scholarly journals mRNA structure regulates protein expression through changes in functional half-life

2019 ◽  
Author(s):  
David M. Mauger ◽  
B. Joseph Cabral ◽  
Vladimir Presnyak ◽  
Stephen V. Su ◽  
David W. Reid ◽  
...  
Keyword(s):  
Secondary Structure ◽  
Protein Expression ◽  
Codon Usage ◽  
Half Life ◽  
High Expression ◽  
Order Structure ◽  
List Type ◽  
Mrna Secondary Structure ◽  
Modified Nucleotides

SummaryMessenger RNAs (mRNAs) encode information in both their primary sequence and their higher order structure. The independent contributions of factors like codon usage and secondary structure to regulating protein expression are difficult to establish as they are often highly correlated in endogenous sequences. Here, we used two approaches, global inclusion of modified nucleotides and rational sequence design of exogenously delivered constructs to understand the role of mRNA secondary structure independent from codon usage. Unexpectedly, highly-expressed mRNAs contained a highly-structured coding sequence (CDS). Modified nucleotides that stabilize mRNA secondary structure enabled high expression across a wide-variety of primary sequences. Using a set of eGFP mRNAs that independently altered codon usage and CDS structure, we find that the structure of the CDS regulates protein expression through changes in functional mRNA half-life (i.e. mRNA being actively translated). This work highlights an underappreciated role of mRNA secondary structure in the regulation of mRNA stability. [150 words]HighlightsProtein expression from modified mRNAs tends to follow the pattern m1 Ψ > U >mo5UProtein expression correlates with mRNA thermodynamic stability: Ψ≈ m1Ψ > U > mo5UA highly structured CDS correlates with high expressionIncreased structured mRNAs extend functional half-life

scholarly journals mRNA structure regulates protein expression through changes in functional half-life

2019 ◽  
Vol 116 (48) ◽  
pp. 24075-24083 ◽  
Author(s):  
David M. Mauger ◽  
B. Joseph Cabral ◽  
Vladimir Presnyak ◽  
Stephen V. Su ◽  
David W. Reid ◽  
...  
Keyword(s):  
Secondary Structure ◽  
Protein Expression ◽  
Codon Usage ◽  
Half Life ◽  
Order Structure ◽  
Messenger Rnas ◽  
Mrna Secondary Structure ◽  
Modified Nucleotides ◽  
Highly Correlated

Messenger RNAs (mRNAs) encode information in both their primary sequence and their higher order structure. The independent contributions of factors like codon usage and secondary structure to regulating protein expression are difficult to establish as they are often highly correlated in endogenous sequences. Here, we used 2 approaches, global inclusion of modified nucleotides and rational sequence design of exogenously delivered constructs, to understand the role of mRNA secondary structure independent from codon usage. Unexpectedly, highly expressed mRNAs contained a highly structured coding sequence (CDS). Modified nucleotides that stabilize mRNA secondary structure enabled high expression across a wide variety of primary sequences. Using a set of eGFP mRNAs with independently altered codon usage and CDS structure, we find that the structure of the CDS regulates protein expression through changes in functional mRNA half-life (i.e., mRNA being actively translated). This work highlights an underappreciated role of mRNA secondary structure in the regulation of mRNA stability.

scholarly journals STIM1 deficiency is linked to Alzheimer’s disease and triggers cell death in SH-SY5Y cells by upregulation of L-type voltage-operated Ca2+ entry

2018 ◽  
Vol 96 (10) ◽  
pp. 1061-1079 ◽  
Author(s):  
Carlos Pascual-Caro ◽  
Maria Berrocal ◽  
Aida M. Lopez-Guerrero ◽  
Alberto Alvarez-Barrientos ◽  
Eulalia Pozo-Guisado ◽  
...  
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Cell Death ◽  
Protein Expression ◽  
Transmembrane Domain ◽  
Neuroblastoma Cell ◽  
Disease Patient ◽  
List Type

Abstract STIM1 is an endoplasmic reticulum protein with a role in Ca2+ mobilization and signaling. As a sensor of intraluminal Ca2+ levels, STIM1 modulates plasma membrane Ca2+ channels to regulate Ca2+ entry. In neuroblastoma SH-SY5Y cells and in familial Alzheimer’s disease patient skin fibroblasts, STIM1 is cleaved at the transmembrane domain by the presenilin-1-associated γ-secretase, leading to dysregulation of Ca2+ homeostasis. In this report, we investigated expression levels of STIM1 in brain tissues (medium frontal gyrus) of pathologically confirmed Alzheimer’s disease patients, and observed that STIM1 protein expression level decreased with the progression of neurodegeneration. To study the role of STIM1 in neurodegeneration, a strategy was designed to knock-out the expression of STIM1 gene in the SH-SY5Y neuroblastoma cell line by CRISPR/Cas9-mediated genome editing, as an in vitro model to examine the phenotype of STIM1-deficient neuronal cells. It was proved that, while STIM1 is not required for the differentiation of SH-SY5Y cells, it is absolutely essential for cell survival in differentiating cells. Differentiated STIM1-KO cells showed a significant decrease of mitochondrial respiratory chain complex I activity, mitochondrial inner membrane depolarization, reduced mitochondrial free Ca2+ concentration, and higher levels of senescence as compared with wild-type cells. In parallel, STIM1-KO cells showed a potentiated Ca2+ entry in response to depolarization, which was sensitive to nifedipine, pointing to L-type voltage-operated Ca2+ channels as mediators of the upregulated Ca2+ entry. The stable knocking-down of CACNA1C transcripts restored mitochondrial function, increased mitochondrial Ca2+ levels, and dropped senescence to basal levels, demonstrating the essential role of the upregulation of voltage-operated Ca2+ entry through Cav1.2 channels in STIM1-deficient SH-SY5Y cell death. Key messages STIM1 protein expression decreases with the progression of neurodegeneration in Alzheimer’s disease. STIM1 is essential for cell viability in differentiated SH-SY5Y cells. STIM1 deficiency triggers voltage-regulated Ca2+ entry-dependent cell death. Mitochondrial dysfunction and senescence are features of STIM1-deficient differentiated cells.

Human Catechol-O-Methyltransferase Haplotypes Modulate Protein Expression by Altering mRNA Secondary Structure

Science ◽  
2006 ◽  
Vol 314 (5807) ◽  
pp. 1930-1933 ◽  
Author(s):  
A. G. Nackley ◽  
S. A. Shabalina ◽  
I. E. Tchivileva ◽  
K. Satterfield ◽  
O. Korchynskyi ◽  
...  
Keyword(s):  
Secondary Structure ◽  
Protein Expression ◽  
Mrna Secondary Structure

Influence of codon usage bias on FGLamide-allatostatin mRNA secondary structure

Peptides ◽  
2011 ◽  
Vol 32 (3) ◽  
pp. 509-517 ◽  
Author(s):  
Francisco Martínez-Pérez ◽  
William G. Bendena ◽  
Belinda S.W. Chang ◽  
Stephen S. Tobe
Keyword(s):  
Secondary Structure ◽  
Codon Usage ◽  
Codon Usage Bias ◽  
Mrna Secondary Structure

scholarly journals 4530 Elucidating the Influence of Chemotherapy (melphalan) and /or C. difficile toxin B Exposure on Beta-catenin Protein Expression in Caco-2 Monolayers

2020 ◽  
Vol 4 (s1) ◽  
pp. 95-95
Author(s):  
Jailene Canales ◽  
Alison Weiss ◽  
Senu Apewokin
Keyword(s):  
Protein Expression ◽  
List Type ◽  
Beta Catenin ◽  
Toxin B ◽  
Protein Levels ◽  
Caco2 Cells ◽  
Adenocarcinoma Cells ◽  
Significant Difference ◽  
Study Population

OBJECTIVES/GOALS: We previously reported that genetic polymorphisms in the beta-catenin gene (CTNNB) are associated with the development of Clostridiodes difficile colitis during autologous stem cell transplantation (https://www-ncbi-nlm-nih-gov.proxy.libraries.uc.edu/pubmed/29594489). To biological validate these findings, we sought to evaluate the development of chemotherapy-associated Clostridiodes difficile infections by assessing the effect of C.difficile toxin B (TcdB) and of using melphalan in beta-catenin protein expression in Caco2 cells. METHODS/STUDY POPULATION: To determine the effect of melphalan and/or C.difficile toxin B on expression of Beta-catenin from human gut epithelial cells: Adenocarcinoma cells (Caco-2) cells were seeded and allowed to grow into monolayersMonolayers were treated with PBS, TcdB, melphalan and/or TcdB + melphalan for 24 hours and then washed with PBSImmunofluorescence was measured on the monolayers to visualize three markers -DAPI-Nuclear Stain (blue),Actin-ccytoskeletal stain (red), B-Catenin (green)Analysis of images with ImageJ (NIH). Statistical analysis of the effect of TcdB and/or melphalan on β-catenin protein levels was determined by One-way ANOVA Cells stained with a primary anti-β catenin antibody and an Alexa-488 secondary antibody were evaluated by flow cytometry to quantify the effect of melphalan and/or C. difficile toxin B on Caco2 cells. RESULTS/ANTICIPATED RESULTS: Immunofluorescent intensity was higher in the control (PSS exposed) cells when compared to melphalan, TcdB and mephalan+TcdB exposed cells (p = 0.026, 0.004 and 0.049 respectively) DISCUSSION/SIGNIFICANCE OF IMPACT: A significant difference was seen in β catenin expression in Caco-2 monolayers exposed to TcdB and/or melphalan. These data support the a role of β-catenin in the pathophysiology of CDI during chemotherapy and support GWAS findings reporting a difference in CDI susceptibility based on β-catenin genotype.

Sign in / Sign up

Export Citation Format

Share Document