scholarly journals Feedback Inhibition of Poly(A)-binding Protein mRNA Translation

2001 ◽  
Vol 276 (50) ◽  
pp. 47352-47360 ◽  
Author(s):  
Jnanankur Bag
Keyword(s):  
Translational Control ◽  
Feedback Inhibition ◽  
Ribosomal Subunit ◽  
Mrna Translation ◽  
Regulatory Sequence ◽  
Free System ◽  
Ribosomal Subunits ◽  
Nuclease Digestion ◽  
Protected Fragment ◽  
Reticulocyte Lysate

An adenine-richciselement at the 5′-untranslated region (UTR) of Pabp1 mRNA is able to inhibit translation of its own mRNA. Similar inhibition of translation of a reporter β-galactosidase mRNA is observed when the adenine-rich auto regulatory sequence (ARS) is placed within the 5′-UTR of this mRNA. For this translational control the distance of the ARS from the 5′ cap is not important. However, it determines the number of 40 S ribosomal subunits bound to the translationally arrested mRNA. Inhibition of mRNA translation by this regulatory sequence occurs at the step of joining of the 60 S ribosomal subunit to the pre-initiation complex. Translational arrest of the ARS containing mRNA in a rabbit reticulocyte lysate cell-free system in the presence of exogenous Pabp1 protects the 5′-flanking region of the ARS from nuclease digestion. This protection depends on the binding of the 40 S ribosomal subunit to the mRNA. The size and the sequence of the nucleotide-protected fragment depends on the location of the ARS within the 5′-UTR. When the ARS is located at a distance of about 78 nucleotides from the 5′ cap, a 40-nucleotide long region adjacent to the ARS is protected. On the other hand, when the ARS is moved further away from the 5′ cap to a distance of ∼267 nucleotides, a 100-nucleotide-long region adjacent to the ARS is protected from nuclease digestion. Nuclease protection is attributed to the presence of one or more stalled 40 S ribosomal subunits near the Pabp1-bound ARS.

scholarly journals Deletion of the PAT1 Gene Affects Translation Initiation and Suppresses a PAB1 Gene Deletion in Yeast

2000 ◽  
Vol 20 (10) ◽  
pp. 3538-3549 ◽  
Author(s):  
Françoise Wyers ◽  
Michèle Minet ◽  
Marie Elisabeth Dufour ◽  
Le Thuy Anh Vo ◽  
François Lacroute
Keyword(s):  
Translation Initiation ◽  
Gene Deletion ◽  
Ribosomal Subunit ◽  
Large Decrease ◽  
Free System ◽  
Ribosomal Subunits ◽  
40S Ribosomal Subunit ◽  
Initiation Of Translation

ABSTRACT The yeast poly(A) binding protein Pab1p mediates the interactions between the 5′ cap structure and the 3′ poly(A) tail of mRNA, whose structures synergistically activate translation in vivo and in vitro. We found that deletion of the PAT1 (YCR077c) gene suppresses a PAB1 gene deletion and that Pat1p is required for the normal initiation of translation. A fraction of Pat1p cosediments with free 40S ribosomal subunits on sucrose gradients. ThePAT1 gene is not essential for viability, although disruption of the gene severely impairs translation initiation in vivo, resulting in the accumulation of 80S ribosomes and in a large decrease in the amounts of heavier polysomes. Pat1p contributes to the efficiency of translation in a yeast cell-free system. However, the synergy between the cap structure and the poly(A) tail is maintained in vitro in the absence of Pat1p. Analysis of translation initiation intermediates on gradients indicates that Pat1p acts at a step before or during the recruitment of the 40S ribosomal subunit by the mRNA, a step which may be independent of that involving Pab1p. We conclude that Pat1p is a new factor involved in protein synthesis and that Pat1p might be required for promoting the formation or the stabilization of the preinitiation translation complexes.

scholarly journals Juxtacap Nucleotide Sequence Modulates eIF4E Binding and Translation

2017 ◽  
Author(s):  
Heather R. Keys ◽  
David M. Sabatini
Keyword(s):  
Translational Control ◽  
Mrna Translation ◽  
Translational Efficiency ◽  
Biological Processes ◽  
Multiprotein Complex ◽  
Free System ◽  
Binding Motifs ◽  
Important Target ◽  
A Cell ◽  
Almost All

ABSTRACTmRNA translation is an energetically costly activity required for almost all biological processes. The multiprotein complex eIF4F, which bridges the 5’ cap and the polyA tail through eIF4E and eIF4G, respectively, is necessary for efficient translation initiation of most mRNAs and is an important target of translational control. Previous work suggests that cap-proximal nucleotides can modulate eIF4E binding to mRNAs, but the effect of specific cap-proximal nucleotide sequences on eIF4E recruitment and the ultimate consequences for translation remain unknown. Using RNA Bind-n-Seq on a model 5’ UTR, we systematically identify eIF4E-intrinsic cap-proximal nucleotide binding preferences. mRNAs with highly-bound motifs are translated well in a cell-free system, whereas those with low-eIF4E-binding motifs are not. However, eIF4E juxtacap motif preferences do not dictate the ribosome occupancy of endogenous mRNAs in cells, suggesting that the effect of juxtacap sequence on eIF4E binding and translation is mRNA-context-dependent. Accordingly, a single downstream point mutation that disrupts a predicted base pair with a preferred juxtacap nucleotide increases translation. The juxtacap sequence is a previously unappreciated determinant of eIF4E recruitment to mRNAs, and we propose that differences in mRNA 5’ end accessibility defined by the juxtacap sequence are important for establishing translational efficiency.

scholarly journals Loss of Translational Control in Yeast Compromised for the Major mRNA Decay Pathway

2004 ◽  
Vol 24 (7) ◽  
pp. 2998-3010 ◽  
Author(s):  
L. E. A. Holmes ◽  
S. G. Campbell ◽  
S. K. De Long ◽  
A. B. Sachs ◽  
M. P. Ashe
Keyword(s):  
Amino Acid ◽  
Translation Initiation ◽  
Translational Control ◽  
Mrna Decay ◽  
Ribosomal Subunit ◽  
Mrna Translation ◽  
Amino Acid Starvation ◽  
Eif2α Phosphorylation ◽  
40S Ribosomal Subunit ◽  
Response To Stress

ABSTRACT The cytoplasmic fate of mRNAs is dictated by the relative activities of the intimately connected mRNA decay and translation initiation pathways. In this study, we have found that yeast strains compromised for stages downstream of deadenylation in the major mRNA decay pathway are incapable of inhibiting global translation initiation in response to stress. In the past, the paradigm of the eIF2α kinase-dependent amino acid starvation pathway in yeast has been used to evaluate this highly conserved stress response in all eukaryotic cells. Using a similar approach we have found that even though the mRNA decay mutants maintain high levels of general translation, they exhibit many of the hallmarks of amino acid starvation, including increased eIF2α phosphorylation and activated GCN4 mRNA translation. Therefore, these mutants appear translationally oblivious to decreased ternary complex abundance, and we propose that this is due to higher rates of mRNA recruitment to the 40S ribosomal subunit.

On the specificity of antisense RNA to arrest in vitro translation of mRNA coding for Drosophila hsp 23

1987 ◽  
Vol 7 (3) ◽  
pp. 239-246 ◽  
Author(s):  
Louis M. Nicole ◽  
Robert M. Tanguay
Keyword(s):  
Antisense Rna ◽  
Mrna Translation ◽  
In Vitro Translation ◽  
Free System ◽  
Small Hsps ◽  
Reticulocyte Lysate ◽  
Shock Proteins ◽  
Translational Level ◽  
T7 Polymerase

The specificity of action of antisense RNA for one of Drosophila low molecular weight heat shock proteins (hsp 23) was tested at the translational level using the rabbit reticulocyte lysate cell-free system. T7 polymerase-driven transcripts of hsp 23 in the antisense orientation were mixed with mRNA from heat-shocked cells under various stringency conditions prior to translation in vitro. Although the four small hsps show considerable sequence homology in their coding sequences, antisense hsp 23 RNA was shown to specifically inhibit hsp 23 mRNA translation under both high (formamide, 45°C and low stringency (37°C conditions. This suggests that the 5′ leader and the ribosome binding region of mRNA are of prime importance in the specificity of action of antisense RNA at the translational level.

Three-dimensional reconstruction of the 40S ribosomal subunit from rabbit reticulocytes

1987 ◽  
Vol 45 ◽  
pp. 936-937
Author(s):  
Neng-Yu Zhang ◽  
Terence Wagenknecht ◽  
Michael Radermacher ◽  
Tom Obrig ◽  
Joachim Frank
Keyword(s):  
Three Dimensional ◽  
Ribosomal Subunit ◽  
Uranyl Acetate ◽  
Reconstruction Method ◽  
Single Exposure ◽  
Electron Dose ◽  
Ribosomal Subunits ◽  
40S Ribosomal Subunit ◽  
Dimensional Reconstruction ◽  
Rabbit Reticulocytes

We have reconstructed the 40S ribosomal subunit at a resolution of 4 nm using the single-exposure pseudo-conical reconstruction method of Radermacher et al.Small (40S) ribosomal subunits were Isolated from rabbit reticulocytes, applied to grids and negatively stained (0.5% uranyl acetate) in a manner that “sandwiches” the specimen between two layers of carbon. Regions of the grid exhibiting uniform and thick staining were identified and photographed twice (magnification 49,000X). The first micrograph was always taken with the specimen tilted by 50° and the second was of the Identical area untilted (Fig. 1). For each of the micrographs the specimen was subjected to an electron dose of 2000-3000 el/nm2.Three hundred thirty particles appearing in the L view (defined in [4]) were selected from both tilted- and untilted-specimen micrographs. The untilted particles were aligned and their rotational alignment produced the azimuthal angles of the tilted particles in the conical tilt series.

scholarly journals The Putative RNA Helicase Dbp6p Functionally Interacts With Rpl3p, Nop8p and the Novel trans-acting Factor Rsa3p During Biogenesis of 60S Ribosomal Subunits in Saccharomyces cerevisiae

Genetics ◽  
2004 ◽  
Vol 166 (4) ◽  
pp. 1687-1699
Author(s):  
Jesús de la Cruz ◽  
Thierry Lacombe ◽  
Olivier Deloche ◽  
Patrick Linder ◽  
Dieter Kressler
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Ribosome Biogenesis ◽  
Null Allele ◽  
Ribosomal Subunit ◽  
Interaction Network ◽  
The Novel ◽  
Rna Helicases ◽  
Ribosomal Subunits ◽  
Synthetic Lethal ◽  
Synthetically Lethal

Abstract Ribosome biogenesis requires at least 18 putative ATP-dependent RNA helicases in Saccharomyces cerevisiae. To explore the functional environment of one of these putative RNA helicases, Dbp6p, we have performed a synthetic lethal screen with dbp6 alleles. We have previously characterized the nonessential Rsa1p, whose null allele is synthetically lethal with dbp6 alleles. Here, we report on the characterization of the four remaining synthetic lethal mutants, which reveals that Dbp6p also functionally interacts with Rpl3p, Nop8p, and the so-far-uncharacterized Rsa3p (ribosome assembly 3). The nonessential Rsa3p is a predominantly nucleolar protein required for optimal biogenesis of 60S ribosomal subunits. Both Dbp6p and Rsa3p are associated with complexes that most likely correspond to early pre-60S ribosomal particles. Moreover, Rsa3p is co-immunoprecipitated with protA-tagged Dbp6p under low salt conditions. In addition, we have established a synthetic interaction network among factors involved in different aspects of 60S-ribosomal-subunit biogenesis. This extensive genetic analysis reveals that the rsa3 null mutant displays some specificity by being synthetically lethal with dbp6 alleles and by showing some synthetic enhancement with the nop8-101 and the rsa1 null allele.

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 The Role of Translation Initiation Regulation in Haematopoiesis

2012 ◽  
Vol 2012 ◽  
pp. 1-10 ◽  
Author(s):  
Godfrey Grech ◽  
Marieke von Lindern
Keyword(s):  
Gene Expression ◽  
Translation Initiation ◽  
Translational Control ◽  
Molecular Mechanisms ◽  
Rna Binding ◽  
Regulation Of Gene Expression ◽  
Mrna Translation ◽  
Translation Control ◽  
Haematological Disorders

Organisation of RNAs into functional subgroups that are translated in response to extrinsic and intrinsic factors underlines a relatively unexplored gene expression modulation that drives cell fate in the same manner as regulation of the transcriptome by transcription factors. Recent studies on the molecular mechanisms of inflammatory responses and haematological disorders indicate clearly that the regulation of mRNA translation at the level of translation initiation, mRNA stability, and protein isoform synthesis is implicated in the tight regulation of gene expression. This paper outlines how these posttranscriptional control mechanisms, including control at the level of translation initiation factors and the role of RNA binding proteins, affect hematopoiesis. The clinical relevance of these mechanisms in haematological disorders indicates clearly the potential therapeutic implications and the need of molecular tools that allow measurement at the level of translational control. Although the importance of miRNAs in translation control is well recognised and studied extensively, this paper will exclude detailed account of this level of control.

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