MYC Recruits SPT5 to RNA Polymerase II to Promote Processive Transcription Elongation.

The MYC oncoprotein binds to promoter-proximal regions of virtually all transcribed genes and enhances RNA polymerase II (Pol II) function, but its precise mode of action is poorly understood. Using mass spectrometry of both MYC and Pol II complexes, we show here that MYC controls the assembly of Pol II with a small set of transcription elongation factors that includes SPT5, a subunit of the elongation factor DSIF. MYC directly binds SPT5, recruits SPT5 to promoters, and enables the CDK7-dependent transfer of SPT5 onto Pol II. Consistent with known functions of SPT5, MYC is required for fast and processive transcription elongation. Intriguingly, the high levels of MYC that are expressed in tumors sequester SPT5 into non-functional complexes, thereby decreasing the expression of growth-suppressive genes. Altogether, these results argue that MYC controls the productive assembly of processive Pol II elongation complexes and provide insight into how oncogenic levels of MYC permit uncontrolled cellular growth.

ZFAND1 Recruits p97 and the 26S Proteasome to Promote the Clearance of Arsenite-Induced Stress Granules.

Stress granules (SGs) are cytoplasmic assemblies of mRNPs stalled in translation initiation. They are induced by various stress conditions, including exposure to the environmental toxin and carcinogen arsenic. While perturbed SG turnover is linked to the pathogenesis of neurodegenerative diseases, the molecular mechanisms underlying SG formation and turnover are still poorly understood. Here, we show that ZFAND1 is an evolutionarily conserved regulator of SG clearance. ZFAND1 interacts with two key factors of protein degradation, the 26S proteasome and the ubiquitin-selective segregase p97, and recruits them to arsenite-induced SGs. In the absence of ZFAND1, SGs lack the 26S proteasome and p97, accumulate defective ribosomal products, and persist after arsenite removal, indicating their transformation into aberrant, disease-linked SGs. Accordingly, ZFAND1 depletion is epistatic to the expression of pathogenic mutant p97 with respect to SG clearance, suggesting that ZFAND1 function is relevant to the multisystem degenerative disorder IBMPFD/ALS.

Grad-seq in a Gram-positive bacterium reveals exonucleolytic sRNA activation in competence control.

RNA-protein interactions are the crucial basis for many steps of bacterial gene expression, including post-transcriptional control by small regulatory RNAs (sRNAs). In stark contrast to recent progress in the analysis of Gram-negative bacteria, knowledge about RNA-protein complexes in Gram-positive species remains scarce. Here, we used the Grad-seq approach to draft a comprehensive landscape of such complexes in Streptococcus pneumoniae, in total determining the sedimentation profiles of ~ 88% of the transcripts and ~ 62% of the proteins of this important human pathogen. Analysis of in-gradient distributions and subsequent tag-based protein capture identified interactions of the exoribonuclease Cbf1/YhaM with sRNAs that control bacterial competence for DNA uptake. Unexpectedly, the nucleolytic activity of Cbf1 stabilizes these sRNAs, thereby promoting their function as repressors of competence. Overall, these results provide the first RNA/protein complexome resource of a Gram-positive species and illustrate how this can be utilized to identify new molecular factors with functions in RNA-based regulation of virulence-relevant pathways.

Use of human predictive patch test (HPPT) data for the classification of skin sensitization hazard and potency.

Since the 1940s, patch tests in healthy volunteers (Human Predictive Patch Tests, HPPTs) have been used to identify chemicals that cause skin sensitization in humans. Recently, we reported the results of a major curation effort to support the development of OECD Guideline 497 on Defined Approaches (DAs) for skin sensitization (OECD in Guideline No. 497: Defined Approaches on Skin Sensitisation, 2021a. https://doi.org/10.1787/b92879a4-en ). In the course of this work, we compiled and published a database of 2277 HPPT results for 1366 unique test substances (Strickland et al. in Arch Toxicol 97:2825-2837, 2023. https://doi.org/10.1007/s00204-023-03530-3 ). Here we report a detailed analysis of the value of HPPT data for classification of chemicals as skin sensitizers under the United Nations' Globally Harmonized System of Classification and Labelling of Chemicals (GHS). As a result, we propose the dose per skin area (DSA) used for classification by the GHS to be replaced by or complemented with a dose descriptor that may better reflect sensitization incidence [e.g., the DSA causing induction of sensitization in one individual (DSA1+) or the DSA leading to an incidence of induction in 5% of the tested individuals (DSA05)]. We also propose standardized concepts and workflows for assessing individual HPPT results, for integrating multiple HPPT results and for using them in concert with Local Lymph Node Assay (LLNA) data in a weight of evidence (WoE) assessment. Overall, our findings show that HPPT results are often not sufficient for deriving unambiguous classifications on their own. However, where they are, the resulting classifications are reliable and reproducible and can be integrated well with those from other skin sensitization data, such as the LLNA.

Estradiol production of granulosa cells is unaffected by the physiological mix of nonesterified fatty acids in follicular fluid.

Ovarian cycle is controlled by circulating levels of the steroid hormone 17-ß-estradiol, which is predominantly synthesized by the granulosa cells (GCs) of ovarian follicles. Our earlier studies showed that unsaturated fatty acids (USFs) downregulate and saturated fatty acids (SFAs) upregulate estradiol production in GCs. However, it was unclear whether pituitary gonadotropins induce accumulation of free fatty acids (FFAs) in the follicular fluid since follicle-stimulating hormone induces and luteinizing hormone inhibits estradiol production in the mammalian ovary. Interestingly, we show here the gas chromatography analysis of follicular fluid revealed no differential accumulation of FFAs between pre- and post-luteinizing hormone surge follicles. We therefore wondered how estradiol production is regulated in the physiological context, as USFs and SFAs are mutually present in the follicular fluid. We thus performed in vitro primary GC cultures with palmitate, palmitoleate, stearate, oleate, linoleate, and alpha-linolenate, representing >80% of the FFA fraction in the follicular fluid, and analyzed 62 different cell culture conditions to understand the regulation of estradiol biosynthesis under diverse FFA combinations. Our analyses showed co-supplementation of SFAs with USFs rescued estradiol production by restoring gonadotropin receptors and aromatase, antagonizing the inhibitory effects of USFs. Furthermore, transcriptome data of oleic acid-treated GCs indicated USFs induce the ERK and Akt signaling pathways. We show SFAs inhibit USF-induced ERK1/2 and Akt activation, wherein ERK1/2 acts as a negative regulator of estradiol synthesis. We propose SFAs are vital components of the follicular fluid, without which gonadotropin signaling and the ovarian cycle would probably be shattered by USFs.

Early milk-feeding regimes in calves exert long-term effects on the development of ovarian granulosa cells.

BACKGROUND: Nutrition has not only an impact on the general wellbeing of an animal but can also affect reproductive processes. In cattle, feeding regimes can influence the age of puberty onset and alter gonadal development. We analyzed effects of different milk replacer (MR) feeding regimes during rearing on ovarian physiology with specific emphasis on the numbers as well as gene expression characteristics of granulosa cells (GCs) at the age of puberty onset. Two groups of calves received either 10% or 20% of bodyweight MR per day during their first 8 weeks. After weaning, both groups were fed the same mixed ration ad libitum until slaughter at 8 months. RESULTS: Animals of the 20% feeding group had a significantly higher body weight, but the proportion of animals having a corpus luteum at the time of slaughter was not different between groups, suggesting a similar onset of puberty. Calves of the 10% group showed a constant GC count regardless of the number of follicles (r = 0.23) whereas in the 20% group increasing numbers of GCs were detected with a higher follicle count (r = 0.71). As a first effort to find a possible molecular explanation for this unexpected limitation of GC numbers in the 10% group, we comparatively analyzed GC transcriptomes in both diet groups. The mRNA microarray analysis revealed a total of 557 differentially expressed genes comparing both groups (fold change > |1.5| and p < 0.05). OAS1X, MX2 and OAS1Z were among the top downregulated genes in the 20% vs. the 10% group, whereas top upregulated genes comprised BOLA and XCL1. All of these genes are known to be regulated by interferon. Subsequent signaling pathway analysis revealed the involvement of several immune response mechanisms in accordance with a number of interferons as upstream regulators. CONCLUSIONS: The results indicate that the plane of MR feeding in early life has an impact on the number and physiology of GCs later in life. This might influence the overall reproductive life initiated by the onset of puberty in cattle. In addition, the observed alterations in GCs of calves fed less MR might be a consequence of interferon regulated immunological pathways.

A database of human predictive patch test data for skin sensitization.

Critical to the evaluation of non-animal tests are reference data with which to assess their relevance. Animal data are typically used because they are generally standardized and available. However, when regulatory agencies aim to protect human health, human reference data provide the benefit of not having to account for possible interspecies variability. To support the evaluation of non-animal approaches for skin sensitization assessment, we collected data from 2277 human predictive patch tests (HPPTs), i.e., human repeat insult patch tests and human maximization tests, for skin sensitization from 1555 publications. We recorded protocol elements and positive or negative outcomes, developed a scoring system to evaluate each test for reliability, and calculated traditional and non-traditional dose metrics. We also traced each test result back to its original report to remove duplicates. The resulting database, which contains information for 1366 unique substances, was characterized for physicochemical properties, chemical structure categories, and protein binding mechanisms. This database is publicly available on the National Toxicology Program Interagency Center for the Evaluation of Alternative Toxicological Methods website and in the Integrated Chemical Environment to serve as a resource for additional evaluation of alternative methods and development of new approach methodologies for skin sensitization assessments.

Translocation of Oocytic HES1 into Surrounding Cumulus Cells in Bovine: Mechanism of Cellular Interaction during IVM?

HES1 (hairy and enhancer of split-1, effector of the NOTCH pathway) plays a role in oocyte maturation and has been detected so far mainly in somatic follicular cells. In this study, we aimed to investigate whether HES1 is present in both compartments of bovine cumulus oocyte complexes (COCs) and whether in vitro maturation itself has an effect on its distribution. We investigated the abundance of HES1 mRNA and protein in bovine COCs characterized by Brilliant-Cresyl-Blue (BCB) stainability by RT-PCR and immunofluorescence before and after in vitro maturation (IVM). To study the interaction of the compartments and the possible translocation of HES1, we injected GFP-HES1 mRNA into oocytes before maturation and analyzed fluorescence recovery after photobleaching (FRAP). The results showed that HES1 mRNA was detectable in oocytes but not in cumulus cells. The number of transcripts increased with maturation, especially in BCB-positive oocytes. In contrast, the protein was mainly visible in cumulus cells both before and after maturation. After GFP-HES1-mRNA injection into oocytes, a signal could be detected not only in the oocytes but also in cumulus cells. Our result shows a nearly exclusive distribution of HES1 mRNA and protein in oocytes and cumulus cells, respectively, that might be explained by the transfer of the protein from the oocyte into cumulus cells.

Lactate-induced effects on bovine granulosa cells are mediated via PKA signaling.

L-lactate acts as a signaling molecule in bovine granulosa cells (GCs). The initiated alterations depend on the transport of L-lactate into the cells via monocarboxylate transporters. In the present study, we further elucidated the intracellular actions of L-lactate and tested whether the PKA signaling pathway is involved. Therefore, we treated cultured bovine GCs with L-lactate and PKA inhibitors H-89 and KT5720, and with an activator of PKA, 6-Bnz-cAMP. L-lactate treatment resulted in decreased estradiol production and downregulation of CYP19A1, FSHR, and LHCGR as well as in the upregulation of the markers of early luteinization PTX3, RGS2, and VNN2. These specific L-lactate effects were almost completely abolished by pre-treatment of the GCs with both inhibitors of PKA signaling. In addition, also the L-lactate-induced upregulation of LDHA and of the monocarboxylate transporters SLC16A1 and SLC16A7 was abolished after PKA inhibition. An activation of the PKA with 6-Bnz-cAMP revealed similar effects on the gene expression like L-lactate alone. In summary, the presented data demonstrate that L-lactate-induced effects on GCs are mediated via PKA signaling thus supporting the role of L-lactate as signaling molecule during the folliculo-luteal transition.

O-GlcNAcylation of PERIOD regulates its interaction with CLOCK and timing of circadian transcriptional repression.

Circadian clocks coordinate time-of-day-specific metabolic and physiological processes to maximize organismal performance and fitness. In addition to light and temperature, which are regarded as strong zeitgebers for circadian clock entrainment, metabolic input has now emerged as an important signal for clock entrainment and modulation. Circadian clock proteins have been identified to be substrates of O-GlcNAcylation, a nutrient sensitive post-translational modification (PTM), and the interplay between clock protein O-GlcNAcylation and other PTMs is now recognized as an important mechanism by which metabolic input regulates circadian physiology. To better understand the role of O-GlcNAcylation in modulating clock protein function within the molecular oscillator, we used mass spectrometry proteomics to identify O-GlcNAcylation sites of PERIOD (PER), a repressor of the circadian transcriptome and a critical biochemical timer of the Drosophila clock. In vivo functional characterization of PER O-GlcNAcylation sites indicates that O-GlcNAcylation at PER(S942) reduces interactions between PER and CLOCK (CLK), the key transcriptional activator of clock-controlled genes. Since we observe a correlation between clock-controlled daytime feeding activity and higher level of PER O-GlcNAcylation, we propose that PER(S942) O-GlcNAcylation during the day functions to prevent premature initiation of circadian repression phase. This is consistent with the period-shortening behavioral phenotype of per(S942A) flies. Taken together, our results support that clock-controlled feeding activity provides metabolic signals to reinforce light entrainment to regulate circadian physiology at the post-translational level. The interplay between O-GlcNAcylation and other PTMs to regulate circadian physiology is expected to be complex and extensive, and reach far beyond the molecular oscillator.

Genome wide effects of oleic acid on cultured bovine granulosa cells: evidence for the activation of pathways favoring folliculo-luteal transition.

BACKGROUND: Metabolic stress, as negative energy balance on one hand or obesity on the other hand can lead to increased levels of free fatty acids in the plasma and follicular fluid of animals and humans. In an earlier study, we showed that increased oleic acid (OA) concentrations affected the function of cultured bovine granulosa cells (GCs). Here, we focus on genome wide effects of increased OA concentrations. RESULTS: Our data showed that 413 genes were affected, of which 197 were down- and 216 up-regulated. Specifically, the expression of FSH-regulated functional key genes, CCND2, LHCGR, INHA and CYP19A1 and 17-ß-estradiol (E2) production were reduced by OA treatment, whereas the expression of the fatty acid transporter CD36 was increased and the morphology of the cells was changed due to lipid droplet accumulation. Bioinformatic analysis revealed that associated pathways of the putative upstream regulators "FSH" and "Cg (choriogonadotropin)" were inhibited and activated, respectively. Down-regulated genes are over-represented in GO terms "reproductive structure/system development", "ovulation cycle process", and "(positive) regulation of gonadotropin secretion", whereas up-regulated genes are involved in "circulatory system development", "vasculature development", "angiogenesis" or "extracellular matrix/structure organization". CONCLUSIONS: From these data we conclude that besides inhibiting GC functionality, increased OA levels seemingly promote angiogenesis and tissue remodelling, thus suggestively initiating a premature fulliculo-luteal transition. In vivo this may lead to impeded folliculogenesis and ovulation, and cause sub-fertility.

The MYC mRNA 3'-UTR couples RNA polymerase II function to glutamine and ribonucleotide levels.

Deregulated expression of MYC enhances glutamine utilization and renders cell survival dependent on glutamine, inducing "glutamine addiction". Surprisingly, colon cancer cells that express high levels of MYC due to WNT pathway mutations are not glutamine-addicted but undergo a reversible cell cycle arrest upon glutamine deprivation. We show here that glutamine deprivation suppresses translation of endogenous MYC via the 3'-UTR of the MYC mRNA, enabling escape from apoptosis. This regulation is mediated by glutamine-dependent changes in adenosine-nucleotide levels. Glutamine deprivation causes a global reduction in promoter association of RNA polymerase II (RNAPII) and slows transcriptional elongation. While activation of MYC restores binding of MYC and RNAPII function on most promoters, restoration of elongation is imperfect and activation of MYC in the absence of glutamine causes stalling of RNAPII on multiple genes, correlating with R-loop formation. Stalling of RNAPII and R-loop formation can cause DNA damage, arguing that the MYC 3'-UTR is critical for maintaining genome stability when ribonucleotide levels are low.

Non-esterified fatty acids in the ovary: friends or foes?

A majority of common metabolic diseases can result in excessive lipolysis, leading to elevated levels of non-esterified fatty acids (NEFAs) in the body fluids. In females, increased NEFA levels in the follicular fluid markedly alter the functions of intrafollicular cells such as granulosa cells (GCs) and oocytes. Therefore, elevated levels of NEFAs have been suggested to be a significant player of subfertility in females of both human and economically important animal species such as cattle, buffalo, sheep, pig, chicken, and dog. However, the effects imposed by saturated and unsaturated fatty acids (SFAs and UFAs) on ovarian follicles are controversial. The present review emphasizes that SFAs induce apoptosis in granulosa and cumulus cells of ovarian follicles in different species. They further could adversely affect oocyte maturation and developmental competence. Many types of UFAs affect steroidogenesis and proliferation processes and could be detrimental for follicular cells, especially when at elevated concentrations. Interestingly, monounsaturated fatty acids (MUFAs) appear to contribute to the etiology of the polycystic ovarian syndrome (PCOS) as they were found to induce the transcription and translation of the androgenic transcription factor SOX9 while downregulating its estrogenic counterpart FOXL2 in GCs. Overall, this review presents our revised understanding of the effects of different fatty acids on the female reproductive success, which may allow other researchers and clinicians to investigate the mechanisms for treating metabolic stress-induced female infertility.

L-lactate induces specific genome wide alterations of gene expression in cultured bovine granulosa cells.

BACKGROUND: Previously, we could show that L-lactate affects cultured bovine granulosa cells (GC) in a specific manner driving the cells into an early pre-ovulatory phenotype. Here we studied genome wide effects in L-lactate-treated GC to further elucidate the underlying mechanisms that are responsible for the L-lactate induced transformation. Cultured estrogen producing GC treated either with L-lactate or vehicle control were subjected to mRNA microarray analysis. RESULTS: The analysis revealed 487 differentially expressed clusters, representing 461 annotated genes. Of these, 333 (= 318 genes) were identified as up- and 154 (= 143 genes) as down-regulated. As the top up-regulated genes we detected TXNIP, H19 and AHSG as well as our previously established marker transcripts RGS2 and PTX3. The top down-regulated genes included VNN1, SLC27A2 and GFRA1, but also MYC and the GC marker transcript CYP19A1. Pathway analysis with differentially expressed genes indicated "cAMP-mediated signaling" and "Axon guidance signaling" among the most affected pathways. Furthermore, estradiol, progesterone and Vegf were identified as potential upstream regulators. An effector network analysis by IPA provided first hints that processes of "angiogenesis" and "vascularization", but also "cell movement" appeared to be activated, whereas "organismal death" was predicted to be inhibited. CONCLUSIONS: Our data clearly show that L-lactate alters gene expression in cultured bovine GC in a broad, but obviously specific manner. Pathway analysis revealed that the mode of L-lactate action in GC initiates angiogenic processes, but also migratory events like cell movement and axonal guidance signaling, thus supporting the transformation of GC into an early luteal phenotype.

Drosophila carboxypeptidase D (SILVER) is a key enzyme in neuropeptide processing required to maintain locomotor activity levels and survival rate.

Neuropeptides are processed from larger preproproteins by a dedicated set of enzymes. The molecular and biochemical mechanisms underlying preproprotein processing and the functional importance of processing enzymes are well-characterised in mammals, but little studied outside this group. In contrast to mammals, Drosophila melanogaster lacks a gene for carboxypeptidase E (CPE), a key enzyme for mammalian peptide processing. By combining peptidomics and neurogenetics, we addressed the role of carboxypeptidase D (dCPD) in global neuropeptide processing and selected peptide-regulated behaviours in Drosophila. We found that a deficiency in dCPD results in C-terminally extended peptides across the peptidome, suggesting that dCPD took over CPE function in the fruit fly. dCPD is widely expressed throughout the nervous system, including peptidergic neurons in the mushroom body and neuroendocrine cells expressing adipokinetic hormone. Conditional hypomorphic mutation in the dCPD-encoding gene silver in the larva causes lethality, and leads to deficits in starvation-induced hyperactivity and appetitive gustatory preference, as well as to reduced viability and activity levels in adults. A phylogenomic analysis suggests that loss of CPE is not common to insects, but only occurred in Hymenoptera and Diptera. Our results show that dCPD is a key enzyme for neuropeptide processing and peptide-regulated behaviour in Drosophila. dCPD thus appears as a suitable target to genetically shut down total neuropeptide production in peptidergic neurons. The persistent occurrence of CPD in insect genomes may point to important further CPD functions beyond neuropeptide processing which cannot be fulfilled by CPE.

Lactate promotes specific differentiation in bovine granulosa cells depending on lactate uptake thus mimicking an early post-LH stage.

BACKGROUND: The LH-induced folliculo-luteal transformation is connected with alterations of the gene expression profile in cells of the granulosa layer. It has been described that hypoxic conditions occur during luteinization, thus favoring the formation of L-lactate within the follicle. Despite being a product of anaerobic respiration, L-lactate has been shown to act as a signaling molecule affecting gene expression in neuronal cells. During the present study, we tested the hypothesis that L-lactate may influence differentiation of follicular granulosa cells (GC). METHODS: In a bovine granulosa cell culture model effects of L- and D-lactate, of increased glucose concentrations and of the lactate transport inhibitor UK5099 were analyzed. Steroid hormone production was analyzed by RIA and the abundance of key transcripts was determined by quantitative real-time RT-PCR. RESULTS: L-lactate decreased the production of estradiol and significantly affected selected genes of the folliculo-luteal transition as well as genes of the lactate metabolism. CYP19A1, FSHR, LHCGR were down-regulated, whereas RGS2, VNN2, PTX3, LDHA and lactate transporters were up-regulated. These effects could be partly or completely reversed by pre-treatment of the cells with UK5099. The non-metabolized enantiomer D-lactate had even more pronounced effects on gene expression, whereas increased glucose concentrations did not affect transcript abundance. CONCLUSIONS: In summary, our data suggest that L-lactate specifically alters physiological and molecular characteristics of GC. These effects critically depend on L-lactate uptake, but are not triggered by increased energy supply. Further, we could show that L-lactate has a positive feedback on the lactate metabolism. Therefore, we hypothesize that L-lactate acts as a signaling molecule in bovine and possibly other monovular species supporting differentiation during the folliculo-luteal transformation.

Linking site-specific loss of histone acetylation to repression of gene expression by the mycotoxin ochratoxin A.

Ochratoxin A (OTA) is a potent renal carcinogen but its mechanism has not been fully resolved. In vitro and in vivo gene expression studies consistently revealed down-regulation of gene expression as the predominant transcriptional response to OTA. Based on the importance of specific histone acetylation marks in regulating gene transcription and our recent finding that OTA inhibits histone acetyltransferases (HATs), leading to loss of acetylation of histones and non-histone proteins, we hypothesized that OTA-mediated repression of gene expression may be causally linked to HAT inhibition and loss of histone acetylation. In this study, we used a novel mass spectrometry approach employing chemical 13C-acetylation of unmodified lysine residues for quantification of post-translational acetylation sites to identify site-specific alterations in histone acetylation in human kidney epithelial cells (HK-2) exposed to OTA. These results showed OTA-mediated hypoacetylation at almost all lysine residues of core histones, including loss of acetylation at H3K9 and H3K14, which are hallmarks of gene activation. ChIP-qPCR used to establish a possible link between H3K9 or H3K14 hypoacetylation and OTA-mediated down-regulation of selected genes (AMIGO2, CLASP2, CTNND1) confirmed OTA-mediated H3K9 hypoacetylation at promoter regions of these genes. Integrated analysis of OTA-mediated genome-wide changes in H3K9 acetylation identified by ChIP-Seq with published gene expression data further demonstrated that among OTA-responsive genes almost 80% of hypoacetylated genes were down-regulated, thus confirming an association between H3K9 acetylation status and gene expression of these genes. However, only 7% of OTA repressed genes showed loss of H3K9 acetylation within promoter regions. Interestingly, however, GO analysis and functional enrichment of down-regulated genes showing loss of H3K9 acetylation at their respective promoter regions revealed enrichment of genes involved in the regulation of transcription, including a number of transcription factors that are predicted to directly or indirectly regulate the expression of 98% of OTA repressed genes. Thus, it is possible that histone acetylation changes in a fairly small set of genes but with key function in transcriptional regulation may trigger a cascade of events that may lead to overall repression of gene expression. Taken together, our data provide evidence for a mechanistic link between loss of H3K9 acetylation as a consequence of OTA-mediated inhibition of HATs and repression of gene expression by OTA, thereby affecting cellular processes critical to tumorigenesis.

Identification of Targets and Interaction Partners of Arginyl-tRNA Protein Transferase in the Moss Physcomitrella patens.

Protein arginylation is a posttranslational modification of both N-terminal amino acids of proteins and sidechain carboxylates and can be crucial for viability and physiology in higher eukaryotes. The lack of arginylation causes severe developmental defects in moss, affects the low oxygen response in Arabidopsis thaliana and is embryo lethal in Drosophila and in mice. Although several studies investigated impact and function of the responsible enzyme, the arginyl-tRNA protein transferase (ATE) in plants, identification of arginylated proteins by mass spectrometry was not hitherto achieved. In the present study, we report the identification of targets and interaction partners of ATE in the model plant Physcomitrella patens by mass spectrometry, employing two different immuno-affinity strategies and a recently established transgenic ATE:GUS reporter line (Schuessele et al., 2016 New Phytol. , DOI: 10.1111/nph.13656). Here we use a commercially available antibody against the fused reporter protein (ß-glucuronidase) to pull down ATE and its interacting proteins and validate its in vivo interaction with a class I small heatshock protein via Förster resonance energy transfer (FRET). Additionally, we apply and modify a method that already successfully identified arginylated proteins from mouse proteomes by using custom-made antibodies specific for N-terminal arginine. As a result, we identify four arginylated proteins from Physcomitrella patens with high confidence.Data are available via ProteomeXchange with identifier PXD003228 and PXD003232.

LARP4B is an AU-rich sequence associated factor that promotes mRNA accumulation and translation.

mRNAs are key molecules in gene expression and subject to diverse regulatory events. Regulation is accomplished by distinct sets of trans-acting factors that interact with mRNAs and form defined mRNA-protein complexes (mRNPs). The resulting "mRNP code" determines the fate of any given mRNA and thus controlling gene expression at the post-transcriptional level. The La-related protein 4B (LARP4B) belongs to an evolutionarily conserved family of RNA-binding proteins characterized by the presence of a La-module implicated in direct RNA binding. Biochemical experiments have shown previously direct interactions of LARP4B with factors of the translation machinery. This finding along with the observation of an association with actively translating ribosomes suggested that LARP4B is a factor contributing to the mRNP code. To gain insight into the function of LARP4B in vivo we tested its mRNA association at the transcriptome level and its impact on the proteome. PAR-CLIP analyses allowed us to identify the in vivo RNA targets of LARP4B. We show that LARP4B binds to a distinct set of cellular mRNAs by contacting their 3' UTRs. Biocomputational analysis combined with in vitro binding assays identified the LARP4B-binding motif on mRNA targets. The reduction of cellular LARP4B levels leads to a marked destabilization of its mRNA targets and consequently their reduced translation. Our data identify LARP4B as a component of the mRNP code that influences the expression of its mRNA targets by affecting their stability.

Cultured bovine granulosa cells rapidly lose important features of their identity and functionality but partially recover under long-term culture conditions.

Cell culture models are essential for the detailed study of molecular processes. We analyze the dynamics of changes in a culture model of bovine granulosa cells. The cells were cultured for up to 8 days and analyzed for steroid production and gene expression. According to the expression of the marker genes CDH1, CDH2 and VIM, the cells maintained their mesenchymal character throughout the time of culture. In contrast, the levels of functionally important transcripts and of estradiol and progesterone production were rapidly down-regulated but showed a substantial up-regulation from day 4. FOXL2, a marker for granulosa cell identity, was also rapidly down-regulated after plating but completely recovered towards the end of culture. In contrast, expression of the Sertoli cell marker SOX9 and the lesion/inflammation marker PTGS2 increased during the first 2 days after plating but gradually decreased later on. We conclude that only long-term culture conditions (>4 days) allow the cells to recover from plating stress and to re-acquire characteristic granulosa cell features.