scholarly journals Secondary Structure of Chloroplast mRNAs In Vivo and In Vitro

Plants ◽  
2020 ◽  
Vol 9 (3) ◽  
pp. 323
Author(s):  
Piotr Gawroński ◽  
Aleksandra Pałac ◽  
Lars B. Scharff
Keyword(s):  
Gene Expression ◽  
Secondary Structure ◽  
Translation Initiation ◽  
Structural Information ◽  
Regulation Of Gene Expression ◽  
Dimethyl Sulfate ◽  
Translation Efficiency ◽  
Mrna Secondary Structure

mRNA secondary structure can influence gene expression, e.g., by influencing translation initiation. The probing of in vivo mRNA secondary structures is therefore necessary to understand what determines the efficiency and regulation of gene expression. Here, in vivo mRNA secondary structure was analyzed using dimethyl sulfate (DMS)-MaPseq and compared to in vitro-folded RNA. We used an approach to analyze specific, full-length transcripts. To test this approach, we chose low, medium, and high abundant mRNAs. We included both monocistronic and multicistronic transcripts. Because of the slightly alkaline pH of the chloroplast stroma, we could probe all four nucleotides with DMS. The structural information gained was evaluated using the known structure of the plastid 16S rRNA. This demonstrated that the results obtained for adenosines and cytidines were more reliable than for guanosines and uridines. The majority of mRNAs analyzed were less structured in vivo than in vitro. The in vivo secondary structure of the translation initiation region of most tested genes appears to be optimized for high translation efficiency.

scholarly journals G-quadruplex RNA motifs influence gene expression in the malaria parasite Plasmodium falciparum

2021 ◽  
Author(s):  
Franck Dumetz ◽  
Eugene Yui-Ching Chow ◽  
Lynne M. Harris ◽  
Mubarak I. Umar ◽  
Anders Jensen ◽  
...  
Keyword(s):  
Gene Expression ◽  
Plasmodium Falciparum ◽  
Malaria Parasite ◽  
Regulation Of Gene Expression ◽  
Low Complexity ◽  
Telomere Maintenance ◽  
Translation Efficiency ◽  
G Quadruplex

ABSTRACTG-quadruplexes are non-helical secondary structures that can fold in vivo in both DNA and RNA. In human cells, they can influence replication, transcription and telomere maintenance in DNA, or translation, transcript processing and stability of RNA. We have previously showed that G-quadruplexes are detectable in the DNA of the malaria parasite Plasmodium falciparum, despite a very highly A/T-biased genome with unusually few guanine-rich sequences. Here, we show that RNA G-quadruplexes can also form in P. falciparum RNA, using rG4-seq for transcriptome-wide structure-specific RNA probing. Many of the motifs, detected here via the rG4seeker pipeline, have non-canonical forms and would not be predicted by standard in silico algorithms. However, in vitro biophysical assays verified the formation of non-canonical motifs. The G-quadruplexes in the P. falciparum transcriptome are frequently clustered in certain genes and associated with regions encoding low-complexity peptide repeats. They are overrepresented in particular classes of genes, notably those that encode PfEMP1 virulence factors, stress response genes and DNA binding proteins. In vitro translation experiments and in vivo measures of translation efficiency showed that G-quadruplexes can influence the translation of P. falciparum mRNAs. Thus, the G-quadruplex is a novel player in post-transcriptional regulation of gene expression in this major human pathogen.

scholarly journals Cytomegalovirus Replicon-Based Regulation of Gene Expression In Vitro and In Vivo

2012 ◽  
Vol 8 (6) ◽  
pp. e1002728 ◽  
Author(s):  
Hermine Mohr ◽  
Christian A. Mohr ◽  
Marlon R. Schneider ◽  
Laura Scrivano ◽  
Barbara Adler ◽  
...  
Keyword(s):  
Gene Expression ◽  
Regulation Of Gene Expression

scholarly journals A Retinoid-Resistant Acute Promyelocytic Leukemia Subclone Expresses a Dominant Negative PML-RARα Mutation

Blood ◽  
1997 ◽  
Vol 89 (12) ◽  
pp. 4282-4289 ◽  
Author(s):  
Wenlin Shao ◽  
Laura Benedetti ◽  
William W. Lamph ◽  
Clara Nervi ◽  
Wilson H. Miller
Keyword(s):  
Gene Expression ◽  
Retinoic Acid ◽  
Ligand Binding ◽  
Cell Line ◽  
Acute Promyelocytic Leukemia ◽  
Regulation Of Gene Expression ◽  
Promyelocytic Leukemia ◽  
Dominant Negative

Abstract The unique t(15; 17) of acute promyelocytic leukemia (APL) fuses the PML gene with the retinoic acid receptor α (RARα) gene. Although retinoic acid (RA) inhibits cell growth and induces differentiation in human APL cells, resistance to RA develops both in vitro and in patients. We have developed RA-resistant subclones of the human APL cell line, NB4, whose nuclear extracts display altered RA binding. In the RA-resistant subclone, R4, we find an absence of ligand binding of PML-RARα associated with a point mutation changing a leucine to proline in the ligand-binding domain of the fusion PML-RARα protein. In contrast to mutations in RARα found in retinoid-resistant HL60 cells, in this NB4 subclone, the coexpressed RARα remains wild-type. In vitro expression of a cloned PML-RARα with the observed mutation in R4 confirms that this amino acid change causes the loss of ligand binding, but the mutant PML-RARα protein retains the ability to heterodimerize with RXRα and thus to bind to retinoid response elements (RAREs). This leads to a dominant negative block of transcription from RAREs that is dose-dependent and not relieved by RA. An unrearranged RARα engineered with this mutation also lost ligand binding and inhibited transcription in a dominant negative manner. We then found that the mutant PML-RARα selectively alters regulation of gene expression in the R4 cell line. R4 cells have lost retinoid-regulation of RXRα and RARβ and the RA-induced loss of PML-RARα protein seen in NB4 cells, but retain retinoid-induction of CD18 and CD38. Thus, the R4 cell line provides data supporting the presence of an RARα-mediated pathway that is independent from gene expression induced or repressed by PML-RARα. The high level of retinoid resistance in vitro and in vivo of cells from some relapsed APL patients suggests similar molecular changes may occur clinically.

scholarly journals Epigenetics and maternal nutrition: nature v. nurture

2010 ◽  
Vol 70 (1) ◽  
pp. 73-81 ◽  
Author(s):  
Rebecca Simmons
Keyword(s):  
Gene Expression ◽  
Maternal Nutrition ◽  
Current Knowledge ◽  
Regulation Of Gene Expression ◽  
Epigenetic Modifications ◽  
Epigenetic Gene Regulation ◽  
Diabetes And Obesity

Under- and over-nutrition during pregnancy has been linked to the later development of diseases such as diabetes and obesity. Epigenetic modifications may be one mechanism by which exposure to an altered intrauterine milieu or metabolic perturbation may influence the phenotype of the organism much later in life. Epigenetic modifications of the genome provide a mechanism that allows the stable propagation of gene expression from one generation of cells to the next. This review highlights our current knowledge of epigenetic gene regulation and the evidence that chromatin remodelling and histone modifications play key roles in adipogenesis and the development of obesity. Epigenetic modifications affecting processes important to glucose regulation and insulin secretion have been described in the pancreatic β-cells and muscle of the intrauterine growth-retarded offspring, characteristics essential to the pathophysiology of type-2 diabetes. Epigenetic regulation of gene expression contributes to both adipocyte determination and differentiation in in vitro models. The contributions of histone acetylation, histone methylation and DNA methylation to the process of adipogenesis in vivo remain to be evaluated.

Applications of cell-penetrating peptides in regulation of gene expression

2007 ◽  
Vol 35 (4) ◽  
pp. 770-774 ◽  
Author(s):  
P. Järver ◽  
K. Langel ◽  
S. El-Andaloussi ◽  
Ü. Langel
Keyword(s):  
Gene Expression ◽  
Lipid Bilayers ◽  
Regulation Of Gene Expression ◽  
Cell Penetrating Peptides ◽  
Remarkable Feature ◽  
Cell Penetrating ◽  
Plasmid Delivery ◽  
Interfering Rna

CPPs (cell-penetrating peptides) can be defined as short peptides that are able to efficiently penetrate cellular lipid bilayers. Because of this remarkable feature, they are excellent candidates regarding alterations in gene expression. CPPs have been utilized in in vivo and in vitro experiments as delivery vectors for different bioactive cargoes. This review focuses on the experiments performed in recent years where CPPs have been used as vectors for multiple effectors of gene expression such as oligonucleotides for antisense, siRNA (small interfering RNA) and decoy dsDNA (double-stranded DNA) applications, and as transfection agents for plasmid delivery.

scholarly journals Signal-Induced Transcriptional Activation by Dif Requires the dTRAP80 Mediator Module

2003 ◽  
Vol 23 (4) ◽  
pp. 1358-1367 ◽  
Author(s):  
Jin Mo Park ◽  
Jung Mo Kim ◽  
Lark Kyun Kim ◽  
Se Nyun Kim ◽  
Jeongsil Kim-Ha ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcriptional Activation ◽  
Specific Binding ◽  
Regulation Of Gene Expression ◽  
Transcriptional Activator ◽  
Mediator Complex ◽  
Activator Proteins ◽  
In Vitro Interactions

ABSTRACT The Mediator complex is the major multiprotein transcriptional coactivator complex in Drosophila melanogaster. Mediator components interact with diverse sets of transcriptional activator proteins to elicit the sophisticated regulation of gene expression. The distinct phenotypes associated with certain mutations in some of the Mediator genes and the specific in vitro interactions of Mediator gene products with transcriptional activator proteins suggest the presence of activator-specific binding subunits within the Mediator complex. However, the physiological relevance of these selective in vitro interactions has not been addressed. Therefore, we analyzed dTRAP80, one of the putative activator-binding subunits of the Mediator, for specificity of binding to a number of natural transcriptional activators from Drosophila. Among the group of activator proteins that requires the Mediator complex for transcriptional activation, only a subset of these proteins interacted with dTRAP80 in vitro and only these dTRAP80-interacting activators were defective for activation under dTRAP80-deficient in vivo conditions. In particular, activation of Drosophila antimicrobial peptide drosomycin gene expression by the NF-κB-like transcription factor Dif during induction of the Toll signaling pathway was dependent on the dTRAP80 module. These results, and the indirect support from the dTRAP80 artificial recruitment assay, indicate that dTRAP80 serves as a genuine activator-binding target responsible for a distinct group of activators.

scholarly journals Gene Codon Composition Determines Differentiation-Dependent Expression of a Viral Capsid Gene in Keratinocytes In Vitro and InVivo

2005 ◽  
Vol 25 (19) ◽  
pp. 8643-8655 ◽  
Author(s):  
Kong-Nan Zhao ◽  
WenYi Gu ◽  
Ning Xia Fang ◽  
Nicholas A. Saunders ◽  
Ian H. Frazer
Keyword(s):  
Gene Expression ◽  
Regulation Of Gene Expression ◽  
Viral Capsid ◽  
Virion Assembly ◽  
Codon Composition ◽  
L1 Protein ◽  
First Time ◽  
Expression Plasmids

ABSTRACT By establishing mouse primary keratinocytes (KCs) in culture, we were able, for the first time, to express papillomavirus major capsid (L1) proteins by transient transfection of authentic or codon-modified L1 gene expression plasmids. We demonstrate in vitro and in vivo that gene codon composition is in part responsible for differentiation-dependent expression of L1 protein in KCs. L1 mRNA was present in similar amounts in differentiated and undifferentiated KCs transfected with authentic or codon-modified L1 genes and had a similar half-life, demonstrating that L1 protein production is posttranscriptionally regulated. We demonstrate further that KCs substantially change their tRNA profiles upon differentiation. Aminoacyl-tRNAs from differentiated KCs but not undifferentiated KCs enhanced the translation of authentic L1 mRNA, suggesting that differentiation-associated change to tRNA profiles enhances L1 expression in differentiated KCs. Thus, our data reveal a novel mechanism for regulation of gene expression utilized by a virus to direct viral capsid protein expression to the site of virion assembly in mature KCs. Analysis of two structural proteins of KCs, involucrin and keratin 14, suggests that translation of their mRNAs is also regulated, in association with KC differentiation in vitro, by a similar mechanism.

scholarly journals Light-Dependent Translation Change of Arabidopsis psbA Correlates with RNA Structure Alterations at the Translation Initiation Region

Cells ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 322
Author(s):  
Piotr Gawroński ◽  
Christel Enroth ◽  
Peter Kindgren ◽  
Sebastian Marquardt ◽  
Stanisław Karpiński ◽  
...  
Keyword(s):  
Secondary Structure ◽  
Translation Initiation ◽  
Rna Structure ◽  
Regulatory Protein ◽  
High Light ◽  
Ribosome Profiling ◽  
General Mechanism ◽  
Mrna Secondary Structure ◽  
Translation Initiation Region

mRNA secondary structure influences translation. Proteins that modulate the mRNA secondary structure around the translation initiation region may regulate translation in plastids. To test this hypothesis, we exposed Arabidopsis thaliana to high light, which induces translation of psbA mRNA encoding the D1 subunit of photosystem II. We assayed translation by ribosome profiling and applied two complementary methods to analyze in vivo RNA secondary structure: DMS-MaPseq and SHAPE-seq. We detected increased accessibility of the translation initiation region of psbA after high light treatment, likely contributing to the observed increase in translation by facilitating translation initiation. Furthermore, we identified the footprint of a putative regulatory protein in the 5′ UTR of psbA at a position where occlusion of the nucleotide sequence would cause the structure of the translation initiation region to open up, thereby facilitating ribosome access. Moreover, we show that other plastid genes with weak Shine-Dalgarno sequences (SD) are likely to exhibit psbA-like regulation, while those with strong SDs do not. This supports the idea that changes in mRNA secondary structure might represent a general mechanism for translational regulation of psbA and other plastid genes.

scholarly journals Selective 40S footprinting reveals that scanning ribosomes remain cap-tethered in human cells

2019 ◽  
Author(s):  
Jonathan Bohlen ◽  
Kai Fenzl ◽  
Günter Kramer ◽  
Bernd Bukau ◽  
Aurelio A. Teleman
Keyword(s):  
Translation Initiation ◽  
Human Cells ◽  
Open Reading Frames ◽  
Translation Regulation ◽  
List Type ◽  
Translation Efficiency ◽  
Initiation Factors ◽  
Upstream Open Reading Frames

SUMMARYTranslation regulation occurs largely during initiation. Currently, translation initiation can be studied in vitro, but these systems lack features present in vivo and on endogenous mRNAs. Here we develop selective 40S footprinting for visualizing initiating 40S ribosomes on endogenous mRNAs in vivo. It pinpoints where on an mRNA initiation factors join the ribosome to act, and where they leave. We discover that in human cells most scanning ribosomes remain attached to the 5’ cap. Consequently, only one ribosome scans a 5’UTR at a time, and 5’UTR length affects translation efficiency. We discover that eIF3B, eIF4G1 and eIF4E remain on translating 80S ribosomes with a decay half-length of ∼12 codons. Hence ribosomes retain these initiation factors while translating short upstream Open Reading Frames (uORFs), providing an explanation for how ribosomes can re-initiate translation after uORFs in humans. This method will be of use for studying translation initiation mechanisms in vivo.HIGHLIGHTSSelective 40S FPing visualizes regulation of translation initiation on mRNAs in vivoScanning ribosomes are cap-tethered in human cellsOnly one ribosome scans a 5’UTR at a time in human cellsRibosomes retain eIFs during early translation, allowing reinitiation after uORFs

scholarly journals Retapamulin-assisted ribosome profiling reveals the alternative bacterial proteome

2019 ◽  
Author(s):  
Sezen Meydan ◽  
James Marks ◽  
Dorota Klepacki ◽  
Virag Sharma ◽  
Pavel V. Baranov ◽  
...  
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Translation Initiation ◽  
Single Gene ◽  
Ribosome Profiling ◽  
Genome Wide ◽  
Translation Start ◽  
Alternative Translation

SUMMARYThe use of alternative translation initiation sites enables production of more than one protein from a single gene, thereby expanding cellular proteome. Although several such examples have been serendipitously found in bacteria, genome-wide mapping of alternative translation start sites has been unattainable. We found that the antibiotic retapamulin specifically arrests initiating ribosomes at start codons of the genes. Retapamulin-enhanced Ribo-seq analysis (Ribo-RET) not only allowed mapping of conventional initiation sites at the beginning of the genes but, strikingly, it also revealed putative internal start sites in a number of Escherichia coli genes. Experiments demonstrated that the internal start codons can be recognized by the ribosomes and direct translation initiation in vitro and in vivo. Proteins, whose synthesis is initiated at an internal in-frame and out-of-frame start sites, can be functionally important and contribute to the ‘alternative’ bacterial proteome. The internal start sites my also play regulatory roles in gene expression.

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