency of the most highly expressed genes present in target cell types. Our tissue-directed codon optimization algorithm utilizes novel codon usage indices generated from target cell gene expression data. As proof of concept, we developed tissue-codon optimized variants of coagulation factor VIII (FVIII) to be utilized in lenti- and adeno-associated viral (LV and AAV, respectively) vectors. These two vectors are the leading platforms for clinical gene therapy of hemophilia A. LV is utilized to target autologous hematopoietic cell types ex vivo while AAV is delivered intravenously to genetically-modify hepatocytes. However to date, development-stage LV and AAV gene therapy products for hemophilia A have been characterized by low-level FVIII transgene product biosynthesis. Initially, we designed human hepatocyte-, monocyte- and standard overall human-optimized FVIII constructs (LCO, MCO and HCO, respectively) to be compared to wild-type FVIII. Upon initial examination, expression of LCO-FVIII was shown to be 3-fold greater than either MCO- or wild-type FVIII from the human hepatoma cell line, HepG2, following transient transfection. In contrast, LCO-FVIII and MCO-FVIII expression was diminished 12 and 4-fold, respectively, compared to wild-type in the ‘neutral’ human embryonic kidney 293T cell line. Furthermore, following hydrodynamic injection of naked plasmid DNA into hemophilia A mice, LCO-FVIII exhibited a sustained 10-fold increase in FVIII expression relative to the HCO-FVIII comparator. In attempt to generate a lead candidate for clinical translation, we utilized several of our most promising vector components to construct a liver-optimized AAV8 vector consisting of LCO-ET3, our previously described high-expression bioengineered FVIII variant, transcriptionally driven by a novel 146bp liver-directed promoter and adjacent MVM intron. Following intravenous delivery into hemophilia A mice, vector doses of 1e12 and 1e11 vector genomes per kg achieved sustained, predictable curative plasma FVIII levels of 200% and 20% of normal human levels, respectively. These initial results support the utility of our novel approach of clinical tissue-directed transgene optimization.

MRNA trans-splicing in gene therapy for genetic diseases.

Spliceosome‐mediated RNA trans‐splicing, or SMaRT, is a promising strategy to design innovative gene therapy solutions for currently intractable genetic diseases. SMaRT relies on the correction of mutations at the post‐transcriptional level by modifying the mRNA sequence. To achieve this, an exogenous RNA is introduced into the target cell, usually by means of gene transfer, to induce a splice event in trans between the exogenous RNA and the target endogenous pre‐mRNA. This produces a chimeric mRNA composed partly of exons of the latter, and partly of exons of the former, encoding a sequence free of mutations. The principal challenge of SMaRT technology is to achieve a reaction as complete as possible, i.e., resulting in 100% repairing of the endogenous mRNA target. The proof of concept of SMaRT feasibility has already been established in several models of genetic diseases caused by recessive mutations. In such cases, in fact, the repair of only a portion of the mutant mRNA pool may be sufficient to obtain a significant therapeutic effect. However in the case of dominant mutations, the target cell must be freed from the majority of mutant mRNA copies, requiring a highly efficient trans‐splicing reaction. This likely explains why only a few examples of SMaRT approaches targeting dominant mutations are reported in the literature. In this review, we explain in details the mechanism of trans‐splicing, review the different strategies that are under evaluation to lead to efficient trans‐splicing, and discuss the advantages and limitations of SMaRT. WIREs RNA 2016, 7:487–498. doi: 10.1002/wrna.1347For further resources related to this article, please visit the WIREs website.

Silencing Status Epilepticus-Induced BDNF Expression with Herpes Simplex Virus Type-1 Based Amplicon Vectors.

Brain-derived neurotrophic factor (BDNF) has been found to produce pro- but also anti-epileptic effects. Thus, its validity as a therapeutic target must be verified using advanced tools designed to block or to enhance its signal. The aim of this study was to develop tools to silence the BDNF signal. We generated Herpes simplex virus type 1 (HSV-1) derived amplicon vectors, i.e. viral particles containing a genome of 152 kb constituted of concatameric repetitions of an expression cassette, enabling the expression of the gene of interest in multiple copies. HSV-1 based amplicon vectors are non-pathogenic and have been successfully employed in the past for gene delivery into the brain of living animals. Therefore, amplicon vectors should represent a logical choice for expressing a silencing cassette, which, in multiple copies, is expected to lead to an efficient knock-down of the target gene expression. Here, we employed two amplicon-based BDNF silencing strategies. The first, antisense, has been chosen to target and degrade the cytoplasmic mRNA pool of BDNF, whereas the second, based on the convergent transcription technology, has been chosen to repress transcription at the BDNF gene. Both these amplicon vectors proved to be effective in down-regulating BDNF expression in vitro, in BDNF-expressing mesoangioblast cells. However, only the antisense strategy was effective in vivo, after inoculation in the hippocampus in a model of status epilepticus in which BDNF mRNA levels are strongly increased. Interestingly, the knocking down of BDNF levels induced with BDNF-antisense was sufficient to produce significant behavioral effects, in spite of the fact that it was produced only in a part of a single hippocampus. In conclusion, this study demonstrates a reliable effect of amplicon vectors in knocking down gene expression in vitro and in vivo. Therefore, this approach may find broad applications in neurobiological studies.

Systemic Reprogramming of Translation Efficiencies on Oxygen Stimulus.

Protein concentrations evolve under greater evolutionary constraint than mRNA levels. Translation efficiency of mRNA represents the chief determinant of basal protein concentrations. This raises a fundamental question of how mRNA and protein levels are coordinated in dynamic systems responding to physiological stimuli. This report examines the contributions of mRNA abundance and translation efficiency to protein output in cells responding to oxygen stimulus. We show that changes in translation efficiencies, and not mRNA levels, represent the major mechanism governing cellular responses to [O2] perturbations. Two distinct cap-dependent protein synthesis machineries select mRNAs for translation: the normoxic eIF4F and the hypoxic eIF4F(H). O2-dependent remodeling of translation efficiencies enables cells to produce adaptive translatomes from preexisting mRNA pools. Differences in mRNA expression observed under different [O2] are likely neutral, given that they occur during evolution. We propose that mRNAs contain translation efficiency determinants for their triage by the translation apparatus on [O2] stimulus.

Functional Cloning Using a <em>Xenopus</em> Oocyte Expression System

Identification of genes responsible for embryonic induction poses a number of challenges; to name a few, secreted molecules of interest may be low in abundance, may not be secreted but tethered to the signaling cell(s), or may require the presence of binding partners or upstream regulatory molecules. Thus in a search for gene products capable of eliciting an early lens-inductive response in competent ectoderm, we utilized an expression cloning system that would allow identification of paracrine or juxtacrine factors as well as transcriptional or other regulatory proteins. Pools of mRNA were injected into Xenopus oocytes, and responding tissue placed directly on the oocytes and co-cultured. Following functional cloning of ldb1 from a neural plate stage cDNA library based on its ability to elicit the expression of the early lens placode marker foxe3 in lens-competent animal cap ectoderm, we characterized the mRNA expression pattern, and assayed developmental progression following overexpression or knockdown of ldb1. This system is suitable in a very wide variety of contexts where identification of an inducer or its upstream regulatory molecules is sought using a functional response in competent tissue.