scholarly journals Dynamics of hunchback translation in real time and at single mRNA resolution in the Drosophila embryo

Development ◽  
2021 ◽  
pp. dev.196121
Author(s):  
Daisy J. Vinter ◽  
Caroline Hoppe ◽  
Thomas G. Minchington ◽  
Catherine Sutcliffe ◽  
Hilary L. Ashe
Keyword(s):  
Single Molecule ◽  
Translational Regulation ◽  
Mrna Translation ◽  
Drosophila Embryo ◽  
Translational Repression ◽  
Expression Domain ◽  
Culture Cells ◽  
Spatiotemporal Regulation ◽  
Developmental Patterning ◽  
Anterior Posterior

The Hunchback (Hb) transcription factor is critical for anterior-posterior patterning of the Drosophila embryo. Despite the maternal hb mRNA acting as a paradigm for translational regulation, due to its repression in the posterior of the embryo, little is known about the translatability of zygotically transcribed hb mRNAs. Here we adapt the SunTag system, developed for imaging translation at single mRNA resolution in tissue culture cells, to the Drosophila embryo to study the translation dynamics of zygotic hb mRNAs. Using single-molecule imaging in fixed and live embryos, we provide evidence for translational repression of zygotic SunTag-hb mRNAs. While the proportion of SunTag-hb mRNAs translated is initially uniform, translation declines from the anterior over time until it becomes restricted to a posterior band in the expression domain. We discuss how regulated hb mRNA translation may help establish the sharp Hb expression boundary, which is a model for precision and noise during developmental patterning. Overall, our data show how use of the SunTag method on fixed and live embryos is a powerful combination for elucidating spatiotemporal regulation of mRNA translation in Drosophila.

scholarly journals Dynamics of hunchback translation in real time and at single mRNA resolution in the Drosophila embryo

2021 ◽  
Author(s):  
Daisy J. Vinter ◽  
Caroline Hoppe ◽  
Thomas G. Minchington ◽  
Catherine Sutcliffe ◽  
Hilary L. Ashe
Keyword(s):  
Translational Regulation ◽  
Mrna Translation ◽  
Drosophila Embryo ◽  
Translational Repression ◽  
Expression Domain ◽  
Culture Cells ◽  
Tissue Culture Cells ◽  
Spatiotemporal Regulation ◽  
Developmental Patterning ◽  
Anterior Posterior

AbstractThe Hunchback (Hb) transcription factor is critical for anterior-posterior patterning of the Drosophila embryo. Despite the maternal hb mRNA acting as a paradigm for translational regulation, due to its repression in the posterior of the embryo, little is known about the translatability of zygotically transcribed hb mRNAs. Here we adapt the SunTag system, developed for imaging translation at single mRNA resolution in tissue culture cells, to the Drosophila embryo to study the translation dynamics of zygotic hb mRNAs. Using singlemolecule imaging in fixed and live embryos, we provide evidence for translational repression of zygotic SunTag-hb mRNAs. While the proportion of SunTag-hb mRNAs translated is initially uniform, translation declines from the anterior over time until it becomes restricted to a posterior band in the expression domain. We discuss how regulated hb mRNA translation may help establish the sharp Hb expression boundary, which is a model for precision and noise during developmental patterning. Overall, our data show how use of the SunTag method on fixed and live embryos is a powerful combination for elucidating spatiotemporal regulation of mRNA translation in Drosophila.

scholarly journals Regulation of mRNA translation during mitosis

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Marvin E Tanenbaum ◽  
Noam Stern-Ginossar ◽  
Jonathan S Weissman ◽  
Ronald D Vale
Keyword(s):  
Protein Function ◽  
Translational Regulation ◽  
Mrna Translation ◽  
Ribosome Profiling ◽  
Translational Repression ◽  
Anaphase Promoting Complex ◽  
Mitotic Entry ◽  
Primary Mechanism ◽  
Specific Regulation ◽  
Translational Mechanisms

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.

scholarly journals Single-molecule imaging of microRNA-mediated gene silencing in cells

2021 ◽  
Author(s):  
Hotaka Kobayashi ◽  
Robert H Singer
Keyword(s):  
Gene Silencing ◽  
Single Molecule ◽  
Nuclear Export ◽  
Mrna Decay ◽  
Ensemble Methods ◽  
Spatiotemporal Analysis ◽  
Translational Repression ◽  
Single Molecule Level ◽  
Spatiotemporal Information ◽  
Spatiotemporal Regulation

MicroRNAs (miRNAs) are small non-coding RNAs, which regulate the expression of thousands of genes; miRNAs silence gene expression from complementary mRNAs through translational repression and mRNA decay. For decades, the function of miRNAs has been studied primarily by ensemble methods, where a bulk collection of molecules is measured outside cells. Thus, the behavior of individual molecules during miRNA-mediated gene silencing, as well as their spatiotemporal regulation inside cells, remains mostly unknown. Here we report single-molecule methods to visualize each step of miRNA-mediated gene silencing in situ inside cells. Simultaneous visualization of single mRNAs, translation, and miRNA-binding revealed that miRNAs preferentially bind to translated mRNAs rather than untranslated mRNAs. Spatiotemporal analysis based on our methods uncovered that miRNAs bind to mRNAs immediately after nuclear export. Subsequently, miRNAs induced translational repression and mRNA decay within 30 and 60 min, respectively, after the binding to mRNAs. This methodology provides a framework for studying mRNA regulation at the single-molecule level with spatiotemporal information inside cells.

scholarly journals Translational regulation of non-autonomous mitochondrial stress response promotes longevity

2019 ◽  
Author(s):  
Jianfeng Lan ◽  
Jarod Rollins ◽  
Di Wu ◽  
Xiao Zang ◽  
Lina Zou ◽  
...  
Keyword(s):  
Stress Response ◽  
Cytochrome C ◽  
Ribosomal Proteins ◽  
Translational Regulation ◽  
Rna Binding ◽  
Mrna Translation ◽  
Translational Repression ◽  
List Type ◽  
Mitochondrial Stress ◽  
Ribosomal S6 Kinase

SummaryInhibition of mRNA translation delays aging, but the underlying mechanisms remain underexplored. Mutations in both DAF-2 (IGF-1 receptor) and RSKS-1 (ribosomal S6 kinase/S6K) cause synergistic lifespan extension in C. elegans. To understand the roles of S6K-mediated translational regulation in this process, we performed genome-wide translational profiling and genetic screens to identify genes that are not only regulated at the translational level in the daf-2 rsks-1 mutant, but also affect lifespan. Inhibition of CYC-2.1 (cytochrome c) in the germline significantly extends lifespan through non-autonomous activation of the mitochondrial unfolded protein response (UPRmt) and AMP-activated kinase (AMPK) in the metabolic tissue. Furthermore, the RNA-binding protein GLD-1-mediated translational repression of cytochrome c in the germline is important for the non-autonomous activation of UPRmt and synergistic longevity of the daf-2 rsks-1 mutant. Together, these results illustrate a translationally regulated non-autonomous mitochondrial stress response mechanism in the modulation of lifespan by insulin-like signaling and S6K.HighlightsLongevity of the daf-2 rsks-1 mutant is mediated by translational repression of ribosomal proteins and CYC-2.1/cytochrome c.Germline inhibition of cyc-2.1 non-autonomously activates UPRmt and AMPK to extend lifespan.GLD-1 represses germline cyc-2.1 translation in the daf-2 rsks-1 mutant.Translational regulation of cyc-2.1 and UPRmt contribute to the synergistic longevity of the daf-2 rsks-1 mutant.

Conserved and divergent expression aspects of the Drosophila segmentation gene hunchback in the short germ band embryo of the flour beetle Tribolium

Development ◽  
1995 ◽  
Vol 121 (12) ◽  
pp. 4227-4236 ◽  
Author(s):  
C. Wolff ◽  
R. Sommer ◽  
R. Schroder ◽  
G. Glaser ◽  
D. Tautz
Keyword(s):  
Translational Control ◽  
Early Stage ◽  
Posterior Pole ◽  
Drosophila Embryo ◽  
Germ Band ◽  
Expression Domain ◽  
Segmentation Gene ◽  
Flour Beetle ◽  
Different Types ◽  
Anterior Posterior

The segmentation gene hunchback (hb) plays a central role in determining the anterior-posterior pattern in the Drosophila embryo. We have cloned the homologue of hb from the flour beetle Tribolium and show that, on the basis of its expression pattern, most of its functions seem to be conserved between these two species. Like Drosophila, Tribolium has a maternal hb expression that appears to be under translational control by a factor at the posterior pole of the embryo. The maternal expression is followed by a zygotic expression in the region of the developing head and thoracic segments. During germ band extension, a posterior expression domain appears that is likely to be homologous to the posterior blastoderm expression of hb in Drosophila. These observations suggest that hb may have the same functions in early Drosophila and Tribolium development, despite the different types of embryogenesis in these two species (long versus short germ development). One differing aspect of hb expression in Tribolium concerns a structure that is not present in Drosophila, namely the serosa. An hb expression domain at the anterior pole precisely demarcates the border between the extraembryonic serosa and the embryonic field in the Tribolium embryo at an early stage, and hb protein remains expressed in the serosa cells until the end of embryogenesis.

scholarly journals Smart-ORF: a single-molecule method for accessing ribosome dynamics in both upstream and main open reading frames

2020 ◽  
Author(s):  
Anthony Gaba ◽  
Hongyun Wang ◽  
Trinisia Fortune ◽  
Xiaohui Qu
Keyword(s):  
Single Molecule ◽  
Translational Regulation ◽  
Mrna Translation ◽  
Open Reading Frames ◽  
Open Reading Frame ◽  
Upstream Open Reading Frame ◽  
Translation Efficiency ◽  
Reading Frame ◽  
Reading Frames

Abstract Upstream open reading frame (uORF) translation disrupts scanning 43S flux on mRNA and modulates main open reading frame (mORF) translation efficiency. Current tools, however, have limited access to ribosome dynamics in both upstream and main ORFs of an mRNA. Here, we develop a new two-color in vitro fluorescence assay, Smart-ORF, that monitors individual uORF and mORF translation events in real-time with single-molecule resolution. We demonstrate the utility of Smart-ORF by applying it to uORF-encoded arginine attenuator peptide (AAP)-mediated translational regulation. The method enabled quantification of uORF and mORF initiation efficiencies, 80S dwell time, polysome formation, and the correlation between uORF and mORF translation dynamics. Smart-ORF revealed that AAP-mediated 80S stalling in the uORF stimulates the uORF initiation efficiency and promotes clustering of slower uORF-translating ribosomes. This technology provides a new tool that can reveal previously uncharacterized dynamics of uORF-containing mRNA translation.

scholarly journals A UV-Induced Mutation in Neurospora That Affects Translational Regulation in Response to Arginine

Genetics ◽  
1996 ◽  
Vol 142 (1) ◽  
pp. 117-127 ◽  
Author(s):  
Michael Freitag ◽  
Nelima Dighde ◽  
Matthew S Sachs
Keyword(s):  
Translational Control ◽  
Translational Regulation ◽  
Fusion Gene ◽  
Mrna Translation ◽  
Small Subunit ◽  
Upstream Open Reading Frame ◽  
Control Mechanisms ◽  
Carbamoyl Phosphate ◽  
Altered Expression ◽  
Reading Frame

The Neurospora crmsu arg-2 gene encodes the small subunit of arginine-specific carbamoyl phosphate synthetase. The levels of arg-2 mRNA and mRNA translation are negatively regulated by arginine. An upstream open reading frame (uORF) in the transcript’s 5′ region has been implicated in arginine-specific control. An arg-2-hph fusion gene encoding hygromycin phosphotransferase conferred arginine-regulated resistance to hygromycin when introduced into N. crassa. We used an arg-2-hph strain to select for UV-induced mutants that grew in the presence of hygromycin and arginine, and we isolated 46 mutants that had either of two phenotypes. One phenotype indicated altered expression of both arg-2-hph and urg-2 genes; the other, altered expression of urg-2-hph but not arg-2. One of the latter mutations, which was genetically closely linked to arg-2-hph, was recovered from the 5′ region of the arg-2-hph gene using PCR. Sequence analyses and transformation experiments revealed a mutation at uORF codon 12 (Asp to Asn) that abrogated negative regulation. Examination of the distribution of ribosomes on arg-2-hph transcripts showed that loss of regulation had a translational component, indicating the uORF sequence was important for Arg-specific translational control. Comparisons with other uORFS suggest common elements in translational control mechanisms.

scholarly journals Multiple Mechanisms Control Phosphorylation of PHAS-I in Five (S/T)P Sites That Govern Translational Repression

2000 ◽  
Vol 20 (10) ◽  
pp. 3558-3567 ◽  
Author(s):  
Isabelle Mothe-Satney ◽  
Daqing Yang ◽  
Patrick Fadden ◽  
Timothy A. J. Haystead ◽  
John C. Lawrence
Keyword(s):  
Mrna Translation ◽  
Hek293 Cells ◽  
Initiation Factor ◽  
Translational Repression ◽  
Phosphorylation Sites ◽  
Translational Repressor ◽  
Eukaryotic Initiation Factor 4E ◽  
Dependent Processes ◽  
Constitutive Phosphorylation

ABSTRACT Control of the translational repressor, PHAS-I, was investigated by expressing proteins with Ser/Thr → Ala mutations in the five (S/T)P phosphorylation sites. Results of experiments with HEK293 cells reveal at least three levels of control. At one extreme is nonregulated phosphorylation, exemplified by constitutive phosphorylation of Ser82. At an intermediate level, amino acids and insulin stimulate the phosphorylation of Thr36, Thr45, and Thr69 via mTOR-dependent processes that function independently of other sites in PHAS-I. At the third level, the extent of phosphorylation of one site modulates the phosphorylation of another. This control is represented by Ser64 phosphorylation, which depends on the phosphorylation of all three TP sites. The five sites have different influences on the electrophoretic properties of PHAS-I and on the affinity of PHAS-I for eukaryotic initiation factor 4E (eIF4E). Phosphorylation of Thr45 or Ser64 results in the most dramatic decreases in eIF4E binding in vitro. However, each of the sites influences mRNA translation, either directly by modulating the binding affinity of PHAS-I and eIF4E or indirectly by affecting the phosphorylation of other sites.

scholarly journals The GNU subunit of PNG kinase, the developmental regulator of mRNA translation, binds BIC-C to localize to RNP granules

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Emir E Avilés-Pagán ◽  
Masatoshi Hara ◽  
Terry L Orr-Weaver
Keyword(s):  
Translational Regulation ◽  
Mrna Translation ◽  
Mature Oocyte ◽  
Early Embryogenesis ◽  
Egg Activation ◽  
Maternal Mrna ◽  
Rnp Granules

Control of mRNA translation is a key mechanism by which the differentiated oocyte transitions to a totipotent embryo. In Drosophila, the PNG kinase complex regulates maternal mRNA translation at the oocyte-to-embryo transition. We previously showed the GNU activating subunit is crucial in regulating PNG and timing its activity to the window between egg activation and early embryogenesis (Hara et al., 2017). In this study, we find associations between GNU and proteins of RNP granules and demonstrate that GNU localizes to cytoplasmic RNP granules in the mature oocyte, identifying GNU as a new component of a subset of RNP granules. Furthermore, we define roles for the domains of GNU. Interactions between GNU and the granule component BIC-C reveal potential conserved functions for translational regulation in metazoan development. We propose that by binding to BIC-C, upon egg activation GNU brings PNG to its initial targets, translational repressors in RNP granules.

scholarly journals P-bodies and the miRNA pathway regulate translational repression of bicoid mRNA during Drosophila melanogaster oogenesis

2018 ◽  
Author(s):  
John M. McLaughlin ◽  
Daniel F.Q. Smith ◽  
Irina E. Catrina ◽  
Diana P. Bratu
Keyword(s):  
Drosophila Melanogaster ◽  
Small Rna ◽  
Translational Regulation ◽  
Translational Repression ◽  
Temporal Regulation ◽  
P Bodies ◽  
Maternal Transcripts ◽  
Spatio Temporal ◽  
Mirna Pathway ◽  
Rna Pathways

ABSTRACTEmbryonic axis patterning in Drosophila melanogaster is partly achieved by mRNAs that are maternally localized to the oocyte; the spatio-temporal regulation of these transcripts’ stability and translation is a characteristic feature of oogenesis. While protein regulatory factors are necessary for the translational regulation of some maternal transcripts (e.g. oskar and gurken), small RNA pathways are also known to regulate mRNA stability and translation in eukaryotes. MicroRNAs (miRNAs) are small RNA regulators of gene expression, widely conserved throughout eukaryotic genomes and essential for animal development. The main D. melanogaster anterior determinant, bicoid, is maternally transcribed, but it is not translated until early embryogenesis. We investigated the possibility that its translational repression during oogenesis is mediated by miRNA activity. We found that the bicoid 3’UTR contains a highly conserved, predicted binding site for miR-305. Our studies reveal that miR-305 regulates the translation of a reporter gene containing the bicoid 3’UTR in cell culture, and that miR-305 only partially contributes to bicoid mRNA translational repression during oogenesis. We also found that Processing bodies (P-bodies) in the egg chamber may play a role in stabilizing bicoid and other maternal transcripts. Here, we offer insights into the possible role of P-bodies and the miRNA pathway in the translational repression of bicoid mRNA during oogenesis.

Sign in / Sign up

Export Citation Format

Share Document