scholarly journals Premature translation termination mediates triosephosphate isomerase mRNA degradation.

1988 ◽  
Vol 8 (2) ◽  
pp. 802-813 ◽  
Author(s):  
I O Daar ◽  
L E Maquat
Keyword(s):  
Amino Acids ◽  
Mrna Stability ◽  
Half Life ◽  
Triosephosphate Isomerase ◽  
Stop Codon ◽  
Translation Termination ◽  
Mrna Structure ◽  
Mrna Half Life ◽  
Premature Translation Termination

We characterized an anemia-inducing mutation in the human gene for triosephosphate isomerase (TPI) that resulted in the production of prematurely terminated protein and mRNA with a reduced cytoplasmic half-life. The mutation converted a CGA arginine codon to a TGA nonsense codon and generated a protein of 188 amino acids, instead of the usual 248 amino acids. To determine how mRNA primary structure and translation influence mRNA stability, in vitro-mutagenized TPI alleles were introduced into cultured L cells and analyzed for their effect on TPI RNA metabolism. Results indicated that mRNA stability is decreased by all nonsense and frameshift mutations. To determine the relative contribution of the changes in mRNA structure and translation to the altered half-life, the effects of individual mutations were compared with the effects of second-site reversions that restored translation termination to normal. All mutations that resulted in premature translation termination reduced the mRNA half-life solely or mainly by altering the length of the mRNA that was translated. The only mutation that altered translation termination and that reduced the mRNA half-life mainly by affecting the mRNA structure was an insertion that shifted termination to a position downstream of the normal stop codon.

Premature translation termination mediates triosephosphate isomerase mRNA degradation

1988 ◽  
Vol 8 (2) ◽  
pp. 802-813 ◽  
Author(s):  
I O Daar ◽  
L E Maquat
Keyword(s):  
Amino Acids ◽  
Mrna Stability ◽  
Half Life ◽  
Triosephosphate Isomerase ◽  
Stop Codon ◽  
Translation Termination ◽  
Mrna Structure ◽  
Mrna Half Life ◽  
Premature Translation Termination

We characterized an anemia-inducing mutation in the human gene for triosephosphate isomerase (TPI) that resulted in the production of prematurely terminated protein and mRNA with a reduced cytoplasmic half-life. The mutation converted a CGA arginine codon to a TGA nonsense codon and generated a protein of 188 amino acids, instead of the usual 248 amino acids. To determine how mRNA primary structure and translation influence mRNA stability, in vitro-mutagenized TPI alleles were introduced into cultured L cells and analyzed for their effect on TPI RNA metabolism. Results indicated that mRNA stability is decreased by all nonsense and frameshift mutations. To determine the relative contribution of the changes in mRNA structure and translation to the altered half-life, the effects of individual mutations were compared with the effects of second-site reversions that restored translation termination to normal. All mutations that resulted in premature translation termination reduced the mRNA half-life solely or mainly by altering the length of the mRNA that was translated. The only mutation that altered translation termination and that reduced the mRNA half-life mainly by affecting the mRNA structure was an insertion that shifted termination to a position downstream of the normal stop codon.

scholarly journals Detailed Dissection and Critical Evaluation of the Pfizer/BioNTech and Moderna mRNA Vaccines

Vaccines ◽  
2021 ◽  
Vol 9 (7) ◽  
pp. 734
Author(s):  
Xuhua Xia
Keyword(s):  
Codon Usage ◽  
Translation Initiation ◽  
Mrna Stability ◽  
Stop Codon ◽  
Translation Termination ◽  
Critical Evaluation ◽  
Immunization Programs ◽  
Translation Elongation ◽  
Different Types ◽  
Similarities And Differences

The design of Pfizer/BioNTech and Moderna mRNA vaccines involves many different types of optimizations. Proper optimization of vaccine mRNA can reduce dosage required for each injection leading to more efficient immunization programs. The mRNA components of the vaccine need to have a 5’-UTR to load ribosomes efficiently onto the mRNA for translation initiation, optimized codon usage for efficient translation elongation, and optimal stop codon for efficient translation termination. Both 5’-UTR and the downstream 3’-UTR should be optimized for mRNA stability. The replacement of uridine by N1-methylpseudourinine () complicates some of these optimization processes because is more versatile in wobbling than U. Different optimizations can conflict with each other, and compromises would need to be made. I highlight the similarities and differences between Pfizer/BioNTech and Moderna mRNA vaccines and discuss the advantage and disadvantage of each to facilitate future vaccine improvement. In particular, I point out a few optimizations in the design of the two mRNA vaccines that have not been performed properly.

scholarly journals CTELS: A Cell-Free System for the Analysis of Translation Termination Rate

Biomolecules ◽  
2020 ◽  
Vol 10 (6) ◽  
pp. 911 ◽  
Author(s):  
Kseniya A. Lashkevich ◽  
Valeriya I. Shlyk ◽  
Artem S. Kushchenko ◽  
Vadim N. Gladyshev ◽  
Elena Z. Alkalaeva ◽  
...  
Keyword(s):  
Stop Codon ◽  
Translation Termination ◽  
Protein Biosynthesis ◽  
Inhibitory Effect ◽  
In Vitro Translation ◽  
Nonsense Suppression ◽  
Translation System ◽  
Free System ◽  
Termination Rate

Translation termination is the final step in protein biosynthesis when the synthesized polypeptide is released from the ribosome. Understanding this complex process is important for treatment of many human disorders caused by nonsense mutations in important genes. Here, we present a new method for the analysis of translation termination rate in cell-free systems, CTELS (for C-terminally extended luciferase-based system). This approach was based on a continuously measured luciferase activity during in vitro translation reaction of two reporter mRNA, one of which encodes a C-terminally extended luciferase. This extension occupies a ribosomal polypeptide tunnel and lets the completely synthesized enzyme be active before translation termination occurs, i.e., when it is still on the ribosome. In contrast, luciferase molecule without the extension emits light only after its release. Comparing the translation dynamics of these two reporters allows visualization of a delay corresponding to the translation termination event. We demonstrated applicability of this approach for investigating the effects of cis- and trans-acting components, including small molecule inhibitors and read-through inducing sequences, on the translation termination rate. With CTELS, we systematically assessed negative effects of decreased 3′ UTR length, specifically on termination. We also showed that blasticidin S implements its inhibitory effect on eukaryotic translation system, mostly by affecting elongation, and that an excess of eRF1 termination factor (both the wild-type and a non-catalytic AGQ mutant) can interfere with elongation. Analysis of read-through mechanics with CTELS revealed a transient stalling event at a “leaky” stop codon context, which likely defines the basis of nonsense suppression.

Glucocorticoids regulate NHE-3 transcription in OKP cells

1996 ◽  
Vol 270 (1) ◽  
pp. F164-F169 ◽  
Author(s):  
M. Baum ◽  
M. Amemiya ◽  
V. Dwarakanath ◽  
R. J. Alpern ◽  
O. W. Moe
Keyword(s):  
Gene Transcription ◽  
Half Life ◽  
Time Course ◽  
In Vitro Transcription ◽  
Mrna Abundance ◽  
Proton Secretion ◽  
Threefold Increase ◽  
Mrna Half Life ◽  
Synthetic Glucocorticoid

OKP cells express NHE-3, an amiloride-resistant Na+/H+ antiporter, which is likely an isoform responsible for apical proton secretion by the proximal tubule. We have previously shown that an amiloride-resistant Na+/H+ antiporter in OKP cells is regulated by dexamethasone, a synthetic glucocorticoid. The purpose of the present study was to examine the mechanism for the glucocorticoid-mediated increase in Na+/H+ antiporter activity. Incubation of OKP cells with 10(-6) M dexamethasone resulted in a two- to threefold increase in NHE-3 mRNA abundance. This increase was seen after 4 h of incubation with dexamethasone, a time course similar to that found for Na+/H+ antiporter activity. To examine the mechanism for the increase in NHE-3 mRNA abundance, mRNA half-life and in vitro transcription experiments were performed. NHE-3 mRNA had a half-life of 8 h in control and dexamethasone-treated cells. The rate of in vitro transcription was 1.8-fold greater when OKP cells were treated with dexamethasone. These data suggest that the glucocorticoid-mediated increase in Na+/H+ antiporter activity is due to an increase in NHE-3 gene transcription.

Transcriptional and post-transcriptional regulation of prolactin during the turkey reproductive cycle

1997 ◽  
Vol 18 (3) ◽  
pp. 223-231 ◽  
Author(s):  
Z Tong ◽  
G R Pitts ◽  
D N Foster ◽  
M E El Halawani
Keyword(s):  
Mrna Stability ◽  
Half Life ◽  
Northern Blot Analysis ◽  
Transcription Inhibitor ◽  
Steady State Levels ◽  
Incubating Birds ◽  
Plasma Prl ◽  
Photoinduced Changes ◽  
Post Transcriptional Regulation ◽  
Mrna Half Life

ABSTRACT The present study examined turkey prolactin (PRL) transcription and PRL mRNA stability during different reproductive stages. Nuclear run-on transcription assays were performed using isolated nuclei from pituitaries of turkeys at different reproductive stages. Meanwhile, cytoplasmic PRL mRNA and plasma PRL were measured by slot blot and RIA respectively. The PRL transcription, pituitary cytoplasmic PRL mRNA abundance and plasma PRL levels increased after photostimulation and peaked at the incubating stage (P<0·05). A decrease in PRL transcription, pituitary cytoplasmic PRL mRNA and plasma PRL (P<0·05) was observed during the transition from incubation to photorefractoriness. Nest-deprivation reduced circulating PRL (P<0·05), whereas pituitary cytoplasmic PRL mRNA and PRL transcription were not significantly altered from those in incubating birds (P>0·05). The half-life of PRL mRNA was determined in pituitaries of non-photostimulated, laying, incubating and photorefractory hens. Primary pituitary cell cultures were treated with the transcription inhibitor actinomycin-D and the decay of the pre-existing PRL mRNA was quantified using Northern blot analysis. The PRL mRNA half-life was 1·5- and 1·4-fold greater in incubating and laying birds respectively than in non-photostimulated turkeys (P<0·05). The half-life of PRL mRNA in photorefractory and incubating hens was similar in spite of great differences in pituitary PRL mRNA steady-state levels and PRL transcription. Our data suggest that photoinduced changes in pituitary PRL mRNA and plasma PRL are due to changes in both PRL transcription and PRL mRNA stability. Nest-deprivation inhibits the PRL releasing mechanism(s) independently of PRL transcription in turkeys.

scholarly journals Regulation of thrombospondin-1 expression through AU-rich elements in the 3′UTR of the mRNA

2011 ◽  
Vol 16 (1) ◽  
Author(s):  
Asa Mcgray ◽  
Timothy Gingerich ◽  
James Petrik ◽  
Jonathan Lamarre
Keyword(s):  
Gene Expression ◽  
Mrna Stability ◽  
Half Life ◽  
Rna Binding ◽  
Binding Protein ◽  
Site Directed Mutagenesis ◽  
Hek293 Cells ◽  
Luciferase Reporter ◽  
Thrombospondin 1 ◽  
Mrna Half Life

AbstractThrombospondin-1 (TSP-1) is a matricellular protein that participates in numerous normal and pathological tissue processes and is rapidly modulated by different stimuli. The presence of 8 highly-conserved AU rich elements (AREs) within the 3′-untranslated region (3′UTR) of the TSP-1 mRNA suggests that post-transcriptional regulation is likely to represent one mechanism by which TSP-1 gene expression is regulated. We investigated the roles of these AREs, and proteins which bind to them, in the control of TSP-1 mRNA stability. The endogenous TSP-1 mRNA half-life is approximately 2.0 hours in HEK293 cells. Luciferase reporter mRNAs containing the TSP-1 3′UTR show a similar rate of decay, suggesting that the 3′UTR influences the decay rate. Site-directed mutagenesis of individual and adjacent AREs prolonged reporter mRNA halflife to between 2.2 and 4.4 hours. Mutation of all AREs increased mRNA half life to 8.8 hours, suggesting that all AREs have some effect, but that specific AREs may have key roles in stability regulation. A labeled RNA oligonucleotide derived from the most influential ARE was utilized to purify TSP-1 AREbinding proteins. The AU-binding protein AUF1 was shown to associate with this motif. These studies reveal that AREs in the 3′UTR control TSP-1 mRNA stability and that the RNA binding protein AUF1 participates in this control. These studies suggest that ARE-dependent control of TSP-1 mRNA stability may represent an important component in the control of TSP-1 gene expression.

EFFECT OF LITHIUM ON DEIODINATING ACTIVITY OF VARIOUS RAT TISSUES IN VITRO

1974 ◽  
Vol 76 (2) ◽  
pp. 260-272 ◽  
Author(s):  
P. T. Männistö
Keyword(s):  
Amino Acids ◽  
Lithium Chloride ◽  
Half Life ◽  
Rat Tissues ◽  
Biological Half Life ◽  
Liver And Kidney ◽  
Licl Treatment

ABSTRACT The effect of lithium chloride (LiCl) on the deiodination of iodotyrosines, on the degradation of 125I-L-thyroxine (125I-L-T4) in vitro and on the disappearance of exogenous 125I-L4 and 125I-rat-TSH in vivo was studied in rats. Iodotyrosine deiodination was studied in vitro with three techniques. The whole thyroid lobes were not satisfactory as substrate. When a diluted mixture of prelabelled iodo-amino acids was used as substrate, thyroid homogenates deiodinated iodotyrosines. The reaction was inhibited by boiling and by 3,5-dinitro-L-tyrosine (DNT), but LiCl (2 × 10−2 m) had no effect. When 125I-3-iodo-L-tyrosine (125I-L-MIT) served as substrate, increasing concentrations of thyroid homogenates showed an increasing deicdinating activity, which was stimulated by NADP (1.5 × 10−4 m). Inhibitors of dehalogenase DNT (10−4 and 10 −3 m) and menandione (10−4 m) inhibited deiodination, but LiCl (5×10−3 − 0.1 m) was again without effect. The degradation of 125I-L-T4 by liver and kidney homogenates was inhibited by LiCl (5 × 10−3 − 0.1 m). The disappearance of 125I-L-T4 was studied in rats treated with LiCl for 1 – 4 or 60 – 64 days in vivo. The half-lives were as follows: at 1 –4 days, the control rats 15.9 ± 1.3 h and the LiCl treated rats 19.1 ± 2.1 h (P < 0.05) and at 60 – 64 days 11.2 ± 2.0 h and 66.8 ± 12.3 h (P < respectively. The prolonged half-life in the LiCl treated rats was not due to the decreased excretion of radioactivity in the urine or faeces. The biological half-life of 125I-rat-TSH (11.4 ± 3.2 min) was not modified by LiCl treatment for 5 days. It can be concluded that the antithyroid effect of LiCl neither originates from the inhibition of iodotyrosine deiodination nor from the change in the half-life of TSH. The half-life of thyroxine is prolonged by LiCl, an effect which is perhaps due to the decreased degradation of thyroxine by tissues.

scholarly journals A Nascent Peptide Code for Translational Control of mRNA Stability in Human Cells

2021 ◽  
Author(s):  
Phillip C. Burke ◽  
Heungwon Park ◽  
Arvind Rasi Subramaniam
Keyword(s):  
Amino Acids ◽  
Mrna Stability ◽  
Translational Control ◽  
Gc Content ◽  
Human Cells ◽  
Sequence Motifs ◽  
Coding Sequence ◽  
Ribosome Stalling ◽  
Nascent Peptide

AbstractStability of eukaryotic mRNAs is associated with their codon, amino acid, and GC content. Yet, coding sequence motifs that predictably alter mRNA stability in human cells remain poorly defined. Here, we develop a massively parallel assay to measure mRNA effects of thousands of synthetic and endogenous coding sequence motifs in human cells. We identify several families of simple dipeptide repeats whose translation triggers acute mRNA instability. Rather than individual amino acids, specific combinations of bulky and positively charged amino acids are critical for the destabilizing effects of dipeptide repeats. Remarkably, dipeptide sequences that form extended β strands in silico and in vitro drive ribosome stalling and mRNA instability in vivo. The resulting nascent peptide code underlies ribosome stalling and mRNA-destabilizing effects of hundreds of endogenous peptide sequences in the human proteome. Our work reveals an intrinsic role for the ribosome as a selectivity filter against the synthesis of bulky and aggregation-prone peptides.

scholarly journals MISTERMINATE Mechanistically Links Mitochondrial Dysfunction with Proteostasis Failure

2019 ◽  
Author(s):  
Zhihao Wu ◽  
Ishaq Tantray ◽  
Junghyun Lim ◽  
Songjie Chen ◽  
Yu Li ◽  
...  
Keyword(s):  
Amino Acids ◽  
Mitochondrial Dysfunction ◽  
Complex I ◽  
Mammalian Cells ◽  
Protein C ◽  
Stop Codon ◽  
Translation Termination ◽  
Disease Model ◽  
C Terminus ◽  
Translational Termination

SUMMARYMitochondrial dysfunction and proteostasis failure frequently coexist as hallmarks of neurodegenerative disease. How these pathologies are related is not well understood. Here we describe a phenomenon termed MISTERMINATE (mitochondrial stress-induced translational termination impairment and protein carboxyl terminal extension), which mechanistically links mitochondrial dysfunction with proteostasis failure. We show that mitochondrial dysfunction impairs translational termination of nuclear-encoded mitochondrial mRNAs including complex-I 30kD subunit (C-I30) mRNA, occurring on mitochondrial surface in Drosophila and mammalian cells. Ribosomes stalled at the normal stop codon continue to add to the C-terminus of C-I30 certain amino acids non-coded by mRNA template. C-terminally-extended C-I30 is toxic when assembled into C-I and forms aggregates in the cytosol. Enhancing co-translational quality control prevents C-I30 C-terminal extension and rescues mitochondrial and neuromuscular degeneration in a Parkinson’s disease model. These findings emphasize the importance of efficient translation termination and reveal unexpected link between mitochondrial health and proteome homeostasis mediated by MISTERMINATE.

scholarly journals Nsp1 of SARS-CoV-2 Stimulates Host Translation Termination

2020 ◽  
Author(s):  
Alexey Shuvalov ◽  
Ekaterina Shuvalova ◽  
Nikita Biziaev ◽  
Elizaveta Sokolova ◽  
Konstantin Evmenov ◽  
...  
Keyword(s):  
Stop Codon ◽  
Translation Termination ◽  
Translation System ◽  
Release Factor ◽  
Viral Mrnas ◽  
Codon Recognition ◽  
Free Translation ◽  
Stop Codon Recognition ◽  
A Cell

ABSTRACTThe Nsp1 protein of SARS-CoV-2 regulates the translation of host and viral mRNAs in cells. Nsp1 inhibits host translation initiation by occluding the entry channel of the 40S ribosome subunit. The structural study of SARS-CoV-2 Nsp1-ribosomal complexes reported post-termination 80S complex containing Nsp1 and the eRF1 and ABCE1 proteins. Considering the presence of Nsp1 in the post-termination 80S ribosomal complex simultaneously with eRF1, we hypothesized that Nsp1 may be involved in translation termination. Using a cell-free translation system and reconstituted in vitro translation system, we show that Nsp1 stimulates translation termination in the stop codon recognition stage at all three stop codons. This stimulation targets the release factor 1 (eRF1) and does not affect the release factor 3 (eRF3). The activity of Nsp1 in translation termination is provided by its N-terminal domain and the minimal required part of eRF1 is NM domain. We assume that biological meaning of Nsp1 activity in translation termination is binding with the 80S ribosomes translating host mRNAs and removal them from the pool of the active ribosomes.

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