Forebrain and midbrain development requires epiblast-restrictedOtx2translational control mediated by its 3′ UTR

Development ◽  
2001 ◽  
Vol 128 (15) ◽  
pp. 2989-3000 ◽  
Author(s):  
Pietro Pilo Boyl ◽  
Massimo Signore ◽  
Dario Acampora ◽  
Juan Pedro Martinez-Barbera ◽  
Cristina Ilengo ◽  
...  
Keyword(s):  
Brain Development ◽  
Translational Control ◽  
Transcriptional Control ◽  
Molecular Level ◽  
Regulatory Element ◽  
Untranslated Regions ◽  
Visceral Endoderm ◽  
Protein Levels ◽  
Neuronal Progenitor ◽  
Anterior Neural Plate

Otx genes play an important role in brain development. Previous mouse models suggested that the untranslated regions (UTRs) of Otx2 mRNA may contain regulatory element(s) required for its post-transcriptional control in epiblast and neuroectoderm. In order to study this, we have perturbed the 3′ UTR of Otx2 by inserting a small fragment of DNA from the λ phage. Otx2λ mutants exhibited proper gastrulation and normal patterning of the early anterior neural plate, but from 8.5 days post coitum they developed severe forebrain and midbrain abnormalities. OTX2 protein levels in Otx2λ mutants were heavily reduced in the epiblast, axial mesendoderm and anterior neuroectoderm but not in the visceral endoderm. At the molecular level, we found out that the ability of the Otx2λ mRNA to form efficient polyribosome complexes was impaired. Sequence analysis of the Otx2-3′ UTR revealed a 140 bp long element that is present only in vertebrate Otx2 genes and conserved in identity by over 80%. Our data provide experimental evidence that murine brain development requires accurate translational control of Otx2 mRNA in epiblast and neuronal progenitor cells. This leads us to hypothesise that this control might have important evolutionary implications.

scholarly journals Tunable protein synthesis by transcript isoforms in human cells

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Stephen N Floor ◽  
Jennifer A Doudna
Keyword(s):  
Translational Control ◽  
Protein Production ◽  
Dynamic Range ◽  
Human Cells ◽  
Untranslated Regions ◽  
Specific Expression ◽  
Transcript Isoforms ◽  
Protein Levels ◽  
Eukaryotic Genes ◽  
Control Protein

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.

scholarly journals Elevated cJUN expression and an ATF/CRE site within the ATF3 promoter contribute to activation of ATF3 transcription by the amino acid response

2013 ◽  
Vol 45 (4) ◽  
pp. 127-137 ◽  
Author(s):  
Lingchen Fu ◽  
Michael S. Kilberg
Keyword(s):  
Amino Acid ◽  
Transcriptional Activation ◽  
Translational Control ◽  
Mammalian Cells ◽  
Transcriptional Control ◽  
Present Report ◽  
Binding Activity ◽  
Maximal Response ◽  
Protein Levels ◽  
Amino Acid Response

Mammalian cells respond to amino acid deprivation through multiple signaling pathways referred to as the amino acid response (AAR). Transcription factors mediate the AAR after their activation by several mechanisms; examples include translational control (activating transcription factor 4, ATF4), phosphorylation (p-cJUN), and transcriptional control (ATF3). ATF4 induces ATF3 transcription through a promoter-localized C/EBP-ATF response element (CARE). The present report characterizes an ATF/CRE site upstream of the CARE that also contributes to AAR-induced ATF3 transcription. ATF4 binds to the ATF/CRE and CARE sequences and both are required for a maximal response to ATF4 induction. ATF3, which antagonizes ATF4 and represses its own gene, also exhibited binding activity to the ATF/CRE and CARE sequences. The AAR resulted in elevated total cJUN and p-cJUN protein levels and both forms exhibited binding activity to the ATF/CRE and CARE ATF3 sequences. Knockdown of AAR-enhanced cJUN expression blocked induction of the ATF3 gene and mutation of either the ATF/CRE or the CARE site prevented the cJUN-dependent increase in ATF3-driven luciferase activity. The results indicate that both increased cJUN and the cis-acting ATF/CRE sequence within the ATF3 promoter contribute to the transcriptional activation of the gene during the AAR.

scholarly journals Tunable protein synthesis by transcript isoforms in human cells

2015 ◽  
Author(s):  
Stephen N Floor ◽  
Jennifer A Doudna
Keyword(s):  
Translational Control ◽  
Protein Production ◽  
Dynamic Range ◽  
Human Cells ◽  
Untranslated Regions ◽  
Specific Expression ◽  
Transcript Isoforms ◽  
Protein Levels ◽  
Eukaryotic Genes ◽  
Control Protein

Eukaryotic genes generate multiple mRNA 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.

scholarly journals Visceral endoderm-restricted translation of Otx1 mediates recovery of Otx2 requirements for specification of anterior neural plate and normal gastrulation

Development ◽  
1998 ◽  
Vol 125 (24) ◽  
pp. 5091-5104 ◽  
Author(s):  
D. Acampora ◽  
V. Avantaggiato ◽  
F. Tuorto ◽  
P. Briata ◽  
G. Corte ◽  
...  
Keyword(s):  
Transcriptional Control ◽  
Expression Patterns ◽  
Epileptic Seizures ◽  
Primitive Streak ◽  
Neural Plate ◽  
Visceral Endoderm ◽  
Temporal Expression ◽  
Biochemical Activity ◽  
The Difference ◽  
Anterior Neural Plate

Otx1 and Otx2, two murine homologs of the Drosophila orthodenticle (otd) gene, contribute to brain morphogenesis. In particular Otx1 null mice are viable and show spontaneous epileptic seizures and abnormalities affecting the dorsal telencephalic cortex. Otx2 null mice die early in development and fail in specification of the rostral neuroectoderm and proper gastrulation. In order to determine whether Otx1(−/−)and Otx2(−/−) highly divergent phenotypes reflect differences in temporal expression or biochemical activity of OTX1 and OTX2 proteins, the Otx2-coding sequence was replaced by a human Otx1 full-coding cDNA. Homozygous mutant embryos recovered anterior neural plate and proper gastrulation but failed to maintain forebrain-midbrain identities, displaying a headless phenotype from 9 days post coitum (d.p.c.) onwards. Unexpectedly, in spite of the RNA distribution in both visceral endoderm (VE) and epiblast, the hOTX1 protein was synthesized only in the VE. This VE-restricted translation was sufficient to recover Otx2 requirements for specification of the anterior neural plate and proper organization of the primitive streak, thus providing evidence that the difference between Otx1 and Otx2 null mice phenotypes originates from their divergent expression patterns. Moreover, our data lead us to hypothesize that the differential post-transcriptional control existing between VE and epiblast cells may potentially contribute to fundamental regulatory mechanisms required for head specification.

Post-Transcriptional Regulation of Cardiac Sarcomere Protein Titin Through 3’ Untranslated Regions

2013 ◽  
Vol 9 ◽  
Author(s):  
Tara A Shrout
Keyword(s):  
Gene Expression ◽  
Transcriptional Control ◽  
Striated Muscle ◽  
Binding Capacity ◽  
Structural Features ◽  
Neonatal Rat ◽  
Regulatory Control ◽  
Untranslated Regions ◽  
Luciferase Reporter ◽  
Regulatory Effects

Titin is the largest known protein in the human body, and forms the backbone of all striated muscle sarcomeres. The elastic nature of titin is an important component of muscle compliance and functionality. A significant amount of energy is expended to synthesize titin, thus we postulate that titin gene expression is under strict regulatory control in order to conserve cellular resources. In general, gene expression is mediated in part by post-transcriptional control elements located within the 5’ and 3’ untranslated regions (UTRs) of mature mRNA. The 3’UTR in particular contains structural features that affect binding capacity to other RNA components, such as MicroRNA, which control mRNA localization, translation, and degradation. The degree and significance of the regulatory effects mediated by two determined variants of titin’s 3’ UTR were evaluated in Neonatal Rat Ventricular Myocyte and Human Embryonic Kidney cell lines. Recombinant plasmids to transfect these cells lines were engineered by insertion of the variant titin 3’UTR 431- and 1047-base pairs sequences into luciferase reporter vectors. Expression due to an unaltered reporter vector served as the control. Quantitative changes in luciferase activity due to the recombinants proportionally represented the effect titin’s respective 3’UTR conferred on downstream post-transcriptional expression relative to the control. The effect due to titin’s shorter 3’UTR sequence was inconclusive; however, results illustrated that titin’s longer 3’UTR sequence caused a 35 percent decrease in protein expression. Secondary structural analysis of the two sequences revealed differential folding patterns that affect the stability and degree of MicroRNA-binding within titin’s variant 3’UTR sequences.

scholarly journals Improved ribosome-footprint and mRNA measurements provide insights into dynamics and regulation of yeast translation

2015 ◽  
Author(s):  
David E Weinberg ◽  
Premal Shah ◽  
Stephen W Eichhorn ◽  
Jeffrey A Hussmann ◽  
Joshua B Plotkin ◽  
...  
Keyword(s):  
Translational Control ◽  
Nucleotide Composition ◽  
Ribosome Profiling ◽  
Open Reading Frames ◽  
Untranslated Regions ◽  
Coding Regions ◽  
Genome Wide ◽  
Upstream Open Reading Frames ◽  
Improved Methods ◽  
Reading Frames

Ribosome-footprint profiling provides genome-wide snapshots of translation, but technical challenges can confound its analysis. Here, we use improved methods to obtain ribosome-footprint profiles and mRNA abundances that more faithfully reflect gene expression in Saccharomyces cerevisiae. Our results support proposals that both the beginning of coding regions and codons matching rare tRNAs are more slowly translated. They also indicate that emergent polypeptides with as few as three basic residues within a 10-residue window tend to slow translation. With the improved mRNA measurements, the variation attributable to translational control in exponentially growing yeast was less than previously reported, and most of this variation could be predicted with a simple model that considered mRNA abundance, upstream open reading frames, cap-proximal structure and nucleotide composition, and lengths of the coding and 5′- untranslated regions. Collectively, our results reveal key features of translational control in yeast and provide a framework for executing and interpreting ribosome- profiling studies.

Abstract 356: KChIP2 Mediates Ion Channel Dysregulation by Novel Transcriptional Regulation of miR-34s

2014 ◽  
Vol 115 (suppl_1) ◽  
Author(s):  
Drew Nassal ◽  
Haiyan Liu ◽  
Xiaoping Wan ◽  
Angelina Ramirez-Navarro ◽  
Eckhard Ficker ◽  
...  
Keyword(s):  
Heart Failure ◽  
Ion Channel ◽  
Transcriptional Control ◽  
Ventricular Myocytes ◽  
Neonatal Rat ◽  
Physical Interaction ◽  
Protein Patterns ◽  
Protein Levels ◽  
Disease States

Introduction: Cardiac ion channel dysregulation is a hallmark of heart failure. Consistently, the disease yields dramatic decline in Ito through loss in Kv4 and its auxiliary partner KChIP2. Notably, transcriptional changes in heart failure can be elicited through KChIP2 silencing without disease signaling, suggesting potential transcriptional capacity for KChIP2. Further, disparity between resulting transcript and protein patterns suggests a mechanism compatible with modified miRNA activity. Considering other members of the KChIP family behave as transcriptional repressors, we hypothesize that KChIP2 regulates discrete miRNAs which in turn regulate cardiac excitability. Methods and Results: A miRNA microarray was conducted on neonatal rat ventricular myocytes (NRVM) following in vitro silencing of KChIP2 by siRNA, identifying the miR-34 family as potential transcriptional targets of KChIP2. Regulation, confirmed by quantitative PCR, showed reduction in miR-34a/b/c when over-expressing KChIP2 and increase following silencing. Luciferase assays were performed on the cloned promoter for miR-34b/c which reinforced direct KChIP2 repression on the miR-34b/c promoter. Furthermore, chromatin immunoprecipitation followed by PCR identified physical interaction of KChIP2 to the promoter site. Previous studies show modified expression of KChIP2 can lead to changes in several ion channel subunits. Therefore, we investigated if this was the consequence of KChIP2 regulation via miR-34. miR-34a/b/c precursors were expressed in NRVM which reduced transcript levels of Nav1.5 and Navβ1, and reduced protein levels for Kv4.3. Reflecting these changes, peak INa was reduced following miR precursor treatment. NRVMs were exposed to 100 μM phenylephrine for 48 hrs, significantly reducing KChIP2, Nav1.5, Navβ1, and Kv4.3, while elevating miR-34b/c. Returning KChIP2 expression by adenovirus normalized these changes back towards baseline, implicating the physiologic relevance of this pathway. Conclusion: These observations describe a novel mechanism where KChIP2 regulates a host of cardiac genes through transcriptional control of miRNAs, potentially explaining electrical remodeling observed in disease states where KChIP2 is reduced.

Vertebrate mRNAs with a 5'-terminal pyrimidine tract are candidates for translational repression in quiescent cells: characterization of the translational cis-regulatory element

1994 ◽  
Vol 14 (6) ◽  
pp. 3822-3833
Author(s):  
D Avni ◽  
S Shama ◽  
F Loreni ◽  
O Meyuhas
Keyword(s):  
Translational Control ◽  
Mammalian Cells ◽  
Regulatory Element ◽  
Elongation Factor ◽  
Translational Efficiency ◽  
Translation Control ◽  
Quiescent Cells ◽  
Terminal Oligopyrimidine ◽  
Mode Of Regulation

The translation of mammalian ribosomal protein (rp) mRNAs is selectively repressed in nongrowing cells. This response is mediated through a regulatory element residing in the 5' untranslated region of these mRNAs and includes a 5' terminal oligopyrimidine tract (5' TOP). To further characterize the translational cis-regulatory element, we monitored the translational behavior of various endogenous and heterologous mRNAs or hybrid transcripts derived from transfected chimeric genes. The translational efficiency of these mRNAs was assessed in cells that either were growing normally or were growth arrested under various physiological conditions. Our experiments have yielded the following results: (i) the translation of mammalian rp mRNAs is properly regulated in amphibian cells, and likewise, amphibian rp mRNA is regulated in mammalian cells, indicating that all of the elements required for translation control of rp mRNAs are conserved among vertebrate classes; (ii) selective translational control is not confined to rp mRNAs, as mRNAs encoding the naturally occurring ubiquitin-rp fusion protein and elongation factor 1 alpha, which contain a 5' TOP, also conform this mode of regulation; (iii) rat rpP2 mRNA contains only five pyrimidines in its 5' TOP, yet this mRNA is translationally controlled in the same fashion as other rp mRNAs with a 5' TOP of eight or more pyrimidines; (iv) full manifestation of this mode of regulation seems to require both the 5' TOP and sequences immediately downstream; and (v) an intact translational regulatory element from rpL32 mRNA fails to exert its regulatory properties even when preceded by a single A residue.

Integrative Data Analysis for Biological Discovery

2011 ◽  
pp. 1058-1065
Author(s):  
Sai Moturu
Keyword(s):  
Molecular Biology ◽  
Biological System ◽  
Molecular Level ◽  
Biological Systems ◽  
Integrated Analysis ◽  
John Muir ◽  
Complex Biological System ◽  
Protein Levels ◽  
Lofty Goal ◽  
Complex Relationships

As John Muir noted, “When we try to pick out anything by itself, we find it hitched to everything else in the Universe” (Muir, 1911). In tune with Muir’s elegantly stated notion, research in molecular biology is progressing toward a systems level approach, with a goal of modeling biological systems at the molecular level. To achieve such a lofty goal, the analysis of multiple datasets is required to form a clearer picture of entire biological systems (Figure 1). Traditional molecular biology studies focus on a specific process in a complex biological system. The availability of high-throughput technologies allows us to sample tens of thousands of features of biological samples at the molecular level. Even so, these are limited to one particular view of a biological system governed by complex relationships and feedback mechanisms on a variety of levels. Integrated analysis of varied biological datasets from the genetic, translational, and protein levels promises more accurate and comprehensive results, which help discover concepts that cannot be found through separate, independent analyses. With this article, we attempt to provide a comprehensive review of the existing body of research in this domain.

MiR-664-2 impacts pubertal development in a precocious-puberty rat model through targeting the NMDA receptor-1†

2019 ◽  
Vol 100 (6) ◽  
pp. 1536-1548 ◽  
Author(s):  
Minda Ju ◽  
Liu Yang ◽  
Jing Zhu ◽  
Zhejun Chen ◽  
Mizhen Zhang ◽  
...  
Keyword(s):  
Precocious Puberty ◽  
Bioinformatics Analysis ◽  
Pubertal Development ◽  
Follicle Stimulating Hormone ◽  
Gonadotropin Releasing Hormone ◽  
Untranslated Regions ◽  
Vaginal Opening ◽  
Protein Levels ◽  
Pulsatile Gnrh ◽  
Puberty Onset

Abstract Precocious puberty (PP) commonly results from premature activation of the hypothalamic–pituitary–gonadal axis (HPGA). Gonadotropin-releasing hormone (GnRH) is the initial trigger for HPGA activation and plays an important role in puberty onset. N-methyl-D-aspartate (NMDA) can promote pulsatile GnRH secretion and accelerates puberty onset. However, the mechanism of N-methyl-D-aspartate receptors (NMDARs) in PP pathogenesis remains obscure. We found that serum GnRH, luteinizing hormone (LH), follicle-stimulating hormone (FSH), estrogen (E2) levels, hypothalamic NMDAR1, and GnRH mRNA expression peaked at the vaginal opening (VO) day. Next, the hypothalamic NMDAR1 mRNA and protein levels in rats treated with danazol, a chemical commonly effecting on the reproductive system, were significantly increased at the VO day (postnatal day 24) compared to controls, accompanied by enhanced serum GnRH, LH, FSH, and E2 levels. Further, microRNA-664-2 (miR-664-2) was selected after bioinformatics analysis and approved in primary hypothalamic neurons, which binds to the 3′-untranslated regions of NMDAR1. Consistently, the miR-664-2 expression in hypothalamus of the Danazol group was decreased compared to Vehicle. Our results suggested that attenuated miR-664-2 might participate in PP pathogenesis through enhancing the NMDAR1 signaling.

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