Extracellular stimuli specifically regulate localized levels of individual neuronal mRNAs

Subcellular regulation of protein synthesis requires the correct localization of messenger RNAs (mRNAs) within the cell. In this study, we investigate whether the axonal localization of neuronal mRNAs is regulated by extracellular stimuli. By profiling axonal levels of 50 mRNAs detected in regenerating adult sensory axons, we show that neurotrophins can increase and decrease levels of axonal mRNAs. Neurotrophins (nerve growth factor, brain-derived neurotrophic factor, and neurotrophin-3) regulate axonal mRNA levels and use distinct downstream signals to localize individual mRNAs. However, myelin-associated glycoprotein and semaphorin 3A regulate axonal levels of different mRNAs and elicit the opposite effect on axonal mRNA levels from those observed with neurotrophins. The axonal mRNAs accumulate at or are depleted from points of ligand stimulation along the axons. The translation product of a chimeric green fluorescent protein–β-actin mRNA showed similar accumulation or depletion adjacent to stimuli that increase or decrease axonal levels of endogenous β-actin mRNA. Thus, extracellular ligands can regulate protein generation within subcellular regions by specifically altering the localized levels of particular mRNAs.

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Targeted gene suppression through double-stranded RNA application using easy-to-use methods in Arabidopsis thaliana

Applied Biological Chemistry ◽

10.1186/s13765-022-00675-0 ◽

2022 ◽

Vol 65 (1) ◽

Author(s):

Minsu Park ◽

Tae Young Um ◽

Geupil Jang ◽

Yang Do Choi ◽

Chanseok Shin

Keyword(s):

Arabidopsis Thaliana ◽

Target Genes ◽

Fluorescent Protein ◽

Specific Interaction ◽

Mrna Levels ◽

Messenger Rnas ◽

Small Interfering Rnas ◽

Double Stranded Rna ◽

Reverse Transcription Pcr ◽

Green Fluorescent

AbstractRNA interference (RNAi) is an RNA-dependent gene silencing process that is regulated by the interaction between the RNA-induced silencing complex (RISC) and double-stranded RNA (dsRNA). Exogenous dsRNAs are imported directly into the cytoplasm, where they are cleaved by Dicer into short dsRNA fragments of 20–25 base pairs. These short dsRNA fragments, called small interfering RNAs (siRNAs) have sequence-specific interaction with target genes. The guide strand, onto which siRNAs are incorporated in the RISC interacts with the target mRNA sequence, thereby inducing cleavage and degradation of target messenger RNAs (mRNAs) by ribonucleases. Recent studies have shown that plant dsRNA treatments can induce RNAi. However, the dsRNA application methods and delivery systems involved have not been well examined. In this study, dsRNA was introduced to Arabidopsis thaliana by two methods: dipping and spray. We synthesized two dsRNAs designed to target mRNAs encoding enhanced green fluorescent protein (EGFP). After applying dsRNAs that target EGFP, we found an obvious reduction in GFP expression. This was determined using fluorescence microscopy and quantitative reverse transcription PCR to assess the mRNA levels of the auxin-sensitive reporter DR5-EGFP Arabidopsis thaliana. Our data revealed that applying target gene-specific exogenous dsRNAs can induce suppression of target genes of interest whether the dipping or spray method is used. This study therefore provides a foundation for understanding how to apply and deliver dsRNAs in plants.

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Relative Strengths and Regulation of Different Promoter-Associated Sequences for Various blaSHV Genes and Their Relationships to β-Lactam Resistance

Journal of Molecular Microbiology and Biotechnology ◽

10.1159/000458708 ◽

2017 ◽

Vol 27 (2) ◽

pp. 91-101 ◽

Cited By ~ 2

Author(s):

Yao Zhai ◽

Zhao Zhang ◽

Zhanwei Wang ◽

Yusheng Chen ◽

Qi Wang ◽

...

Keyword(s):

Escherichia Coli ◽

Green Fluorescent Protein ◽

Mrna Expression ◽

Inhibitory Concentration ◽

Fluorescent Protein ◽

Mrna Levels ◽

Associated Sequences ◽

Regulation Function ◽

Green Fluorescent ◽

Mrna Expression Levels

Aims: This work investigated the relative strengths of different blaSHV promoter-associated sequences and their regulation function in blaSHV expression and β-lactam resistance. Methods: Recombinant plasmids with the promoter-associated sequences (P-W, P-S, P-IS, and P-WPD), tac promoter, and combined fragments of promoter and blaSHV were separately constructed and transformed into Escherichia coli DH5α. The relative strengths of the promoters indicated by the intensities of green fluorescent protein and the mRNA expression levels of blaSHV were compared. The minimum inhibitory concentration and extended spectrum β-lactamase phenotypes were evaluated. Results: The relative strengths were ranked as P-tac > P-WPD > P-IS > P-S > P-W. The mRNA expression and β-lactam resistance levels of the different promoter-associated sequence groups were generally consistent with the strength rank, but the extent of gfp and blaSHV mRNA levels varied significantly in each group. The β-lactam resistance levels were inconsistent with the strength rank in certain blaSHV groups. In relation to the different promoter-associated sequences, blaSHV-ESBLs displayed significantly different change modes of β-lactam resistance compared with blaSHV-non-ESBLs. Conclusion: The mRNA expression and β-lactam resistance of the blaSHV showed consistencies and inconsistencies with the strengths of the promoter-associated sequences. The mechanisms accounting for these discrepancies need further investigation.

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Habenular Kiss1 Neurons Modulate the Serotonergic System in the Brain of Zebrafish

Endocrinology ◽

10.1210/en.2012-1062 ◽

2012 ◽

Vol 153 (5) ◽

pp. 2398-2407 ◽

Cited By ~ 40

Author(s):

Satoshi Ogawa ◽

Kai We Ng ◽

Priveena Nair Ramadasan ◽

Fatima Megala Nathan ◽

Ishwar S. Parhar

Keyword(s):

Fluorescent Protein ◽

Serotonergic System ◽

Raphe Nuclei ◽

Transgenic Zebrafish ◽

Mrna Levels ◽

Interpeduncular Nucleus ◽

Hypothalamic Nuclei ◽

Green Fluorescent ◽

Raphé Nuclei ◽

The Brain

The Kiss1/KISS1 gene has recently been implicated as a potent hypothalamic regulator of reproductive functions, in particular, the onset of puberty in mammals. In zebrafish (Danio rerio), there are two kiss1 homologues (kiss1 and kiss2) expressed in the brain: Kiss2-expressing neurons in the hypothalamic nuclei are considered potent regulators of reproduction, whereas the role of Kiss1-expressing neurons in the habenula remains unknown. We first analyzed the expression of kiss1 mRNA in a transgenic zebrafish, in which the habenula-interpeduncular nucleus (IPN) pathway is labelled with green fluorescent protein, and our application of a biocytin neural tracer into the habenula showed the presence of neuronal projections of Kiss1 neurons to the ventral IPN. Therefore, we speculated that kiss1 neurons might regulate the serotonergic system in the raphe. However, laser microdissection followed by real-time PCR revealed the expression of Kiss1 receptor (kissr1) mRNA in the habenula and the ventral IPN but not in the dorsal IPN or the serotonergic neurons in the raphe nuclei. Dual-fluorescent in situ hybridization revealed the coexpression of kiss1 and kissr1 mRNA in the habenula. Administration of Kiss1 significantly decreased the level of kiss1 mRNA (0.3- to 0.5-fold, P < 0.001), but the level of c-fos mRNA was increased (∼3-fold, P < 0.05) in the ventral habenula, suggesting that there is autocrine regulation of the kiss1 gene. Kiss1 administration significantly increased the c-fos mRNA levels in the raphe nuclei (2.5-fold, P < 0.001) and genes involved in the regulation of serotonin levels (pet1 and slc6a4a; 3.3- and 2.2-fold, P < 0.01). These findings suggest that the autocrine-regulated habenular Kiss1 neurons indirectly regulate the serotonergic system in the raphe nuclei through the IPN in the zebrafish.

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Localized Gene Expression in Pseudomonas aeruginosa Biofilms

Applied and Environmental Microbiology ◽

10.1128/aem.00710-08 ◽

2008 ◽

Vol 74 (14) ◽

pp. 4463-4471 ◽

Cited By ~ 109

Author(s):

Ailyn P. Lenz ◽

Kerry S. Williamson ◽

Betsey Pitts ◽

Philip S. Stewart ◽

Michael J. Franklin

Keyword(s):

Gene Expression ◽

Pseudomonas Aeruginosa ◽

Green Fluorescent Protein ◽

16S Rrna ◽

Fluorescent Protein ◽

Mrna Levels ◽

Protein Fluorescence ◽

Bacterial Gene ◽

Qrt Pcr ◽

Green Fluorescent

ABSTRACT Gene expression in biofilms is dependent on bacterial responses to the local environmental conditions. Most techniques for studying bacterial gene expression in biofilms characterize average values across the entire population. Here, we describe the use of laser capture microdissection microscopy (LCMM) combined with multiplex quantitative real-time reverse transcriptase PCR (qRT-PCR) to isolate and quantify RNA transcripts from small groups of cells at spatially resolved sites within biofilms. The approach was first tested and analytical parameters were determined for Pseudomonas aeruginosa containing an isopropyl-β-d-thiogalactopyranoside-inducible gene for the green fluorescent protein (gfp). The results show that the amounts of gfp mRNA were greatest in the top zones of the biofilms, and that gfp mRNA levels correlated with the zone of active green fluorescent protein fluorescence. The method then was used to quantify transcripts from wild-type P. aeruginosa biofilms for a housekeeping gene, acpP; the 16S rRNA; and two genes regulated by quorum sensing, phzA1 and aprA. The results demonstrated that the amount of acpP mRNA was greatest in the top 30 μm of the biofilm, with little or no mRNA for this gene at the base of the biofilms. In contrast, 16S rRNA amounts were relatively uniform throughout biofilm strata. Using this strategy, the RNA amounts of individual genes were determined, and therefore the results are dependent on both gene expression and the half-life of the transcripts. Therefore, the uniform amount of rRNA throughout the biofilms likely is due to the stability of the rRNA within ribosomes. The levels of aprA mRNA showed stratification, with the largest amounts in the upper 30-μm zone of these biofilms. The results demonstrate that mRNA levels for individual genes are not uniformly distributed throughout biofilms but may vary by orders of magnitude over small distances. The LCMM/qRT-PCR technique can be used to resolve and quantify this RNA variability at high spatial resolution.

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Induction of Transgene Suppression in Plants via External Application of Synthetic dsRNA

International Journal of Molecular Sciences ◽

10.3390/ijms20071585 ◽

2019 ◽

Vol 20 (7) ◽

pp. 1585 ◽

Cited By ~ 10

Author(s):

Alexandra Dubrovina ◽

Olga Aleynova ◽

Alexander Kalachev ◽

Andrey Suprun ◽

Zlata Ogneva ◽

...

Keyword(s):

Fluorescent Protein ◽

Crop Improvement ◽

Plant Genome ◽

Mrna Levels ◽

Small Interfering Rnas ◽

External Application ◽

Transcript Levels ◽

Exogenous Application ◽

Green Fluorescent

Recent investigations show that exogenously applied small interfering RNAs (siRNA) and long double-stranded RNA (dsRNA) precursors can be taken up and translocated in plants to induce RNA interference (RNAi) in the plant or in its fungal pathogen. The question of whether genes in the plant genome can undergo suppression as a result of exogenous RNA application on plant surface is almost unexplored. This study analyzed whether it is possible to influence transcript levels of transgenes, as more prone sequences to silencing, in Arabidopsis genome by direct exogenous application of target long dsRNAs. The data revealed that in vitro synthesized dsRNAs designed to target the gene coding regions of enhanced green fluorescent protein (EGFP) or neomycin phosphotransferase II (NPTII) suppressed their transcript levels in Arabidopsis. The fact that, simple exogenous application of polynucleotides can affect mRNA levels of plant transgenes, opens new opportunities for the development of new scientific techniques and crop improvement strategies.

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Characterization of cis-acting element in renal NaPi-2 cotransporter mRNA that determines mRNA stability

AJP Renal Physiology ◽

10.1152/ajprenal.00332.2002 ◽

2003 ◽

Vol 284 (4) ◽

pp. F663-F670 ◽

Cited By ~ 10

Author(s):

Yulia Moz ◽

Justin Silver ◽

Tally Naveh-Many

Keyword(s):

Fluorescent Protein ◽

Mrna Levels ◽

In Vitro Degradation ◽

Cis Acting ◽

Cis Element ◽

Protein Binding Region ◽

Green Fluorescent ◽

Subsequent Stabilization

Hypophosphatemia leads to an increase in Na+-Pi cotransporter (NaPi-2) mRNA levels. This increase is posttranscriptional and correlates with a more stable transcript mediated by the terminal 698 nt of the NaPi-2 mRNA. A 71-nt binding element was identified with renal proteins from rats fed control and low-Pi (−Pi) diet. The binding of −Pi renal proteins to this transcript was increased compared with control proteins. The functionality of the ciselement was demonstrated by an in vitro degradation assay. −Pi renal proteins stabilized transcripts that included the cis element compared with control renal extracts. The full-length NaPi-2 transcript, but not control transcripts, was stabilized by −Pi extracts. Insertion of the binding element into green fluorescent protein (GFP) as a reporter gene decreased chimeric GFP mRNA levels in transfection experiments. Our results suggest that the protein-binding region of the NaPi-2 mRNA functions as a cis-acting instability element. In hypophosphatemia there is increased binding to the cis-acting element and subsequent stabilization of NaPi-2 mRNA.

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Living Colors in the Gray Mold Pathogen Botrytis cinerea: Codon-Optimized Genes Encoding Green Fluorescent Protein and mCherry, Which Exhibit Bright Fluorescence

Applied and Environmental Microbiology ◽

10.1128/aem.02644-10 ◽

2011 ◽

Vol 77 (9) ◽

pp. 2887-2897 ◽

Cited By ~ 45

Author(s):

Michaela Leroch ◽

Dennis Mernke ◽

Dieter Koppenhoefer ◽

Prisca Schneider ◽

Andreas Mosbach ◽

...

Keyword(s):

Green Fluorescent Protein ◽

Botrytis Cinerea ◽

Fluorescent Protein ◽

Gc Content ◽

Gray Mold ◽

Mrna Levels ◽

Content Type ◽

Mold Fungus ◽

Encoding Gene ◽

Green Fluorescent

ABSTRACTThe green fluorescent protein (GFP) and its variants have been widely used in modern biology as reporters that allow a variety of live-cell imaging techniques. So far, GFP has rarely been used in the gray mold fungusBotrytis cinereabecause of low fluorescence intensity. The codon usage ofB. cinereagenes strongly deviates from that of commonly used GFP-encoding genes and reveals a lower GC content than other fungi. In this study, we report the development and use of a codon-optimized version of theB. cinereaenhanced GFP (eGFP)-encoding gene (Bcgfp) for improved expression inB. cinerea. Both the codon optimization and, to a smaller extent, the insertion of an intron resulted in higher mRNA levels and increased fluorescence. Bcgfpwas used for localization of nuclei in germinating spores and for visualizing host penetration. We further demonstrate the use of promoter-Bcgfpfusions for quantitative evaluation of various toxic compounds as inducers of theatrBgene encoding an ABC-type drug efflux transporter ofB. cinerea. In addition, a codon-optimized mCherry-encoding gene was constructed which yielded bright red fluorescence inB. cinerea.

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147 GENE KNOCKDOWN OF MATERNAL- AND EMBRYONIC-EXPRESSED GREEN FLUORESCENT PROTEIN IN MURINE EMBRYOS BY THE INJECTION OF A SHORT INTERFERING RNA (siRNA)

Reproduction Fertility and Development ◽

10.1071/rdv19n1ab147 ◽

2007 ◽

Vol 19 (1) ◽

pp. 191

Author(s):

K. Iqbal ◽

W. A. Kues ◽

J. W. Carnwath ◽

H. Niemann

Keyword(s):

Green Fluorescent Protein ◽

Gene Silencing ◽

Fluorescent Protein ◽

Mrna Levels ◽

Knock Down ◽

Gfp Gene ◽

Mammalian Embryos ◽

Murine Embryos ◽

Green Fluorescent

RNA interference (RNAi) is now widely used for gene silencing in various biological systems. Injection of long double-stranded RNAs has been shown to specifically knock down gene expression in mammalian embryos. The utilization of short interfering RNAs (siRNA) to target specific embryonic genes would make this approach flexible and efficient enough for studying physiological functions in development. To demonstrate the feasibility of a single class of siRNA molecules for achieving long-lasting effects after injection into mammalian zygotes, we used siRNAs to knock down expression of the green fluorescent protein (GFP) in transgenic murine embryos of the OG2 transgenic line. Homozygous OG2-animals, carrying the Oct4-GFP transgene, were mated with NMRI animals to produce OG2 hemizygous zygotes, which show a parentally dependent expression pattern of the marker gene. Hemizygous zygotes with a maternally inherited Oct4-GFP gene continuously express the GFP marker; hemizygous zygotes with a paternally inherited Oct4-GFP start transcription of the GFP gene at the 4–8 cell stage, i.e., after onset of embryonic genomic activation. Thus efficacy and duration of gene silencing could be tested under 2 different conditions where (i) GFP mRNA was already present at the time point of injection, and (ii) GFP transcription started 2–3 cell cycles after siRNA injection. Zygotes were microinjected with either a 22-basepair GFP-siRNA or a control siRNA and then cultured in vitro. The siRNAs were conjugated with the fluorochome rhodamine to allow monitoring of injection and subsequent degradation of siRNAs. At the end of the in vitro culture, the developmental stage, the number of nuclei, GFP fluorescence (Table 1), and the GFP mRNA levels were determined by RT-PCR. In conclusion, results demonstrate that injection of a synthetic siRNA is sufficient to knock down a target gene transcript with either maternal or embryonic expression in mammalian embryos. Importantly, maternally derived GFP proteins showed a delayed functional knockdown of GFP for 2 days. The advantages of this approach are that (i) off-target effects of long double-stranded RNAs can be avoided, (ii) siRNAs against any known transcript can be rapidly designed and synthesized, and (iii) developmentally important genes can be silenced in embryos for at least 5 days following injection. Table 1.siRNA microinjection into zygotes with maternally and paternally inherited GFP

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Calreticulin modulates cell adhesiveness via regulation of vinculin expression.

The Journal of Cell Biology ◽

10.1083/jcb.135.6.1913 ◽

1996 ◽

Vol 135 (6) ◽

pp. 1913-1923 ◽

Cited By ~ 92

Author(s):

M Opas ◽

M Szewczenko-Pawlikowski ◽

G K Jass ◽

N Mesaeli ◽

M Michalak

Keyword(s):

Fluorescent Protein ◽

High Capacity ◽

Fibroblast Cell ◽

Cell Junctions ◽

Mrna Levels ◽

Regulation Of Expression ◽

Low Concentrations ◽

Green Fluorescent ◽

In Cells ◽

Cell Cell

Calreticulin is an ubiquitous and highly conserved high capacity Ca(2+)-binding protein that plays a major role in Ca2+ storage within the lumen of the ER. Here, using L fibroblast cell lines expressing different levels of calreticulin, we show that calreticulin plays a role in the control of cell adhesiveness via regulation of expression of vinculin, a cytoskeletal protein essential for cell-substratum and cell-cell attachments. Both vinculin protein and mRNA levels are increased in cells overexpressing calreticulin and are downregulated in cells expressing reduced level of calreticulin. Abundance of actin, talin, alpha 5 and beta 1 integrins, pp125 focal adhesion kinase, and alpha-catenin is not affected by the differential calreticulin expression. Overexpression of calreticulin increases both cell-substratum and cell-cell adhesiveness of L fibroblasts that, most surprisingly, establish vinculin-rich cell-cell junctions. Upregulation of calreticulin also affects adhesion-dependent phenomena such as cell motility (which decreases) and cell spreading (which increases). Downregulation of calreticulin brings about inverse effects. Cell adhesiveness is Ca2+ regulated. The level of calreticulin expression, however, has no effect on either the resting cytoplasmic Ca2+ concentration or the magnitude of FGF-induced Ca2+ transients. Calreticulin, however, participates in Ca2+ homeostasis as its level of expression affects cell viability at low concentrations of extracellular Ca2+. Consequently, we infer that it is not the Ca2+ storage function of calreticulin that affects cell adhesiveness. Neither endogenous calreticulin nor overexpressed green fluorescent protein-calreticulin construct can be detected outside of the ER. Since all of the adhesion-related effects of differential calreticulin expression can be explained by its regulation of vinculin expression, we conclude that it is the ER-resident calreticulin that affects cellular adhesiveness.

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Intravenous infusion of iPSC-derived neural precursor cells increases acid β-glucosidase function in the brain and lessens the neuronopathic phenotype in a mouse model of Gaucher disease

Human Molecular Genetics ◽

10.1093/hmg/ddz184 ◽

2019 ◽

Vol 28 (20) ◽

pp. 3406-3421 ◽

Cited By ~ 1

Author(s):

Yanyan Peng ◽

Benjamin Liou ◽

Venette Inskeep ◽

Rachel Blackwood ◽

Christopher N Mayhew ◽

...

Keyword(s):

Mouse Model ◽

Gaucher Disease ◽

Fluorescent Protein ◽

Therapeutic Potential ◽

Wild Type Mouse ◽

Induced Pluripotent Stem Cell ◽

Precursor Cells ◽

Mrna Levels ◽

Green Fluorescent ◽

The Brain

Abstract Gaucher disease (GD) is caused by GBA1 mutations leading to functional deficiency of acid-β-glucosidase (GCase). No effective treatment is available for neuronopathic GD (nGD). A subclass of neural stem and precursor cells (NPCs) expresses VLA4 (integrin α4β1, very late antigen-4) that facilitates NPC entry into the brain following intravenous (IV) infusion. Here, the therapeutic potential of IV VLA4+NPCs was assessed for nGD using wild-type mouse green fluorescent protein (GFP)-positive multipotent induced pluripotent stem cell (iPSC)-derived VLA4+NPCs. VLA4+NPCs successfully engrafted in the nGD (4L;C*) mouse brain. GFP-positive cells differentiated into neurons, astrocytes and oligodendrocytes in the brainstem, midbrain and thalamus of the transplanted mice and significantly improved sensorimotor function and prolonged life span compared to vehicle-treated 4L;C* mice. VLA4+NPC transplantation significantly decreased levels of CD68 and glial fibrillary acidic protein, as well as TNFα mRNA levels in the brain, indicating reduced neuroinflammation. Furthermore, decreased Fluoro-Jade C and NeuroSilver staining suggested inhibition of neurodegeneration. VLA4+NPC-engrafted 4L;C* midbrains showed 35% increased GCase activity, reduced substrate [glucosylceramide (GC, −34%) and glucosylsphingosine (GS, −11%)] levels and improved mitochondrial oxygen consumption rates in comparison to vehicle-4L;C* mice. VLA4+NPC engraftment in 4L;C* brain also led to enhanced expression of neurotrophic factors that have roles in neuronal survival and the promotion of neurogenesis. This study provides evidence that iPSC-derived NPC transplantation has efficacy in an nGD mouse model and provides proof of concept for autologous NPC therapy in nGD.

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