scholarly journals UU/UA Dinucleotide Frequency Reduction in Coding Regions Results in Increased mRNA Stability and Protein Expression

2012 ◽  
Vol 20 (5) ◽  
pp. 954-959 ◽  
Author(s):  
Maher Al-Saif ◽  
Khalid SA Khabar
Keyword(s):  
Protein Expression ◽  
Mrna Stability ◽  
Dinucleotide Frequency ◽  
Coding Regions

scholarly journals Csde1 binds transcripts involved in protein homeostasis and controls their expression in erythropoiesis

2017 ◽  
Author(s):  
Kat S Moore ◽  
Nurcan Yagci ◽  
Floris van Alphen ◽  
Nahuel A Paolini ◽  
Rastislav Horos ◽  
...  
Keyword(s):  
Protein Expression ◽  
Mrna Expression ◽  
Mrna Stability ◽  
Cold Shock ◽  
Rna Binding ◽  
Mrna Translation ◽  
Mitochondrial Respiratory Chain ◽  
Protein Homeostasis ◽  
Diamond Blackfan Anemia ◽  
Cold Shock Domain

AbstractExpression of the RNA-binding protein Csde1 (Cold shock domain protein e1) is strongly upregulated during erythropoiesis compared to other hematopoietic lineages. In the severe congenital anemia Diamond Blackfan Anemia (DBA), however, Csde1 expression is impaired. Reduced expression of Csde1 in healthy erythroblasts impaired their proliferation and differentiation, which suggests an important role for Csde1 in erythropoiesis. To investigate the cellular pathways controlled by Csde1 in erythropoiesis, we identified the transcripts that physically associate with Csde1 in erythroid cells. These mainly encoded proteins involved in ribogenesis, mRNA translation and protein degradation, but also proteins associated with the mitochondrial respiratory chain and mitosis. Crispr/Cas9-mediated deletion of the first cold shock domain of Csde1 affected RNA expression and/or protein expression of Csde1-bound transcripts. For instance, protein expression of Pabpc1 was enhanced while Pabpc1 mRNA expression was reduced indicating more efficient translation of Pabpc1 followed by negative feedback on mRNA stability. Overall, the effect of reduced Csde1 function on mRNA stability and translation of Csde1-bound transcripts was modest. Clones with complete loss of Csde1, however, could not be generated. We suggest that Csde1 is involved in feed-back control in protein homeostasis and that it dampens stochastic changes in mRNA expression.

scholarly journals MAPK signaling pathways regulate IL-8 mRNA stability and IL-8 protein expression in cystic fibrosis lung epithelial cell lines

2011 ◽  
Vol 300 (1) ◽  
pp. L81-L87 ◽  
Author(s):  
Sharmistha Bhattacharyya ◽  
Usha Gutti ◽  
Jose Mercado ◽  
Chad Moore ◽  
Harvey B. Pollard ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Epithelial Cells ◽  
Protein Expression ◽  
Signaling Pathways ◽  
Mrna Stability ◽  
Mapk Signaling ◽  
Lung Epithelial Cells ◽  
Lung Epithelial ◽  
The Stability ◽  
Mapk Signaling Pathways

Cystic fibrosis (CF) is characterized by a massive proinflammatory phenotype in the lung, caused by mutations in the CFTR gene. IL-8 and other proinflammatory mediators are elevated in the CF airway, and the immediate mechanism may depend on disease-specific stabilization of IL-8 mRNA in CF lung epithelial cells. MAPK signaling pathways impact directly on IL-8 protein expression in CF cells, and we have hypothesized that the mechanism may also involve stabilization of the IL-8 mRNA. To test this hypothesis, we have examined the effects of pharmacological and molecular inhibitors of p38, and downstream MK2, ERK1/2, and JNK, on stability of IL-8 mRNA in CF lung epithelial cells. We previously showed that tristetraprolin (TTP) was constitutively low in CF and that raising TTP destabilized the IL-8 mRNA. We therefore also tested these effects on CF lung epithelial cells stably expressing TTP. TTP binds to AU-rich elements in the 3′-UTR of the IL-8 mRNA. We find that inhibition of p38 and ERK1/2 reduces the stability of IL-8 mRNA in parental CF cells. However, neither intervention further lowers TTP-dependent destabilization of IL-8 mRNA. By contrast, inhibition of the JNK-2 pathway has no effect on IL-8 mRNA stability in parental CF cell, but rather increases the stability of the message in cells expressing high levels of TTP. However, we find that inhibition of ERK1/2 or p38 leads to suppression of the effect of JNK-2 inhibition on IL-8 mRNA stability. These data thus lend support to our hypothesis that constitutive MAPK signaling and proteasomal activity might also contribute, along with aberrantly lower TTP, to the proinflammatory phenotype in CF lung epithelial cells by increasing IL-8 mRNA stability and IL-8 protein expression.

scholarly journals Coding regions affect mRNA stability in human cells

RNA ◽  
2019 ◽  
Vol 25 (12) ◽  
pp. 1751-1764 ◽  
Author(s):  
Ashrut Narula ◽  
James Ellis ◽  
J. Matthew Taliaferro ◽  
Olivia S. Rissland
Keyword(s):  
Mrna Stability ◽  
Human Cells ◽  
Coding Regions

scholarly journals Direct Binding of Ataxin-2 to Distinct Elements in 3′ UTRs Promotes mRNA Stability and Protein Expression

2014 ◽  
Vol 55 (2) ◽  
pp. 186-198 ◽  
Author(s):  
Moe Yokoshi ◽  
Quan Li ◽  
Munetaka Yamamoto ◽  
Hitomi Okada ◽  
Yutaka Suzuki ◽  
...  
Keyword(s):  
Protein Expression ◽  
Mrna Stability ◽  
Direct Binding ◽  
Ataxin 2

scholarly journals Protein Binding to Cis-Motifs in mRNAs Coding Sequence Is Common and Regulates Transcript Stability and the Rate of Translation

Cells ◽  
2021 ◽  
Vol 10 (11) ◽  
pp. 2910
Author(s):  
Ewa A. Grzybowska ◽  
Maciej Wakula
Keyword(s):  
Protein Binding ◽  
High Throughput ◽  
Mrna Stability ◽  
Regulatory Network ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Translational Efficiency ◽  
Coding Sequence ◽  
Transcript Stability ◽  
Coding Regions

Protein binding to the non-coding regions of mRNAs is relatively well characterized and its functionality has been described in many examples. New results obtained by high-throughput methods indicate that binding to the coding sequence (CDS) by RNA-binding proteins is also quite common, but the functions thereof are more obscure. As described in this review, CDS binding has a role in the regulation of mRNA stability, but it has also a more intriguing role in the regulation of translational efficiency. Global approaches, which suggest the significance of CDS binding along with specific examples of CDS-binding RBPs and their modes of action, are outlined here, pointing to the existence of a relatively less-known regulatory network controlling mRNA stability and translation on yet another level.

scholarly journals Exchange of endogenous and heterogeneous yeast terminators in Pichia pastoris to tune mRNA stability and gene expression

2020 ◽  
Vol 48 (22) ◽  
pp. 13000-13012
Author(s):  
Yoichiro Ito ◽  
Goro Terai ◽  
Misa Ishigami ◽  
Noriko Hashiba ◽  
Yasuyuki Nakamura ◽  
...  
Keyword(s):  
Metabolic Engineering ◽  
Pichia Pastoris ◽  
Protein Expression ◽  
Mrna Stability ◽  
The Novel ◽  
Yeast Saccharomyces Cerevisiae ◽  
Expression Levels ◽  
Tunable Range ◽  
The Stability ◽  
Pathway Flux

Abstract In the yeast Saccharomyces cerevisiae, terminator sequences not only terminate transcription but also affect expression levels of the protein-encoded upstream of the terminator. The non-conventional yeast Pichia pastoris (syn. Komagataella phaffii) has frequently been used as a platform for metabolic engineering but knowledge regarding P. pastoris terminators is limited. To explore terminator sequences available to tune protein expression levels in P. pastoris, we created a ‘terminator catalog’ by testing 72 sequences, including terminators from S. cerevisiae or P. pastoris and synthetic terminators. Altogether, we found that the terminators have a tunable range of 17-fold. We also found that S. cerevisiae terminator sequences maintain function when transferred to P. pastoris. Successful tuning of protein expression levels was shown not only for the reporter gene used to define the catalog but also using betaxanthin production as an example application in pathway flux regulation. Moreover, we found experimental evidence that protein expression levels result from mRNA abundance and in silico evidence that levels reflect the stability of mRNA 3′-UTR secondary structure. In combination with promoter selection, the novel terminator catalog constitutes a basic toolbox for tuning protein expression levels in metabolic engineering and synthetic biology in P. pastoris.

scholarly journals Combinatorial optimization of mRNA structure, stability, and translation for RNA-based therapeutics

2021 ◽  
Author(s):  
Kathrin Leppek ◽  
Gun Woo Byeon ◽  
Wipapat Kladwang ◽  
Hannah K. Wayment-Steele ◽  
Craig H. Kerr ◽  
...  
Keyword(s):  
Protein Expression ◽  
Mrna Stability ◽  
Hydrolytic Degradation ◽  
Structure Stability ◽  
Solution Stability ◽  
Experimental Platform ◽  
Mrna Structure ◽  
Cell Stability ◽  
Nucleoside Modification ◽  
Protein Output

SUMMARYTherapeutic mRNAs and vaccines are being developed for a broad range of human diseases, including COVID-19. However, their optimization is hindered by mRNA instability and inefficient protein expression. Here, we describe design principles that overcome these barriers. We develop a new RNA sequencing-based platform called PERSIST-seq to systematically delineate in-cell mRNA stability, ribosome load, as well as in-solution stability of a library of diverse mRNAs. We find that, surprisingly, in-cell stability is a greater driver of protein output than high ribosome load. We further introduce a method called In-line-seq, applied to thousands of diverse RNAs, that reveals sequence and structure-based rules for mitigating hydrolytic degradation. Our findings show that “superfolder” mRNAs can be designed to improve both stability and expression that are further enhanced through pseudouridine nucleoside modification. Together, our study demonstrates simultaneous improvement of mRNA stability and protein expression and provides a computational-experimental platform for the enhancement of mRNA medicines.

scholarly journals Rapid generation of hypomorphic mutations

2017 ◽  
Vol 8 (1) ◽  
Author(s):  
Laura L. Arthur ◽  
Joyce J. Chung ◽  
Preetam Janakirama ◽  
Kathryn M. Keefer ◽  
Igor Kolotilin ◽  
...  
Keyword(s):  
Protein Expression ◽  
Mrna Stability ◽  
Model Organisms ◽  
Promoter Strength ◽  
Temporal Expression ◽  
Allelic Series ◽  
Translation Elongation ◽  
Gene Coding ◽  
Adenosine Nucleotides ◽  
Rapid Generation

Abstract Hypomorphic mutations are a valuable tool for both genetic analysis of gene function and for synthetic biology applications. However, current methods to generate hypomorphic mutations are limited to a specific organism, change gene expression unpredictably, or depend on changes in spatial-temporal expression of the targeted gene. Here we present a simple and predictable method to generate hypomorphic mutations in model organisms by targeting translation elongation. Adding consecutive adenosine nucleotides, so-called polyA tracks, to the gene coding sequence of interest will decrease translation elongation efficiency, and in all tested cell cultures and model organisms, this decreases mRNA stability and protein expression. We show that protein expression is adjustable independent of promoter strength and can be further modulated by changing sequence features of the polyA tracks. These characteristics make this method highly predictable and tractable for generation of programmable allelic series with a range of expression levels.

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