Post-transcriptional Gene Regulation Research Articles

MicroRNA-34 Family in Cancers: Role, Mechanism, and Therapeutic Potential.

MicroRNA (miRNA) are small noncoding RNAs that play vital roles in post-transcriptional gene regulation by inhibiting mRNA translation or promoting mRNA degradation. The dysregulation of miRNA has been implicated in numerous human diseases, including cancers. miR-34 family members (miR-34s), including miR-34a, miR-34b, and miR-34c, have emerged as the most extensively studied tumor-suppressive miRNAs. In this comprehensive review, we aim to provide an overview of the major signaling pathways and gene networks regulated by miR-34s in various cancers and highlight the critical tumor suppressor role of miR-34s. Furthermore, we will discuss the potential of using miR-34 mimics as a novel therapeutic approach against cancer, while also addressing the challenges associated with their development and delivery. It is anticipated that gaining a deeper understanding of the functions and mechanisms of miR-34s in cancer will greatly contribute to the development of effective miR-34-based cancer therapeutics.

Post-transcriptional gene silencing in a dynamic RNP world.

MicroRNA (miRNA)-guided gene silencing is a key regulatory process in various organisms and linked to many human diseases. MiRNAs are processed from precursor molecules and associate with Argonaute proteins to repress the expression of complementary target mRNAs. Excellent work by numerous labs has contributed to a detailed understanding of the mechanisms of miRNA function. However, miRNA effects have mostly been analyzed and viewed as isolated events and their natural environment as part of complex RNA-protein particles (RNPs) is often neglected. RNA binding proteins (RBPs) regulate key enzymes of the miRNA processing machinery and furthermore RBPs or readers of RNA modifications may modulate miRNA activity on mRNAs. Such proteins may function similarly to miRNAs and add their own contributions to the overall expression level of a particular gene. Therefore, post-transcriptional gene regulation might be more the sum of individual regulatory events and should be viewed as part of a dynamic and complex RNP world.

Exploring the pan-cancer landscape of posttranscriptional regulation

Understanding the mechanisms underlying cancer gene expression is critical for precision oncology. Posttranscriptional regulation is a key determinant of protein abundance and cancer cell behavior. However, to what extent posttranscriptional regulatory mechanisms impact protein levels and cancer progression is an ongoing question. Here, we exploit cancer proteogenomics data to systematically compare mRNA-protein correlations across 14 different human cancer types. We identify two clusters of genes with particularly low mRNA-protein correlations across all cancer types, shed light on the role of posttranscriptional regulation of cancer driver genes and drug targets, and unveil a cohort of 55 mutations that alter systems-wide posttranscriptional regulation. Surprisingly, we find that decreased levels of posttranscriptional control in patients correlate with shorter overall survival across multiple cancer types, prompting further mechanistic studies into how posttranscriptional regulation affects patient outcomes. Our findings underscore the importance of a comprehensive understanding of the posttranscriptional regulatory landscape for predicting cancer progression.

The m6A reader IGF2BP1 manipulates BUB1B expression to affect malignant behaviors, stem cell properties, and immune resistance of non-small-cell lung cancer stem cells.

N6-methyladenosine (m6A) modification is the most common internal modification in eukaryotic mRNA and an important mechanism for post-transcriptional regulation of genes. This study focuses on the role of the m6A reader insulin-like growth factor 2 mRNA binding protein 1 (IGF2BP1) in the malignant behaviors of non-small-cell lung cancer (NSCLC) cells and especially the cancer stem cells (CSCs). We obtained IGF2BP1 as an aberrantly upregulated gene linking to poor survival of patients with NSCLC by bioinformatics, and then confirmed increased IGF2BP1 expression in NSCLC tissues and cells, especially in the enriched CSCs. Knockdown of IGF2BP1 suppressed proliferation, mobility and epithelial-mesenchymal transition activity of NSCLC cells and CSCs, and it reduced stemness, self-renewal ability, xenograft tumorigenesis and immune resistance of the CSCs. IGF2BP1 was predicted to have a positive correlation with BUB1 mitotic checkpoint serine/threonine kinase B (BUB1B), and it upregulated BUB1B expression through m6A modification. Further overexpression of BUB1B in CSCs counteracted the effects of IGF2BP1 silencing and restored the malignant phenotype, self-renewal, and immune resistance of CSCs in vitro and in vivo. Taken together, this work demonstrates that IGF2BP1 manipulates BUB1B expression to affect malignant behaviors, stem cell properties and immune resistance of NSCLC stem cells.

The molecular genetics of nELAVL in brain development and disease.

Embryonic development requires tight control of gene expression levels, activity, and localisation. This control is coordinated by multiple levels of regulation on DNA, RNA and protein. RNA-binding proteins (RBPs) are recognised as key regulators of post-transcriptional gene regulation, where their binding controls splicing, polyadenylation, nuclear export, mRNA stability, translation rate and decay. In brain development, the ELAVL family of RNA binding proteins undertake essential functions across spatiotemporal windows to help regulate and specify transcriptomic programmes for cell specialisation. Despite their recognised importance in neural tissues, their molecular roles and connections to pathology are less explored. Here we provide an overview of the neuronal ELAVL family, noting commonalities and differences amongst different species, their molecular characteristics, and roles in the cell. We bring together the available molecular genetics evidence to link different ELAVL proteins to phenotypes and disease, in both the brain and beyond, including ELAVL2, which is the least studied ELAVL family member. We find that ELAVL-related pathology shares a common neurological theme, but different ELAVL proteins are more strongly connected to different phenotypes, reflecting their specialised expression across time and space.

Identification of ARF transcription factor gene family and its defense responses to bacterial infection and salicylic acid treatment in sugarcane.

Auxin response factor (ARF) is a critical regulator in the auxin signaling pathway, involved in a variety of plant biological processes. Here, gene members of 24 SpapARFs and 39 SpnpARFs were identified in two genomes of Saccharum spontaneum clones AP85-441 and Np-X, respectively. Phylogenetic analysis showed that all ARF genes were clustered into four clades, which is identical to those ARF genes in maize (Zea mays) and sorghum (Sorghum bicolor). The gene structure and domain composition of this ARF family are conserved to a large degree across plant species. The SpapARF and SpnpARF genes were unevenly distributed on chromosomes 1-8 and 1-10 in the two genomes of AP85-441 and Np-X, respectively. Segmental duplication events may also contribute to this gene family expansion in S. spontaneum. The post-transcriptional regulation of ARF genes likely involves sugarcane against various stressors through a miRNA-medicated pathway. Expression levels of six representative ShARF genes were analyzed by qRT-PCR assays on two sugarcane cultivars [LCP85-384 (resistant to leaf scald) and ROC20 (susceptible to leaf scald)] triggered by Acidovorax avenae subsp. avenae (Aaa) and Xanthomonas albilineans (Xa) infections and salicylic acid (SA) treatment. ShARF04 functioned as a positive regulator under Xa and Aaa stress, whereas it was a negative regulator under SA treatment. ShARF07/17 genes played positive roles against both pathogenic bacteria and SA stresses. Additionally, ShARF22 was negatively modulated by Xa and Aaa stimuli in both cultivars, particularly LCP85-384. These findings imply that sugarcane ARFs exhibit functional redundancy and divergence against stressful conditions. This work lays the foundation for further research on ARF gene functions in sugarcane against diverse environmental stressors.

Homeobox-D 1 and FTO form a transcriptional-epigenetic feedback loop to promote head and neck cancer proliferation.

Transcription factors (TFs) and N6-methyladenosine (m6A) modifiers are involved in tumor progression through transcriptional regulation and posttranscriptional regulation of genes, respectively. However, the crosstalk and role of these two types of gene expression regulators in head and neck squamous cell carcinoma (HNSC) remains poorly understood. In this study, we demonstrate that the TF homeobox-D1 (HOXD1) and the m6A demethylase fat mass and obesity-associated protein (FTO) form a positive feedback loop to promote cell proliferation and survival in HNSC. Clinically, HOXD1 expression is dysregulated in multiple cancer types and is associated with worse prognosis in patients with HNSC, stomach adenocarcinoma, uterine corpus endometrial carcinoma, and pheochromocytoma and paraganglioma. Mechanistically, FTO is overexpressed in HNSC tumor samples and positively regulates HOXD1 expression in an m6A-dependent manner. Functionally, deficiency of HOXD1 relieved the resistance of HNSC cells to apoptosis and arrested tumor cells at the G0/G1 phase, thereby inhibiting cell growth, whereas overexpression of HOXD1 caused the opposite effect. Furthermore, HOXD1 activates the transcription of the oncogenic factor FTO by directly targeting its promoter. Downregulation of FTO mimicked the biological effect of HOXD1 knockdown on HNSC. Importantly, overexpression of HOXD1 significantly rescued the proliferation inhibition and apoptosis promotion of HNSC cells induced by deficiency of FTO. Together, our findings reveal HOXD1 as a novel prognostic predictor and a potential target for HNSC, providing mechanistic insights into the role of the HOXD1-FTO circuit in this cancer.

Keratin 19 binds and regulates cytoplasmic HNRNPK mRNA targets in triple-negative breast cancer.

Heterogeneous nuclear ribonucleoprotein K (HNRNPK) regulates pre-mRNA processing and long non-coding RNA localization in the nucleus. It was previously shown that shuttling of HNRNPK to the cytoplasm promotes cell proliferation and cancer metastasis. However, the mechanism of HNRNPK cytoplasmic localization, its cytoplasmic RNA ligands, and impact on post-transcriptional gene regulation remain uncharacterized. Here we show that the intermediate filament protein Keratin 19 (K19) directly interacts with HNRNPK and sequesters it in the cytoplasm. Correspondingly, in K19 knockout breast cancer cells, HNRNPK does not localize in the cytoplasm, resulting in reduced cell proliferation. We comprehensively mapped HNRNPK binding sites on mRNAs and showed that, in the cytoplasm, K19-mediated HNRNPK-retention increases the abundance of target mRNAs bound to the 3' untranslated region (3'UTR) at the expected cytidine-rich (C-rich) sequence elements. Furthermore, these mRNAs protected by HNRNPK in the cytoplasm are typically involved in cancer progression and include the p53 signaling pathway that is dysregulated upon HNRNPK knockdown (HNRNPK KD) or K19 knockout (KRT19 KO). This study identifies how a cytoskeletal protein can directly regulate gene expression by controlling the subcellular localization of RNA-binding proteins to support pathways involved in cancer progression.

Abstract P3052: RNA Binding Protein Csdc2 Is Required For Metabolic And Cardiac Stress Responses

Post transcriptional gene regulation (PTGR) mediated by networks of microRNAs and RNA binding proteins (RBP's) controls mRNA processing, localization, and expression. Hundreds of microRNAs and RBPs are expressed in heart, and much remains unknown regarding PTGR’s influence on cardiovascular health and disease. Through bioinformatic analyses, we identified Csdc2 as a cardiac-enriched RBP that is consistently downregulated in failing hearts. Csdc2’s paralog, Carshp1, controls metabolic gene expression in liver through PPARα. We hypothesize that Csdc2 plays key roles in cardio-metabolic stress. To address this, we first assessed mRNA-seq changes in neonatal rat cardiomyocytes after overexpression and knockdown of Csdc2. Pathway analyses revealed reciprocal dysregulation of fatty acid metabolism, ion channels, and cytoskeletal and sarcomere genes. We generated constitutive Csdc2-KO mice, which are viable with no overt baseline phenotypes. Cardiac phenotyping revealed that Csdc2-KO mice have slightly decreased PR interval (~10%, p=1.3e-4, n=16-22) but maintain normal cardiac structure/function. To assess Csdc2’s potential role in metabolic stress, mice were fed a high-fat-diet for 10 weeks and monitored for lean/fat mass and insulin/glucose tolerances. Csdc2-KO mice gained 20% more weight than wildtype littermates (p=0.04, n=13-18) and developed insulin resistance (p<0.01). To test if Csdc2 loss influences cardiac stress, normal chow cohorts were subjected to microsurgery-induced myocardial infarction (MI); Csdc2-KO mice showed ~2-fold increase in mortality within two weeks (70% versus 40% for wildtype). Echocardiographic measures support that Csdc2-KO hearts have larger infarcts, dilation without compensatory remodeling and worse systolic function. Together, these data highlight Csdc2 as a disease-relevant RBP that controls cardio-metabolic stress responses.

Abstract P3051: Master Splicing Regulator Rbpms2 Is Not Required For Murine Cardiac Development Or Contractile Function

Post-transcriptional gene regulation (PTGR) through RNA-binding proteins (RBP) is an understudied area of cardiac biology. Emerging research describes important roles for Rbpms and Rbpms2 in RNA splicing, cardiomyocyte diploidy and proliferation, cytoskeletal reorganization and translation initiation during cardiomyocyte differentiation and cardiac development. However, the role of Rbpms2 in adult heart is largely unknown. We identified Rbpms2 as a cardiovascular-enriched RBP that is consistently downregulated in failing hearts from humans and mice. Though prior research has focused on roles in splicing, a large portion Rbpms2 protein is extranuclear, and >35% of empirically determined Rbpms binding sites localize in 3’UTRs, suggesting alternative PTGR roles. To assess Rbpms2’s role in cardiomyocyte PTGR, we assessed mRNA-seq changes in neonatal rat cardiomyocytes after overexpression and knockdown of Rbpms2. Pathway analyses revealed significant upregulation of genes involved in cell cycle control, and downregulation of genes involved in viral host defense pathway after Rbpms2 knockdown. We next characterized Rbpms2 knockout (KO) mice, which are viable without overt phenotypes, to provide initial descriptions of adult mouse cardiac function after Rbpms2 deletion. Baseline echocardiography data of male mice at 6 months of age shows normal cardiac size, structure, and function without significant difference between KO and wildtype (WT) mice (n=10). To assess the role of Rbpms2 in cardiac hypertrophy, we induced pressure overload via transverse aortic constriction (TAC) on mix-sex WT and Rbpms2-KO mice, which resulted in robust hypertrophic responses and reduced ejection fraction in both groups, but no significant differences between WT and KO mice (n=11 female, n=7-10 male). Overall, our findings are surprising given recently published data showing that Rbpms2 is a master regulator of RNA splicing affecting sarcomere organization and cardiomyocyte contractile function in zebrafish and stem cell-derived cardiomyocytes, supporting the need for follow-up studies on cardiac Rbpms2. It is possible that Rbpms compensates for the loss of Rbpms2 in the mouse heart and would require double KOs to assess synergistic phenotypes.

Abstract P2103: Cardiac Fibroblast-specific Role Of PTBP1 In Maintaining Heart Function

Cardiac fibroblasts (CFs) are the primary nonmyocyte in the heart that maintain cardiac homeostasis by regulating myocardial function and cardiac remodeling. CFs secrete an extracellular matrix to stabilize the myocardial wall while maintaining the heart’s mechanical integrity during development and pathogenesis. Developmental and pathogenic transitions are directed through CFs by transcriptional and post-transcriptional modifications, such as alternative splicing. Alternative splicing is a central element in post-transcriptional gene regulation and proteomic diversity. Impaired and aberrant splicing is known to cause developmental defects and drive cardiac pathogenesis. RNA binding proteins (RBPs), which function as alternative splicing factors, are upregulated in cardiovascular disease states. While nearly a third of all CF gene regulation changes during cardiac fibrosis are subject to transcriptional regulation via RBPs, the role of CF specific splicing RBPs in the adult heart and during cardiac fibrosis is unknown. Our group previously identified high expression of well-known RBP and splicing factor polypyrimidine tract-binding protein (PTBP1) in CFs when compared to cardiomyocytes in the adult heart. With single cell transcriptomics we found PTBP1 regulates CF identity and plasticity, suggesting an emergent role of PTBP1 within CF biology. We hypothesize PTBP1 is essential for maintaining cardiac fibroblast function in the adult heart. Preliminary in vitro data suggests partial Ptbp1 knockdown impacts mechanical function by decreasing overall CF migration. To further characterize PTBP1 in vivo , we have validated our novel mouse model to specifically delete PTBP1 from CFs in adult mice. Following PTBP1 deletion, we will utilize an established model of fibrosis by Angiotensin II osmotic minipumps to characterize the CF specific PTBP1 loss during fibrosis. We will evaluate CF biology with our in vitro and in vivo models to examine PTBP1’s physiological and mechanistic role in maintaining CF biology during cardiac homeostasis and cardiac fibrosis pathogenesis.

Abstract P3041: Stage-specific Alternative Rna Splicing Attunes Cardiomyocyte Proliferation And Contraction

During heart development, cardiomyocytes display reduced proliferation and enhanced contractility to support cardiac functional maturation, but the mechanistic basis is not fully understood. Alternative RNA splicing is an important mechanism of RNA posttranscriptional modification and gene regulation in the heart. In studying a cardiac RNA binding protein, RBPMS (RNA-binding protein with multiple splicing), we found that RBPMS mediates distinct RNA splicing profiles in adult hearts versus neonatal hearts, indicating its stage-specific modulation of alternative RNA splicing. We showed that RBPMS regulates the splicing of cytoskeletal genes to enable proliferation of neonatal cardiomyocytes. In adult hearts, RBPMS regulates the splicing of sarcomere genes to support cardiac contraction. Cardiac-specific loss of RBPMS causes severe dilated cardiomyopathy and early lethality in adult mice. Using both in vivo and in vitro approaches, we found that the absence of RBPMS caused aberrant splicing of some key sarcomeric components, including Titin and Nexilin, causing cardiomyocyte contractile defects. Intriguingly, we also showed that RBPMS interacts with other cardiac RNA binding proteins, such as RBM20, suggesting their synergistic effects on cardiomyocyte physiology. Our work provides novel insights into how RNA binding proteins regulate cardiomyocyte proliferation and contraction by mediating alternative RNA splicing.

Extracellular vesicles derived from Spirometra erinaceieuropaei plerocercoids inhibit activation of murine macrophage RAW264.7 cells.

Parasitic helminths modify host immune reactions to promote long-term parasitism. We previously purified a glycoprotein, plerocercoid-immunosuppressive factor (P-ISF), from the excretory/secretory products of Spirometra erinaceieuropaei plerocercoids and reported its cDNA and genomic DNA sequences. In this study, we isolated extracellular vesicles (EVs) from the excretory/secretory products of S. erinaceieuropaei plerocercoids and found that they suppressed the production of nitric oxide and the gene expression of tumor necrosis factor-α, interleukin-1β, and interleukin-6 in lipopolysaccharide-stimulated macrophages. EVs are membrane-bound vesicles 50-250nm in diameter and are localized in the whole bodies of plerocercoids. EVs from plerocercoids encapsulate a variety of unidentified proteins and microRNAs (miRNAs), which are non-coding RNAs that play essential roles in post-transcriptional gene regulation. The miRNAs of the EVs were analyzed, and 334,137 sequencing reads were mapped to the genomes of other organisms. A total of 26 different miRNA families were identified, including miR-71, miR-10-5p, miR-223, and let-7-5p, which have been reported to have immunosuppressive effects. We confirmed that P-ISF was present in the supernatant but not in the EVs by western blotting with an anti-P-ISF antibody. These results suggest that S. erinaceieuropaei plerocercoids suppress host immunity by releasing P-ISF and EVs.

Long-Term LDL-Apheresis Treatment and Dynamics of Circulating miRNAs in Patients with Severe Familial Hypercholesterolemia.

Lipoprotein apheresis (LA) is a therapeutic option for patients with severe hypercholesterolemia who have persistently elevated LDL-C levels despite attempts at drug therapy. MicroRNAs (miRNAs), important posttranscriptional gene regulators, are involved in the pathogenesis of atherosclerosis. Our study aimed to monitor the dynamics of twenty preselected circulating miRNAs in patients under long-term apheresis treatment. Plasma samples from 12 FH patients (men = 50%, age = 55.3 ± 12.2 years; mean LA overall treatment time = 13.1 ± 7.8 years) were collected before each apheresis therapy every sixth month over the course of four years of treatment. Eight complete follow-up (FU) samples were measured in each patient. Dynamic changes in the relative quantity of 6 miRNAs (miR-92a, miR-21, miR-126, miR-122, miR-26a, and miR-185; all p < 0.04) during FU were identified. Overall apheresis treatment time influenced circulating miR-146a levels (p < 0.04). In LDLR mutation homozygotes (N = 5), compared to heterozygotes (N = 7), we found higher plasma levels of miR-181, miR-126, miR-155, and miR-92a (all p < 0.03). Treatment with PCSK9 inhibitors (N = 6) affected the plasma levels of 7 miRNAs (miR-126, miR-122, miR-26a, miR-155, miR-125a, miR-92a, and miR-27a; all p < 0.04). Long-term monitoring has shown that LA in patients with severe familial hypercholesterolemia influences plasma circulating miRNAs involved in endothelial dysfunction, cholesterol homeostasis, inflammation, and plaque development. The longer the treatment using LA, the better the miRNA milieu depicting the potential cardiovascular risk.

In Silico and Experimental Outcomes of the Expression of miR508-5p and miR-635 in Tumor Tissues of Patients with Breast Cancer

Background: Breast cancer (BC) is a malignant tumor that occurs in the epithelial tissue of the breast gland and has become the most common malignancy in women. Various studies have reported the effect of epigenetic changes, including DNA methylation and microRNAs, on breast carcinogenesis. microRNAs play an important role in the post-transcriptional regulation of genes and are important regulators of oncogenic pathways. Studying microRNAs in BC facilitates the development of targeted therapies and early detection of this cancer. Objectives: This study aimed to evaluate the expression level of miR-508-5p and miR-635 in BC tumor tissues compared to healthy marginal tissues. Methods: In silico analysis confirmed microarray datasets (GSE40525, GSE44124 and GSE45666) downloaded from the GEO database. The analysis was defined using the Affy packages in R software to screen remarkably dysregulated miRNAs attended by utilized to predict the potential biological processes and molecular pathways of miR-508-5p and miR-635. Experimental statistical significance of differences in miRNA relative expression results was analyzed by pair-wise fixed reallocation randomization test as a statistical model included in the REST (relative expression software tool). Results: GEO microarray data set, similar to qPCR results, showed that miR-508-5p was downregulated in the sample group by a mean factor of 0.327 (S.E.M range is 0.031-2.000). Moreover, miR-635 was upregulated in the sample group by a mean factor of 2.361 (S.E.M range is 0.250-16.000). Conclusion: miR-508-5p was downregulated, while miR-635 was upregulated in BC tissues. They may be proposed as diagnostic and therapeutic biomarkers for patients with BC.

Mapping microRNA expression quantitative trait loci in the prenatal human brain implicates miR-1908-5p expression in bipolar disorder and other brain-related traits.

MicroRNA (miRNA) are small non-coding RNA involved in post-transcriptional gene regulation. Given their known involvement in early neurodevelopment processes, we here sought to identify common genetic variants associated with altered miRNA expression in the prenatal human brain. We performed small RNA sequencing on brain tissue from 112 genome-wide genotyped fetuses from the second trimester of gestation, identifying high-confidence (false discovery rate < 0.05) expression quantitative trait loci for 30 mature miRNA. Integrating our findings with genome-wide association study data for brain-related disorders, we implicate increased prenatal expression of miR-1908-5p as a risk mechanism for bipolar disorder and find that predicted mRNA targets of miR-1908-5p that are expressed in the fetal brain are enriched for common variant genetic association with the condition. Extending these analyses to other brain-related traits, we find that common genetic variation associated with increased miR-1908-5p expression in fetal brain is additionally associated with depressive symptoms, irritability, increased right cerebellum exterior volume and increased sleep duration in the general population. Our findings provide support to the view that altered miRNA expression can influence susceptibility to neuropsychiatric illness and suggest an early neurodevelopmental risk mechanism for bipolar disorder.

Platelet miRNAs: differential expression in coronary artery disease and associations with course of left ventricular systolic function

BackgroundMicroRNAs are paramount in post transcriptional gene regulation. We investigated platelet miRNAs in patients with CAD and examined potential associations with course of left ventricular ejection fraction (LVEF%).Materials and methodsIn a first cohort, 62 MiRNAs were measured in platelets of 100 patients suffering from CAD. Expression profiles of individuals with chronic coronary syndrome (CCS) and MI were compared (CCS n = 67, MI n = 33). Also, associations between miRNA profiles and change in left ventricular ejection fraction (LVEF%) were investigated. In a second cohort of patients suffering from CCS (n = 10), MI (n = 11) or no CAD (n = 13), we measured miRNA expression in platelets, platelet supernatant and serum. This was carried out before and after in vitro platelet activation with CRP.ResultsPlatelet miRNAs 103a-3p and 155-5p demonstrated higher expression in patients with CCS then in individuals with MI. Furthermore, multiple miRNAs were significantly higher expressed in matched controls compared to MI patients. 8 miRNAs showed higher expression in patients with improving LVEF% after a 1-year follow-up. In our second cohort, we found higher concentrations of 6 miRNAs in the platelet supernatant of patients with CCS, MI and no CAD after in vitro platelet activation. Most of these miRNAs showed a higher abundance in serum of MI patients as compared to CCS.ConclusionSeveral miRNAs show higher expression in platelets of CCS compared to MI. After in vitro platelet activation, a release of multiple miRNAs out of the thrombocyte was observed. Furthermore, upregulation of serum miRNAs was found in MI patients when compared to CCS patients and individuals without CAD. Hence, platelets could present a source of upregulated circulating miRNAs in MI and additionally affect course of LVEF%.

Mapping genetic variants for nonsense-mediated mRNA decay regulation across human tissues

BackgroundNonsense-mediated mRNA decay (NMD) was originally conceived as an mRNA surveillance mechanism to prevent the production of potentially deleterious truncated proteins. Research also shows NMD is an important post-transcriptional gene regulation mechanism selectively targeting many non-aberrant mRNAs. However, how natural genetic variants affect NMD and modulate gene expression remains elusive.ResultsHere we elucidate NMD regulation of individual genes across human tissues through genetical genomics. Genetic variants corresponding to NMD regulation are identified based on GTEx data through unique and robust transcript expression modeling. We identify genetic variants that influence the percentage of NMD-targeted transcripts (pNMD-QTLs), as well as genetic variants regulating the decay efficiency of NMD-targeted transcripts (dNMD-QTLs). Many such variants are missed in traditional expression quantitative trait locus (eQTL) mapping. NMD-QTLs show strong tissue specificity especially in the brain. They are more likely to overlap with disease single-nucleotide polymorphisms (SNPs). Compared to eQTLs, NMD-QTLs are more likely to be located within gene bodies and exons, especially the penultimate exons from the 3′ end. Furthermore, NMD-QTLs are more likely to be found in the binding sites of miRNAs and RNA binding proteins.ConclusionsWe reveal the genome-wide landscape of genetic variants associated with NMD regulation across human tissues. Our analysis results indicate important roles of NMD in the brain. The preferential genomic positions of NMD-QTLs suggest key attributes for NMD regulation. Furthermore, the overlap with disease-associated SNPs and post-transcriptional regulatory elements implicates regulatory roles of NMD-QTLs in disease manifestation and their interactions with other post-transcriptional regulators.

Expression of targets of the RNA-binding protein AUF-1 in human airway epithelium indicates its role in cellular senescence and inflammation.

The RNA-binding protein AU-rich-element factor-1 (AUF-1) participates to posttranscriptional regulation of genes involved in inflammation and cellular senescence, two pathogenic mechanisms of chronic obstructive pulmonary disease (COPD). Decreased AUF-1 expression was described in bronchiolar epithelium of COPD patients versus controls and in vitro cytokine- and cigarette smoke-challenged human airway epithelial cells, prompting the identification of epithelial AUF-1-targeted transcripts and function, and investigation on the mechanism of its loss. RNA immunoprecipitation-sequencing (RIP-Seq) identified, in the human airway epithelial cell line BEAS-2B, 494 AUF-1-bound mRNAs enriched in their 3'-untranslated regions for a Guanine-Cytosine (GC)-rich binding motif. AUF-1 association with selected transcripts and with a synthetic GC-rich motif were validated by biotin pulldown. AUF-1-targets' steady-state levels were equally affected by partial or near-total AUF-1 loss induced by cytomix (TNFα/IL1β/IFNγ/10 nM each) and siRNA, respectively, with differential transcript decay rates. Cytomix-mediated decrease in AUF-1 levels in BEAS-2B and primary human small-airways epithelium (HSAEC) was replicated by treatment with the senescence- inducer compound etoposide and associated with readouts of cell-cycle arrest, increase in lysosomal damage and senescence-associated secretory phenotype (SASP) factors, and with AUF-1 transfer in extracellular vesicles, detected by transmission electron microscopy and immunoblotting. Extensive in-silico and genome ontology analysis found, consistent with AUF-1 functions, enriched RIP-Seq-derived AUF-1-targets in COPD-related pathways involved in inflammation, senescence, gene regulation and also in the public SASP proteome atlas; AUF-1 target signature was also significantly represented in multiple transcriptomic COPD databases generated from primary HSAEC, from lung tissue and from single-cell RNA-sequencing, displaying a predominant downregulation of expression. Loss of intracellular AUF-1 may alter posttranscriptional regulation of targets particularly relevant for protection of genomic integrity and gene regulation, thus concurring to airway epithelial inflammatory responses related to oxidative stress and accelerated aging. Exosomal-associated AUF-1 may in turn preserve bound RNA targets and sustain their function, participating to spreading of inflammation and senescence to neighbouring cells.

PEPseq quantifies transcriptome-wide changes in protein occupancy and reveals selective translational repression after translational stress.

Post-transcriptional gene regulation is accomplished by the interplay of the transcriptome with RNA-binding proteins, which occurs in a dynamic manner in response to altered cellular conditions. Recording the combined occupancy of all proteins binding to the transcriptome offers the opportunity to interrogate if a particular treatment leads to any interaction changes, pointing to sites in RNA that undergo post-transcriptional regulation. Here, we establish a method to monitor protein occupancy in a transcriptome-wide fashion by RNA sequencing. To this end, peptide-enhanced pull-down for RNA sequencing (or PEPseq) uses metabolic RNA labelling with 4-thiouridine (4SU) for light-induced protein-RNA crosslinking, and N-hydroxysuccinimide (NHS) chemistry to isolate protein-crosslinked RNA fragments across all long RNA biotypes. We use PEPseq to investigate changes in protein occupancy during the onset of arsenite-induced translational stress in human cells and reveal an increase of protein interactions in the coding region of a distinct set of mRNAs, including mRNAs coding for the majority of cytosolic ribosomal proteins. We use quantitative proteomics to demonstrate that translation of these mRNAs remains repressed during the initial hours of recovery after arsenite stress. Thus, we present PEPseq as a discovery platform for the unbiased investigation of post-transcriptional regulation.