Post-ejaculation thermal stress causes changes to the RNA profile of sperm in an external fertilizer

Sperm cells experience considerable post-ejaculation environmental variation. However, little is known about whether this affects their molecular composition, probably owing to the assumption that sperm are transcriptionally quiescent. Nevertheless, recent evidence shows sperm have distinct RNA profiles that affect fertilization and embryo viability. Moreover, RNAs are expected to be highly sensitive to extracellular changes. One such group of RNAs are heat shock protein (hsp) transcripts, which function in stress responses and are enriched in sperm. Here, we exploit the experimental tractability of the mussel Mytilus galloprovincialis by exposing paired samples of ejaculated sperm to ambient (19°C) and increased (25°C) temperatures, then measure (i) sperm motility phenotypes, and (ii) messenger RNA (mRNA) levels of two target genes ( hsp70 and hsp90 ) and several putative reference genes. We find no phenotypic changes in motility, but reduced mRNA levels for hsp90 and the putative reference gene gapdh at 25°C. This could reflect either decay of specific RNAs, or changes in translation and degradation rates of transcripts to maintain sperm function under stress. These findings represent, to our knowledge, the first evidence for changes in sperm RNA profiles owing to post-ejaculation environments, and suggest that sperm may be more vulnerable to stress from rising temperatures than currently thought.

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The Cotton miR477-CBP60A Module Participates in Plant Defense Against Verticillium dahlia

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-10-19-0302-r ◽

2020 ◽

Vol 33 (4) ◽

pp. 624-636 ◽

Cited By ~ 1

Author(s):

Guang Hu ◽

Mengyan Hao ◽

Le Wang ◽

Jianfen Liu ◽

Zhennan Zhang ◽

...

Keyword(s):

Plant Resistance ◽

Stress Responses ◽

Messenger Rna ◽

Target Genes ◽

Root Surface ◽

Site Analysis ◽

Oxidation Reduction ◽

Differentially Expressed Mirnas ◽

Verticillium Dahlia ◽

Induction Stage

Previous reports have shown that, when Verticillium dahliae localizes at the root surface, many microRNAs (miRNAs) were identified at the early induction stage. Here, we constructed two groups from two timepoints of small RNA (sRNA) in cotton root responses to V. dahliae at the later induction stage, pathogen localizing in the interior of root tissue. We identified 71 known and 378 novel miRNAs from six libraries of the pathogen-induced and the control sRNAs. Combined with degradome and sRNA sequencing, 178 corresponding miRNA target genes were identified, in which 40 target genes from differentially expressed miRNAs were primarily associated with oxidation-reduction and stress responses. More importantly, we characterized the cotton miR477-CBP60A module in the later response of the plant to V. dahliae infection. A β-glucuronidase fusion reporter and cleavage site analysis showed that ghr-miR477 directly cleaved the messenger RNA of GhCBP60A in the posttranscriptional process. The ghr-miR477-silencing decreased plant resistance to this fungus, while the knockdown of GhCBP60A increased plant resistance, which regulated GhICS1 expression to determine salicylic acid level. Our data documented that numerous later-inducible miRNAs in the plant response to V. dahliae, suggesting that these miRNAs play important roles in plant resistance to vascular disease.

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Adapted approach to profile genes while reconciling Vegf-a mRNA expression in the developing and injured lung

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00053.2015 ◽

2015 ◽

Vol 308 (12) ◽

pp. L1202-L1211 ◽

Cited By ~ 5

Author(s):

Daniel D. Lee ◽

Margaret A. Schwarz

Keyword(s):

Messenger Rna ◽

Housekeeping Genes ◽

Newborn Mouse ◽

Mrna Levels ◽

Developmental Studies ◽

Rna Integrity ◽

Amplicon Length ◽

Highly Sensitive ◽

Injured Lung ◽

Mouse Lungs

During lung development and injury, messenger RNA (mRNA) transcript levels of genes fluctuate over both space and time. Quantitative PCR (qPCR) is a highly sensitive, widely used technique to measure the mRNA levels. The sensitivity of this technique can be disadvantageous and errors amplified when each qPCR assay is not validated. In contrast to other organs, lungs have high RNase activity, resulting in less than optimal RNA integrity. We implemented a strategy to address these limitations in developing and injured lungs. Parameters were established and a filter designed that optimized amplicon length and included or excluded samples based on RNA integrity. This approach was illustrated and validated by measuring mRNA levels including Vegf-a in newborn mouse lungs that were injured by 85% oxygen (hyperoxia) for 12 days and compared with control (normoxia). We demonstrate that, in contrast to contradictory Vegf-a expression when normalized to the least suitable housekeeping genes, application of this filter and normalization to most suitable three housekeeping genes, Hprt, Eef2, and Rpl13a, gave reproducible Vegf-a expression, thus corroborating the sample filter. Accordingly, both short amplicon length and proper normalization to ranked, evaluated genes minimized erroneous fluctuation and qPCR amplification issues associated with nonideal RNA integrity in injured and developing lungs. Furthermore, our work uncovers how RNA integrity, purity, amplicon length, and discovery of stable candidate reference genes enhance precision of qPCR results and utilizes the advantages of qPCR in developmental studies.

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Identification and characterization of microRNAs and their target genes from Nile tilapia (Oreochromis niloticus)

Zeitschrift für Naturforschung C ◽

10.1515/znc-2015-0104 ◽

2016 ◽

Vol 71 (7-8) ◽

pp. 215-223 ◽

Cited By ~ 2

Author(s):

Yong Huang ◽

Xiu Ying Ma ◽

You Bing Yang ◽

Hong Tao Ren ◽

Xi Hong Sun ◽

...

Keyword(s):

Stress Responses ◽

Nile Tilapia ◽

Target Genes ◽

Expressed Sequence Tag ◽

Mrna Levels ◽

Stem Loop ◽

Mirna Genes ◽

Nile Tilapia Oreochromis Niloticus ◽

Survey Sequence

Abstract MicroRNAs (miRNAs) are a class of small single-stranded, endogenous 21–22 nt non-coding RNAs that regulate their target mRNA levels by causing either inactivation or degradation of the mRNAs. In recent years, miRNA genes have been identified from mammals, insects, worms, plants, and viruses. In this research, bioinformatics approaches were used to predict potential miRNAs and their targets in Nile tilapia from the expressed sequence tag (EST) and genomic survey sequence (GSS) database, respectively, based on the conservation of miRNAs in many animal species. A total of 19 potential miRNAs were detected following a range of strict filtering criteria. To test the validity of the bioinformatics method, seven predicted Nile tilapia miRNA genes were selected for further biological validation, and their mature miRNA transcripts were successfully detected by stem–loop RT-PCR experiments. Using these potential miRNAs, we found 56 potential targets in this species. Most of the target mRNAs appear to be involved in development, metabolism, signal transduction, transcription regulation and stress responses. Overall, our findings will provide an important foundation for further research on miRNAs function in the Nile tilapia.

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RNAi-Mediated Resistance Against Viruses in Perennial Fruit Plants

Plants ◽

10.3390/plants8100359 ◽

2019 ◽

Vol 8 (10) ◽

pp. 359 ◽

Cited By ~ 1

Author(s):

Khushwant Singh ◽

Chris Dardick ◽

Jiban Kumar Kundu

Keyword(s):

Rna Interference ◽

Stress Responses ◽

Small Rnas ◽

Messenger Rna ◽

High Throughput Sequencing ◽

Target Genes ◽

Current Knowledge ◽

Plant Immunity ◽

Plant Viruses ◽

Perennial Plants

Small RNAs (sRNAs) are 20–30-nucleotide-long, regulatory, noncoding RNAs that induce silencing of target genes at the transcriptional and posttranscriptional levels. They are key components for cellular functions during plant development, hormone signaling, and stress responses. Generated from the cleavage of double-stranded RNAs (dsRNAs) or RNAs with hairpin structures by Dicer-like proteins (DCLs), they are loaded onto Argonaute (AGO) protein complexes to induce gene silencing of their complementary targets by promoting messenger RNA (mRNA) cleavage or degradation, translation inhibition, DNA methylation, and/or histone modifications. This mechanism of regulating RNA activity, collectively referred to as RNA interference (RNAi), which is an evolutionarily conserved process in eukaryotes. Plant RNAi pathways play a fundamental role in plant immunity against viruses and have been exploited via genetic engineering to control disease. Plant viruses of RNA origin that contain double-stranded RNA are targeted by the RNA-silencing machinery to produce virus-derived small RNAs (vsRNAs). Some vsRNAs serve as an effector to repress host immunity by capturing host RNAi pathways. High-throughput sequencing (HTS) strategies have been used to identify endogenous sRNA profiles, the “sRNAome”, and analyze expression in various perennial plants. Therefore, the review examines the current knowledge of sRNAs in perennial plants and fruits, describes the development and implementation of RNA interference (RNAi) in providing resistance against economically important viruses, and explores sRNA targets that are important in regulating a variety of biological processes.

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Protective role of estrogen against excessive erythrocytosis in Monge’s disease

Experimental & Molecular Medicine ◽

10.1038/s12276-020-00550-2 ◽

2021 ◽

Vol 53 (1) ◽

pp. 125-135

Author(s):

Priti Azad ◽

Francisco C. Villafuerte ◽

Daniela Bermudez ◽

Gargi Patel ◽

Gabriel G. Haddad

Keyword(s):

High Altitude ◽

Messenger Rna ◽

Target Genes ◽

Protective Role ◽

Mrna Levels ◽

Protein Levels ◽

Hypoxic Conditions ◽

Andean Population

AbstractMonge’s disease (chronic mountain sickness (CMS)) is a maladaptive condition caused by chronic (years) exposure to high-altitude hypoxia. One of the defining features of CMS is excessive erythrocytosis with extremely high hematocrit levels. In the Andean population, CMS prevalence is vastly different between males and females, being rare in females. Furthermore, there is a sharp increase in CMS incidence in females after menopause. In this study, we assessed the role of sex hormones (testosterone, progesterone, and estrogen) in CMS and non-CMS cells using a well-characterized in vitro erythroid platform. While we found that there was a mild (nonsignificant) increase in RBC production with testosterone, we observed that estrogen, in physiologic concentrations, reduced sharply CD235a+ cells (glycophorin A; a marker of RBC), from 56% in the untreated CMS cells to 10% in the treated CMS cells, in a stage-specific and dose-responsive manner. At the molecular level, we determined that estrogen has a direct effect on GATA1, remarkably decreasing the messenger RNA (mRNA) and protein levels of GATA1 (p < 0.01) and its target genes (Alas2, BclxL, and Epor, p < 0.001). These changes result in a significant increase in apoptosis of erythroid cells. We also demonstrate that estrogen regulates erythropoiesis in CMS patients through estrogen beta signaling and that its inhibition can diminish the effects of estrogen by significantly increasing HIF1, VEGF, and GATA1 mRNA levels. Taken altogether, our results indicate that estrogen has a major impact on the regulation of erythropoiesis, particularly under chronic hypoxic conditions, and has the potential to treat blood diseases, such as high altitude severe erythrocytosis.

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InsP7is a small-molecule regulator of NUDT3-mediated mRNA decapping and processing-body dynamics

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1922284117 ◽

2020 ◽

Vol 117 (32) ◽

pp. 19245-19253 ◽

Cited By ~ 1

Author(s):

Soumyadip Sahu ◽

Zhenzhen Wang ◽

Xinfu Jiao ◽

Chunfang Gu ◽

Nikolaus Jork ◽

...

Keyword(s):

Stress Responses ◽

Messenger Rna ◽

Control Process ◽

Stem Cell Differentiation ◽

Wild Type ◽

Inositol Pyrophosphate ◽

Body Dynamics ◽

Processing Body ◽

The Stability

Regulation of enzymatic 5′ decapping of messenger RNA (mRNA), which normally commits transcripts to their destruction, has the capacity to dynamically reshape the transcriptome. For example, protection from 5′ decapping promotes accumulation of mRNAs into processing (P) bodies—membraneless, biomolecular condensates. Such compartmentalization of mRNAs temporarily removes them from the translatable pool; these repressed transcripts are stabilized and stored until P-body dissolution permits transcript reentry into the cytosol. Here, we describe regulation of mRNA stability and P-body dynamics by the inositol pyrophosphate signaling molecule 5-InsP7(5-diphosphoinositol pentakisphosphate). First, we demonstrate 5-InsP7inhibits decapping by recombinant NUDT3 (Nudix [nucleoside diphosphate linked moiety X]-type hydrolase 3) in vitro. Next, in intact HEK293 and HCT116 cells, we monitored the stability of a cadre of NUDT3 mRNA substrates following CRISPR-Cas9 knockout ofPPIP5Ks(diphosphoinositol pentakisphosphate 5-kinases type 1 and 2, i.e.,PPIP5KKO), which elevates cellular 5-InsP7levels by two- to threefold (i.e., within the physiological rheostatic range). ThePPIP5KKO cells exhibited elevated levels of NUDT3 mRNA substrates and increased P-body abundance. Pharmacological and genetic attenuation of 5-InsP7synthesis in the KO background reverted both NUDT3 mRNA substrate levels and P-body counts to those of wild-type cells. Furthermore, liposomal delivery of a metabolically resistant 5-InsP7analog into wild-type cells elevated levels of NUDT3 mRNA substrates and raised P-body abundance. In the context that cellular 5-InsP7levels normally fluctuate in response to changes in the bioenergetic environment, regulation of mRNA structure by this inositol pyrophosphate represents an epitranscriptomic control process. The associated impact on P-body dynamics has relevance to regulation of stem cell differentiation, stress responses, and, potentially, amelioration of neurodegenerative diseases and aging.

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Use of Oxidative Stress Responses to Determine the Efficacy of Inactivation Treatments on Cryptosporidium Oocysts

Microorganisms ◽

10.3390/microorganisms9071463 ◽

2021 ◽

Vol 9 (7) ◽

pp. 1463

Author(s):

Tamirat Tefera Temesgen ◽

Kristoffer Relling Tysnes ◽

Lucy Jane Robertson

Keyword(s):

Oxidative Stress ◽

Stress Responses ◽

Target Genes ◽

Cryptosporidium Oocyst ◽

Drinking Water Treatment ◽

Proof Of Concept ◽

Environmental Matrices ◽

Oocyst Wall ◽

Novel Approach ◽

Cryptosporidium Oocysts

Cryptosporidium oocysts are known for being very robust, and their prolonged survival in the environment has resulted in outbreaks of cryptosporidiosis associated with the consumption of contaminated water or food. Although inactivation methods used for drinking water treatment, such as UV irradiation, can inactivate Cryptosporidium oocysts, they are not necessarily suitable for use with other environmental matrices, such as food. In order to identify alternative ways to inactivate Cryptosporidium oocysts, improved methods for viability assessment are needed. Here we describe a proof of concept for a novel approach for determining how effective inactivation treatments are at killing pathogens, such as the parasite Cryptosporidium. RNA sequencing was used to identify potential up-regulated target genes induced by oxidative stress, and a reverse transcription quantitative PCR (RT-qPCR) protocol was developed to assess their up-regulation following exposure to different induction treatments. Accordingly, RT-qPCR protocols targeting thioredoxin and Cryptosporidium oocyst wall protein 7 (COWP7) genes were evaluated on mixtures of viable and inactivated oocysts, and on oocysts subjected to various potential inactivation treatments such as freezing and chlorination. The results from the present proof-of-concept experiments indicate that this could be a useful tool in efforts towards assessing potential technologies for inactivating Cryptosporidium in different environmental matrices. Furthermore, this approach could also be used for similar investigations with other pathogens.

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Alternative splicing landscapes in Arabidopsis thaliana across tissues and stress conditions highlight major functional differences with animals

Genome Biology ◽

10.1186/s13059-020-02258-y ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Guiomar Martín ◽

Yamile Márquez ◽

Federica Mantica ◽

Paula Duque ◽

Manuel Irimia

Keyword(s):

Gene Expression ◽

Arabidopsis Thaliana ◽

Alternative Splicing ◽

Stress Responses ◽

Target Genes ◽

Intron Retention ◽

Single Gene ◽

Exon Skipping ◽

Environmental Response ◽

Biotic Stresses

Abstract Background Alternative splicing (AS) is a widespread regulatory mechanism in multicellular organisms. Numerous transcriptomic and single-gene studies in plants have investigated AS in response to specific conditions, especially environmental stress, unveiling substantial amounts of intron retention that modulate gene expression. However, a comprehensive study contrasting stress-response and tissue-specific AS patterns and directly comparing them with those of animal models is still missing. Results We generate a massive resource for Arabidopsis thaliana, PastDB, comprising AS and gene expression quantifications across tissues, development and environmental conditions, including abiotic and biotic stresses. Harmonized analysis of these datasets reveals that A. thaliana shows high levels of AS, similar to fruitflies, and that, compared to animals, disproportionately uses AS for stress responses. We identify core sets of genes regulated specifically by either AS or transcription upon stresses or among tissues, a regulatory specialization that is tightly mirrored by the genomic features of these genes. Unexpectedly, non-intron retention events, including exon skipping, are overrepresented across regulated AS sets in A. thaliana, being also largely involved in modulating gene expression through NMD and uORF inclusion. Conclusions Non-intron retention events have likely been functionally underrated in plants. AS constitutes a distinct regulatory layer controlling gene expression upon internal and external stimuli whose target genes and master regulators are hardwired at the genomic level to specifically undergo post-transcriptional regulation. Given the higher relevance of AS in the response to different stresses when compared to animals, this molecular hardwiring is likely required for a proper environmental response in A. thaliana.

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Extract of Wax Gourd Peel Prevents High-Fat Diet-Induced Hyperlipidemia in C57BL/6 Mice via the Inhibition of the PPARγPathway

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2013/342561 ◽

2013 ◽

Vol 2013 ◽

pp. 1-11 ◽

Cited By ~ 8

Author(s):

Ming Gu ◽

Shengjie Fan ◽

Gaigai Liu ◽

Lu Guo ◽

Xiaobo Ding ◽

...

Keyword(s):

Blood Glucose ◽

High Fat Diet ◽

Target Genes ◽

Metabolic Diseases ◽

Body Weight Gain ◽

Fasting Blood Glucose ◽

Peroxisome Proliferator ◽

Mrna Levels ◽

High Fat ◽

Wax Gourd

Wax gourd is a popular vegetable in East Asia. In traditional Chinese medicine, wax gourd peel is used to prevent and treat metabolic diseases such as hyperlipidemia, hyperglycemia, obesity, and cardiovascular disease. However, there is no experimental evidence to support these applications. Here, we examined the effect of the extract of wax gourd peel (EWGP) on metabolic disorders in diet-induced C57BL/6 obese mice. In the preventive experiment, EWGP blocked body weight gain and lowered serum total cholesterol (TC), low-density lipoprotein cholesterol (LDL-c), liver TG and TC contents, and fasting blood glucose in mice fed with a high-fat diet. In the therapeutic study, we induced obesity in the mice and treated with EWGP for two weeks. We found that EWGP treatment reduced serum and liver triglyceride (TG) contents and fasting blood glucose and improved glucose tolerance in the mice. Reporter assay and gene expression analysis showed that EWGP could inhibit peroxisome proliferator-activated receptorγ(PPARγ) transactivities and could decrease mRNA levels of PPARγand its target genes. We also found that HMG-CoA reductase (HMGCR) was downregulated in the mouse liver by EWGP. Our data suggest that EWGP lowers hyperlipidemia of C57BL/6 mice induced by high-fat diet via the inhibition of PPARγand HMGCR signaling.

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Integrated whole transcriptome and small RNA analysis revealed multiple regulatory networks in colorectal cancer

Scientific Reports ◽

10.1038/s41598-021-93531-y ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Hibah Shaath ◽

Salman M. Toor ◽

Mohamed Abu Nada ◽

Eyad Elkord ◽

Nehad M. Alajez

Keyword(s):

Colorectal Cancer ◽

Messenger Rna ◽

Regulatory Networks ◽

Potential Interaction ◽

Therapeutic Interventions ◽

Signaling Networks ◽

Protein Coding ◽

Paired Samples ◽

And Migration ◽

Whole Transcriptome

AbstractColorectal cancer (CRC) remains a global disease burden and a leading cause of cancer related deaths worldwide. The identification of aberrantly expressed messenger RNA (mRNA), long non-coding RNA (lncRNA), and microRNA (miRNA), and the resulting molecular interactions and signaling networks is essential for better understanding of CRC, identification of novel diagnostic biomarkers and potential development of therapeutic interventions. Herein, we performed microRNA (miRNA) sequencing on fifteen CRC and their non-tumor adjacent tissues and whole transcriptome RNA-Seq on six paired samples from the same cohort and identified alterations in miRNA, mRNA, and lncRNA expression. Computational analyses using Ingenuity Pathway Analysis (IPA) identified multiple activated signaling networks in CRC, including ERBB2, RABL6, FOXM1, and NFKB networks, while functional annotation highlighted activation of cell proliferation and migration as the hallmark of CRC. IPA in combination with in silico prediction algorithms and experimentally validated databases gave insight into the complex associations and interactions between downregulated miRNAs and upregulated mRNAs in CRC and vice versa. Additionally, potential interaction between differentially expressed lncRNAs such as H19, SNHG5, and GATA2-AS1 with multiple miRNAs has been revealed. Taken together, our data provides thorough analysis of dysregulated protein-coding and non-coding RNAs in CRC highlighting numerous associations and regulatory networks thus providing better understanding of CRC.

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