mRNA Abundance Research Articles

DHX36 binding induces RNA structurome remodeling and regulates RNA abundance via m6A reader YTHDF1.

RNA structure constitutes a new layer of gene regulatory mechanisms. RNA binding proteins can modulate RNA secondary structures, thus participating in post-transcriptional regulation. The DEAH-box helicase 36 (DHX36) is known to bind and unwind RNA G-quadruplex (rG4) structure but the transcriptome-wide RNA structure remodeling induced by DHX36 binding and the impact on RNA fate remain poorly understood. Here, we investigate the RNA structurome alteration induced by DHX36 depletion. Our findings reveal that DHX36 binding induces structural remodeling not only at the localized binding sites but also on the entire mRNA transcript most pronounced in 3'UTR regions. DHX36 binding increases structural accessibility at 3'UTRs which is correlated with decreased post-transcriptional mRNA abundance. Further analyses and experiments uncover that DHX36 binding sites are enriched for N6-methyladenosine (m6A) modification and YTHDF1 binding; and DHX36 induced structural changes may facilitate YTHDF1 binding to m6A sites leading to RNA degradation. Altogether, our findings uncover the structural remodeling effect of DHX36 binding and its impact on RNA abundance through regulating m6A dependent YTHDF1 binding.

Kidney mRNA-protein expression correlation: what can we learn from the Human Protein Atlas?

The Human Protein Atlas, with more than 10 million immunohistochemical images showing tissue- and cell-specific protein expression levels and subcellular localization information, is widely used in kidney research. The Human Protein Atlas contains comprehensive data on multi-tissue transcript and protein abundance, allowing for comparisons across tissues. However, while visual and intuitive to interpret, immunohistochemistry is limited by its semi-quantitative nature. This can lead to mismatches in protein expression measurements across different platforms. We performed a comparison of the Human Protein Atlas' kidney-specific RNA sequencing and immunohistochemistry data to determine whether the mRNA and protein abundance levels are concordant. Our study shows that there is a discordance between mRNA and protein expression in the kidney based on the Human Protein Atlas data. Using an external validation mass spectrometry dataset, we show that more than 500 proteins undetected by immunohistochemistry are robustly measured by mass spectrometry. The Human Protein Atlas transcriptome data, on the other hand, exhibit similar transcript detection levels as other kidney RNA-seq datasets. Discordance in mRNA-protein expression could be due to both biological and technical reasons, such as transcriptional dynamics, translation rates, protein half-lives, and measurement errors. This is further complicated by the heterogeneity of the kidney tissue itself, which can increase the discordance if the cell populations or tissue compartment samples do not match. As such, shedding light on the mRNA-protein relationship of the kidney-specific Human Protein Atlas data can provide context to our scientific inferences onrenal gene and protein quantification.

Haloxylon salicornicum Phytochemicals Suppress NF-κB, iNOS and Pro-inflammatory Cytokines in Lipopolysaccharide-induced Macrophages.

Haloxylon salicornicum is traditionally used for the treatment of several disorders associated with inflammation. Despite it is a defense response against tissue injury and infections, inflammation can become a chronic condition that can negatively impact the body. This study investigated the effect of H. salicornicum phytochemicals nuclear factor-kappaB (NF-κB), inducible nitric oxide synthase (iNOS) and cytokines release by lipopolysaccharide (LPS)-challenged macrophages in vitro. The binding affinity of the tested phytochemical towards NF-κB and iNOS was investigated using molecular docking. Ten compounds (four coumarins, three sterols and three flavonoids) were isolated from the ethanolic extract of H. salicornicum. Treatment of LPS-challenged macrophages with the compounds resulted in remarkable decrease in NF-κB p65 and iNOS mRNA abundance. All compounds suppressed the production of nitric oxide (NO) and the pro-inflammatory cytokines (tumor necrosis factor (TNF)-α and interleukin (IL)-6) from macrophages challenged with LPS. Molecular docking revealed the ability of the isolated phytochemicals to bind NF-κB p65 and iNOS. In conclusion, H. salicornicum is a rich source of phytochemicals with anti-inflammatory properties. The anti-inflammatory efficacy of H. salicornicum phytoconstituents is mediated via their ability to modulate NF-κB and iNOS, and suppress the release of NO, TNF-α, and IL-6 from macrophages.

GLUT1 and prorenin receptor mediate differential regulation of TGF-β and CTGF in renal inner medullary collecting duct cells during high glucose conditions

BackgroundDuring diabetes, prorenin is highly produced by the renal collecting ducts. The binding of prorenin to (pro)renin receptor (PRR) on the apical plasma membrane triggers intracellular profibrotic genes, including TGF-β and CTGF. However, the underlying mechanisms contributing to the stimulation of these pathways remain unclear. Hence, we hypothesize that the glucose transporter-1 (GLUT1) favors the PRR-dependent stimulation of TGF-β and CTGF in the distal nephron segments during high glucose (HG) conditions.MethodsTo test this hypothesis, primary cultured renal inner medullary collecting duct (IMCD) cells were treated with normal glucose (NG, 5 mM) or high glucose (HG, 25 mM) for 48 h in the presence or absence of the GLUT1-specific inhibitor BAY 876 (2 nM). Additionally, IMCD cells were treated with the PRR antagonist PRO20. The expression of TGF-β and CTGF was quantified by immunoblot and qRT-PCR.ResultsHG increased GLUT1 mRNA and protein abundance, while BAY 876 inhibited these responses. HG treatment upregulated PRR, but the concomitant treatment with BAY 876 partially prevented this effect. TGF-β and CTGF expressions were augmented in IMCD cells treated with HG. However, PRO20 prevented the increases in TGF-β but not those of CTGF. GLUT1 inhibition partially prevented the increases in reactive oxygen species (ROS) during HG while PRO20 did not. ROS scavenging impaired CTGF upregulation during HG conditions. Additionally, long-term exposure to HG increases lipid peroxidation and reduced cell viability.ConclusionsThe data indicate that glucose transportation via GLUT1 is implicated in the PRR-dependent upregulation of TGF-β while CTGF is mediated mainly via a mechanism depending on ROS formation in renal medullary collecting duct cells.

KIF1A promotes neuroendocrine differentiation in prostate cancer by regulating the OGT-mediated O-GlcNAcylation.

Neuroendocrine prostate cancer (NEPC) arises from prostate adenocarcinoma after endocrine treatment failure and implies lethality and limited therapeutic options. Deciphering the molecular mechanisms underlying transdifferentiation from adenocarcinoma to NEPC may provide valuable therapeutic strategies. We performed a pan-cancer differential mRNA abundance analysis and identified that Kinesin-like protein (KIF1A) was highly expressed in NEPC. KIF1A knockdown impaired neuroendocrine(NE) features, including NE marker gene expression, stemness, and epithelial-mesenchymal transition (EMT), whereas KIF1A overexpression promoted these processes. Targeting KIF1A inhibited the growth of NE differentiated prostate cancer (PCa) cells in vitro and in vivo. Mechanistically, KIF1A bound with O-linked N-acetylglucosamine transferase (OGT) and regulated its protein expression and activity. Nuclear accumulation of OGT induced by KIF1A overexpression promoted intranuclear O-GlcNAcylation of β-catenin and OCT4 in nucleus. More importantly, our data revealed that OGT was critical for KIF1A induced NE differentiation and aggressive tumor growth. An OGT inhibitor, OSMI-1, can significantly inhibited NE differentiated PCa cell proliferation in vitro and tumor growth in vivo. Our findings showed that KIF1A promotes NE differentiation to NEPC by regulating the OGT-mediated O-GlcNAcylation. Targeting O-GlcNAcylation may impede the development of NEPC for a group of PCa patients with elevated KIF1A expression.

Positive impact of dietary marine sulfated polysaccharides derived from macroalgae during a necrotic enteritis challenge

Caused by the Gram-positive bacteria Clostridium perfringens, necrotic enteritis (NE) is an enteric disease with significant economic implications in broiler production. This study employed an experimental NE model involving co-infection with Eimeria maxima and C. perfringens to assess whether sulfate polysaccharides extracted from marine macroalgae could mitigate the adverse effects of NE in broilers. A total of 600 day (d)-old Ross 708 male broilers were randomly assigned to one of four treatment groups: NC (negative control, fed a corn-soybean meal diet); PC (positive control, NC + 15 ppm Avilamycin and 125 ppm Amprolium); AGS (Algimun® Standard, NC + Algimun added at 0.1 % of the diet); and AGH (Algimun High, NC + Algimun added at 0.2 % of the diet). Average daily feed intake and weight gain were calculated and adjusted for daily mortality on d 14, 21, 28, and 42. On d 21, four birds/pen were examined for intestinal NE lesions. On d 14, 21, and 42, serum and jejunum samples from one bird/pen were collected to measure the concentration of the biomarker calprotectin and mRNA abundance of cytokines and tight junction proteins. Data were analyzed using JMP and significance between treatments identified by LSD (P ≤ 0.05). NE lesion scores on d 21, and mortality and FCR during d 0 to 42 were lower in PC, AGS, and AGH compared to NC birds. AGS birds had the lowest levels for serum calprotectin on d 21 while PC, AGS, and AGH had lower levels than the NC group on d 42. On d 14 and d 42, mRNA abundance of CLDN1, 3, ZO1, IL1β, IFNγ, IL10, and IL12B was greater in AGS and AGH birds compared to NC. Also, AGH had a greater abundance of TNFα on d 14 and d 42 compared to NC. Further, mRNA abundance of CLDN3, ZO1, 2, OCLDN, IL1β, IL10, IL12B, IFNγ, and ANXA1 was greater in PC on d 21 compared to NC and AGS. Based on these findings, dietary supplementation of this macroalgae-derived sulfated polysaccharides yielded a similar response to an antibiotic growth promoter presenting potential as an alternative additive.

Exploration of Some Nutrient Absorption-Related Genes and Intestinal Histology in Japanese Quails (Coturnix japonica) Throughout Their Productive Lifespan.

Quail is an efficient poultry species known for many years, and its productivity is highly affected by gastrointestinal function, health, digestion and commensal bacteria. Specific histological parameters and nutrient absorption-related genes could be used to study intestinal functionality and efficiency. This study aimed to evaluate the mRNA abundance of nutrient absorption-related genes and intestinal histology changes in laying Japanese quail during productive lifespan. A total of 135 quails from a commercial flock at different egg production periods (early production [EP, 6 weeks of age], pick production [PP, 21 weeks of age] and post pick production [PPP, 40 weeks of age]) were randomly selected (n=45/group) and kept for 6 weeks. Production performance was evaluated during last 2 weeks of the experimental period, and tissue samples were taken from the duodenum, jejunum and ileum for histological analysis and mRNA abundance purposes at the end of the experiment. The mRNA abundance of the CALB1, SGLT1, SGLT5, EAAT3 and NaPi-IIb genes were compared among groups. Production period did not affect the eggshell thickness, goblet cell number and mRNA abundance of CALB1, EAAT3 and NaPi-IIb (p>0.05). The PPP quails had the highest egg weight and FCR compared to EP and PP quails (p<0.001). Egg production and egg mass of the EP and PP quails were higher than those of PPP quails (p<0.001). Moreover, EP quails had the highest villus height (VH)/crypt depth (CD) ratio in the duodenum and jejunum and the lowest VH:CD in the ileum compared with the PPP quails (p<0.01). The lowest SGLT1 mRNA abundance was noted in the PP quails, whereas EP quails had the highest SGLT5 mRNA abundance (p<0.05). In conclusion, although histology parameter changes were in-line with the lowest performance observed in the PPP quails, the current results failed to show a relationship between mRNA abundance changes and production performance attributes at different productive lifespans.

Comparison of chromatin accessibility remodeling of granulosa cells in patients with endometrioma or pelvic/tubal infertility.

To elucidatethe epigenetic alteration associated with impaired oogenesis in endometrioma using multi-omic approaches. ATAC-seq was performed on the granulosa cells (GCs) of 6 patients (3 with endometrioma and 3 without). Follicular samples from another 20 patients (10 with endometrioma and 10 without) were collected for mRNA-seq analysis of GCs and extracellular vesicles (EVs) of follicular fluid. qRT-PCR validated candidate genes in GCs from 44 newly enrolled patients (19 with endometrioma and 25 without). mRNA abundance was compared with the Mann-Whitney test. Pearson's correlation analyzed relationships between candidate genes and oocyte parameters. Chromatin accessibility and gene expression profiles of GCs from endometrioma patients differed significantly from the pelvic/tubal infertility group. RNA-seq revealed most differentially expressed genes were downregulated (6216/7325) and enriched in the cellular localization pathway. Multi-omics analyses identified 22 significantly downregulated genes in the GCs of endometrioma patients, including PPIF (P < 0.0001) and VEGFA (P = 0.0148). Both genes were further confirmed by qRT-PCR. PPIF (r = 0.46, p = 0.043) and VEGFA (r = 0.45, p = 0.048) correlated with the total number of retrieved oocytes. GC chromatin remodeling may disrupt GC and EV transcriptomes, interfering with somatic cell-oocyte communication and leading to compromised oogenesis in endometrioma patients.

PTH-dependent stabilization of RANKL mRNA is associated with increased phosphorylation of the KH-Type Splicing Regulatory Protein

Parathyroid hormone (PTH) receptor agonists promote bone formation but also increase osteoclastogenesis, in part by increasing expression of the receptor activator of nuclear factor kappa-Β ligand (RANKL). In addition to activation of transcription, regulation of mRNA stability is another important molecular mechanism controlling mRNA abundance. PTH treatment for 6 hrs. resulted in a 7.4-fold elevation in RANKL mRNA expression in UAMS-32P cells, despite prior inhibition of cellular transcription by thiophosphoryl (TPL). RANKL mRNA, like other TNF family members, contains AU-Rich Elements (AREs) in the 3’ UTR. AU-Rich Element Binding Proteins (ABPs including KSRP, TTP, AUF1 and HuR) bind to AREs and regulate mRNA stability. There was significantly more KSRP bound to RANKL mRNA than any of the other ABPs. PTH did not increase the amount of ABPs bound to the RANKL transcript. However, the level of cellular phosphorylated KSRP was significantly increased in UAMS-32P cells pre-treated with TPL followed by PTH exposure, compared to cells treated with vehicle following TPL. The extent of phosphorylation of cellular AUF1, HuR, and TTP did not increase with PTH treatment. There were no significant changes in the cellular content of total Pin1 and phospho-Pin1 protein with PTH treatment. We conclude that increases in cellular phospho-KSRP following PTH treatment, together with fact that the total amount of the KSRP bound to the RANKL mRNA did not change with PTH-treatment, may indicate that phospho-KSRP plays some role in stabilizing the RANKL transcript.

Distinct Strategies Regulate Correlated Ion Channel mRNAs and Ionic Currents in Continually versus Episodically Active Neurons.

Relationships among membrane currents allow central pattern generator (CPG) neurons to reliably drive motor programs. We hypothesize that continually active CPG neurons utilize activity-dependent feedback to correlate expression of ion channel genes to balance essential membrane currents. However, episodically activated neurons experience absences of activity-dependent feedback and, thus, presumably employ other strategies to coregulate the balance of ionic currents necessary to generate appropriate output after periods of quiescence. To investigate this, we compared continually active pyloric dilator (PD) neurons with episodically active lateral gastric (LG) CPG neurons of the stomatogastric ganglion (STG) in male Cancer borealis crabs. After experimentally activating LG for 8 h, we measured three potassium currents and abundances of their corresponding channel mRNAs. We found that ionic current relationships were correlated in LG's silent state, but ion channel mRNA relationships were correlated in the active state. In continuously active PD neurons, ion channel mRNAs and ionic currents are simultaneously correlated. Therefore, two distinct relationships exist between channel mRNA abundance and the ionic current encoded in these cells: in PD, a direct correlation exists between Shal channel mRNA levels and the A-type potassium current it carries. Conversely, such channel mRNA-current relationships are not detected and appear to be temporally uncoupled in LG neurons. Our results suggest that ongoing feedback maintains membrane current and channel mRNA relationships in continually active PD neurons, while in LG neurons, episodic activity serves to establish channel mRNA relationships necessary to produce the ionic current profile necessary for the next bout of activity.

In-depth analysis of miRNA binding sites reveals the complex response of uterine epithelium to miR-26a-5p and miR-125b-5p during early pregnancy

Post-transcriptional regulation of gene expression by miRNAs likely makes significant contributions to mRNA abundance at the embryo-maternal interface. In this study, we investigated how miR-26a-5p and miR-125b-5p contribute to molecular changes occurring in the uterine luminal epithelium, which serves as the first site of signal exchange between the mother and developing embryo. To measure de novo protein synthesis after miRNA delivery to primary uterine luminal epithelial cells, we employed pulsed stable isotope labeling by amino acids (pSILAC). We found that both miRNAs alter the proteome of luminal epithelial cells, impacting numerous cellular functions, immune responses, as well as intracellular and second messenger signaling pathways. Additionally, we identified several features of miRNA-mRNA interactions that may influence the targeting efficiency of miR-26a-5p and miR-125b-5p. Overall, our study suggests a complex interaction of miR-26a-5p and miR-125b-5p with their respective targets. However, both appear to cooperatively function in modulating the cellular environment of the luminal epithelium, facilitating the morphological and molecular changes that occur during the intensive communication between the embryo and uterus at pregnancy.

M6Am sequesters PCF11 to suppress premature termination and drive neuroblastoma differentiation

N6,2'-O-dimethyladenosine (m6Am) is an abundant mRNA modification that impacts multiple diseases, but its function remains controversial because the m6Am reader is unknown. Using quantitative proteomics, we identified transcriptional terminator premature cleavage factor II (PCF11) as a m6Am-specific reader in human cells. Direct quantification of mature versus nascent RNAs reveals that m6Am does not regulate mRNA stability but promotes nascent transcription. Mechanistically, m6Am functions by sequestering PCF11 away from proximal RNA polymerase II (RNA Pol II). This suppresses PCF11 from dissociating RNA Pol II near transcription start sites, thereby promoting full-length transcription of m6Am-modified RNAs. m6Am's unique relationship with PCF11 means m6Am function is enhanced when PCF11 is reduced, which occurs during all-trans-retinoic-acid (ATRA)-induced neuroblastoma-differentiation therapy. Here, m6Am promotes expression of ATF3, which represses neuroblastoma biomarker MYCN. Depleting m6Am suppresses MYCN repression in ATRA-treated neuroblastoma and maintains their tumor-stem-like properties. Collectively, we characterize m6Am as an anti-terminator RNA modification that suppresses premature termination and modulates neuroblastoma's therapeutic response.

Evolutionary divergent clusters of transcribed extinct truncated retroposons drive low mRNA expression and developmental regulation in the protozoan Leishmania

BackgroundThe Leishmania genome harbors formerly active short interspersed degenerated retroposons (SIDERs) representing the largest family of repetitive elements among trypanosomatids. Their substantial expansion in Leishmania is a strong predictor of important biological functions. In this study, we combined multilevel bioinformatic predictions with high-throughput genomic and transcriptomic analyses to gain novel insights into the diversified roles retroposons of the SIDER2 subfamily play in Leishmania genome evolution and expression.ResultsWe show that SIDER2 retroposons form various evolutionary divergent clusters, each harboring homologous SIDER2 sequences usually located nearby in the linear sequence of chromosomes. This intriguing genomic organization underscores the importance of SIDER2 proximity in shaping chromosome dynamics and co-regulation. Accordingly, we show that transcripts belonging to the same SIDER2 cluster can display similar levels of expression. SIDER2 retroposons are mostly transcribed as part of 3'UTRs and account for 13% of the Leishmania transcriptome. Genome-wide expression profiling studies underscore SIDER2 association generally with low mRNA expression. The remarkable link of SIDER2 retroposons with downregulation of gene expression supports their co-option as major regulators of mRNA abundance. SIDER2 sequences also add to the diversification of the Leishmania gene expression repertoire since ~ 35% of SIDER2-containing transcripts can be differentially regulated throughout the parasite development, with a few encoding key virulence factors. In addition, we provide evidence for a functional bias of SIDER2-containing transcripts with protein kinase and transmembrane transporter activities being most represented.ConclusionsAltogether, these findings provide important conceptual advances into evolutionary innovations of transcribed extinct retroposons acting as major RNA cis-regulators.

Evaluation of the Dietary Arginine Supplementation on Yellow Catfish: From a Low-Temperature Farming Perspective

The yellow catfish is an economically significant freshwater fish with increasing importance in aquaculture. However, the low temperature environments prevalent in certain regions pose challenges to its growth, development, and overall health. This study aimed to explore the impact of dietary arginine (Arg) addition on the growth, digestive capacity, and intestinal antioxidant response in fish under low temperature acclimation (18 °C). Total 720 fish were randomly distributed into six groups, each containing 120 fish. Over the course of eight weeks, each group was fed with diets about varying Arg concentrations (1.79–3.26 g/kg). The results indicated that Arg supplementation resulted in an increase in specific growth rate (SGR), feed intake (FI), feed efficiency (FE), as well as pancreatic enzyme activities in both pancreas and intestine. Conversely, malondialdehyde (MDA) and protein carbonyl (PC) contents initially decreased but increased with higher Arg concentrations. Glutathione peroxidase 1a (GPX1a) showed a positive correlation with nuclear factor-erythroid 2-related factor-2 (Nrf2), showing its role in antioxidative capacity. Furthermore, this study revealed that Arg significantly enhanced the activities of anti-superoxide anion, anti-hydroxyl radical, and anti-oxidative enzymes, along with the relative mRNA abundance of Copper-Zinc superoxide dismutase (CuZnSOD), catalase, GPX1a, glutamate-cysteine ligase catalytic subunit (GCLC), and Nrf2 in the intestine. It was determined that yellow catfish weighing between 61.0 g and 89.0 g require an intake of 26.8 g of Arg per kilogram of diet based on polynomial regression analysis of specific growth rate (SGR), which is equivalent to 37.0 g of dietary protein, under sub-low temperature conditions of 18 °C.

Loss of IDH1 and IDH2 in macrophages induces pathological phenotypes in cardiomyocytes

Abstract Introduction The clonal expansion of hematopoietic stem cells can be induced by a variety of age-related somatic mutations, a phenomenon known as "clonal hematopoiesis of indeterminate potential" (CHIP). CHIP is associated with an increased incidence and poor outcomes in patients with cardiovascular disease. The mechanisms underlying the crosstalk between CHIP cells and cardiac tissue are yet to be elucidated. Rare mutations in the metabolism-regulating isocitrate dehydrogenases encoding genes IDH1 and IDH2 have been identified as AML-like and cardiovascular high-risk CHIP-driving mutations in patient cohorts. However, the potential paracrine effects of these mutations in immune cells on cardiac tissue are unknown. Therefore, we assessed the impact of IDH1 and IDH2 mutations on the paracrine activities of macrophages. Methods and Results To gain first insights into the functional consequences of the IDH mutations, we used the AlphaMissense algorithm, which predicts the impact of mutations on protein function. IDH1 and IDH2 mutations identified in our patient cohorts were predicted to induce pathological alterations of protein function with a score &amp;gt;0.99. Therefore, we assessed the effects of silencing of the two genes in primary human CD14+ M0 macrophages on the paracrine activity on neonatal rat cardiomyocytes. Transfection with siRNA pools targeting IDH1 and IDH2 to mimic CHIP-driving conditions reduced mRNA abundance by 78% and 73%, respectively (both p&amp;lt;0.05). To assess the impact of IDH-silencing on cardiovascular cells, cardiomyocytes were treated with the supernatant of the macrophages. Interestingly, supernatants of IDH1 silenced macrophages significantly increased apoptosis of cardiomyocytes (8.6-fold increased expression of cleaved caspase-3; p&amp;lt;0.05) and reduced beating frequency. In addition, supernatants of IDH1 silenced macrophages induced a 1.5-fold increase in cardiomyocyte hypertrophy (p&amp;lt;0.01), whereas silencing of IDH2 showed no effect on cardiomyocyte hypertrophy and apoptosis. However, supernatants of both IDH1 and IDH2 silenced macrophages resulted in significantly increased numbers of bi- and multinucleated cardiomyocytes. Conclusion: Loss of isocitrate dehydrogenases in human macrophages affects cardiomyocyte functions in a paracrine fashion. Particularly silencing of IDH1 in macrophages introduced cardiomyocyte hypertrophy, apoptosis, and bi- and multinucleation, and reduced contractile function. Ongoing studies will address how IDH1 and IDH2 silencing-induced alterations in macrophages control their paracrine activity on cardiomyocytes.

MRNA ADENOSINE METHYLASE promotes drought tolerance through N6-methyladenosine-dependent and independent impacts on mRNA regulation in Arabidopsis.

Among many mRNA modifications, adenine methylation at the N6 position (N6-methyladenosine, m6A) is known to affect mRNA biology extensively. The influence of m6A has yet to be assessed under drought, one of the most impactful abiotic stresses. We show that Arabidopsis thaliana (L.) Heynh. (Arabidopsis) plants lacking mRNA ADENOSINE METHYLASE (MTA) are drought-sensitive. Subsequently, we comprehensively assess the impacts of MTA-dependent m6A changes during drought on mRNA abundance, stability, and translation in Arabidopsis. During drought, there is a global trend toward hypermethylation of many protein-coding transcripts that does not occur in mta. We also observe complex regulation of m6A at a transcript-specific level, possibly reflecting compensation by other m6A components. Importantly, a subset of transcripts that are hypermethylated in an MTA-dependent manner exhibited reduced turnover and translation in mta, compared with wild-type (WT) plants, during drought. Additionally, MTA impacts transcript stability and translation independently of m6A. We also correlate drought-associated deposition of m6A with increased translation of modulators of drought response, such as RD29A, COR47, COR413, ALDH2B, ERD7, and ABF4 in WT, which is impaired in mta. m6A is dynamic during drought and, alongside MTA, promotes tolerance by regulating drought-responsive changes in transcript turnover and translation.

Single-cell chemoproteomics identifies metastatic activity signatures in breast cancer.

Protein activity state, rather than protein or mRNA abundance, is a biologically regulated and relevant input to many processes in signaling, differentiation, development, and diseases such as cancer. While there are numerous methods to detect and quantify mRNA and protein abundance in biological samples, there are no general approaches to detect and quantify endogenous protein activity with single-cell resolution. Here, we report the development of a chemoproteomic platform, single-cell activity-dependent proximity ligation, which uses automated, microfluidics-based single-cell capture and nanoliter volume manipulations to convert the interactions of family-wide chemical activity probes with native protein targets into multiplexed, amplifiable oligonucleotide barcodes. We demonstrate accurate, reproducible, and multiplexed quantitation of a six-enzyme (Ag-6) panel with known ties to cancer cell aggressiveness directly in single cells. We further identified increased Ag-6 enzyme activity across breast cancer cell lines of increasing metastatic potential, as well as in primary patient-derived tumor cells and organoids from patients with breast cancer.

Toxoplasma gondii infection induces the expression of the chemokine CXCL16 in macrophages to promote chemoattraction of CXCR6+ cells.

CXCL16 is a multifaceted chemokine expressed by macrophages and other immune cells in response to viral and bacterial pathogens. However, few studies have investigated its role in parasitic infections. The obligate intracellular parasite Toxoplasma gondii (T. gondii) is the causative agent of toxoplasmosis, an infection with potentially deleterious consequences in immunocompromised individuals and the developing fetus of acutely infected pregnant women. Chemokines are critical mediators of host defense and, as such, dysregulation of their expression is a subversion strategy often employed by the parasite to ensure its survival. Herein, we report that types I and II T. gondii strains upregulated the expression of both transmembrane and soluble forms of CXCL16 in infected bone marrow-derived macrophages (BMDM). Exposure to soluble T. gondii antigens (STAg) and to excreted-secreted proteins (TgESP) led to the induction of CXCL16. Cxcl16 mRNA abundance and CXCL16 protein levels increased in a time-dependent manner upon T. gondii infection. Importantly, conditioned medium (CM) collected from T. gondii-infected wild-type (WT) macrophage cultures promoted the migration of RAW264.7 cells expressing CXCR6, the cognate receptor of CXCL16, an effect that was significantly reduced by a neutralizing anti-CXCL16 antibody or use of CM from CXCL16 knockout (KO) macrophages. Lastly, T. gondii-driven CXCL16 expression appeared to modulate cytokine-induced (IL-4 + IL-13) alternative macrophage activation and M2 phenotypic marker expression. Further investigation is required to determine whether this chemokine contributes to the pathogenesis of toxoplasmosis and to elucidate the underlying molecular mechanisms.

Localized synthesis of molecular chaperones sustains neuronal proteostasis.

Neurons are challenged to maintain proteostasis in neuronal projections, particularly with the physiological stress at synapses to support intercellular communication underlying important functions such as memory and movement control. Proteostasis is maintained through regulated protein synthesis and degradation and chaperone-assisted protein folding. Using high-resolution fluorescent microscopy, we discovered that neurons localize a subset of chaperone mRNAs to their dendrites, particularly more proximal regions, and increase this asymmetric localization following proteotoxic stress through microtubule-based transport from the soma. The most abundant chaperone mRNA in dendrites encodes the constitutive heat shock protein 70, HSPA8. Proteotoxic stress in cultured neurons, induced by inhibiting proteasome activity or inducing oxidative stress, enhanced transport of Hspa8 mRNAs to dendrites and the percentage of mRNAs engaged in translation on mono and polyribosomes. Knocking down the ALS-related protein Fused in Sarcoma (FUS) and a dominant mutation in the heterogenous nuclear ribonucleoprotein A2/B1 (HNRNPA2B1) impaired stress-mediated localization of Hspa8 mRNA to dendrites in cultured murine motor neurons and human iPSC-derived neurons, respectively, revealing the importance of these RNA-binding proteins in maintaining proteostasis. These results reveal the increased dendritic localization and translation of the constitutive HSP70 Hspa8 mRNA as a crucial neuronal stress response to uphold proteostasis and prevent neurodegeneration.

Effects of PPARG on the proliferation, apoptosis, and estrogen secretion in goat granulosa cells

As a member of peroxisome proliferator-activated receptor (PPAR) family, PPARG has been reported to be involved in glucolipid metabolism in various species. However, the function of PPARG in estradiol (E2) synthesis, apoptosis, and proliferation in goat ovarian granulosa cells (GCs) is unclear. In this study, we found that goat PPARG was expressed in GCs of all grades of follicles, and localized in the cytoplasm and nucleus of GCs. Transfection of small interfering RNA-PPARG2 (si-PPARG2) decreased E2 synthesis and the abundances of HSD3B and CYP19A1 mRNA and protein. It also promoted cell apoptosis with significant increases in the ratio of BAX/BCL-2 and Caspase3 mRNA and protein. Meanwhile, cell cycle was inhibited by si-PPARG2 transfection, accompanied by decreased mRNA levels of CDK4, CKD6, MYC, CCND1, CCND2, PCNA, and CCNB, increased mRNA level of P53, and decreased protein levels of CDK4, MYC, and CCND1. Furthermore, PPARG interference affected the mRNA and protein abundances of CREB as well as the phosphorylation of CREB but not PKA. In conclusion, our data suggest that PPARG plays an important role in regulating E2 synthesis, cell apoptosis, and proliferation of goat GCs, including the CREB expression and phosphorylation. These results provide evidences for the in-depth study of PPARG in the regulation of goat GCs function.