Expression patterns of m6A RNA methylation regulators under apoptotic conditions in various human cancer cell lines.

Background/aim: Cancer is a complex disease that involves both genetic and epigenetic factors. While emerging evidence clearly suggests that changes in epitranscriptomics play a crucial role in cancer pathogenesis, a comprehensive understanding of the writers, erasers, and readers of epitranscriptomic processes, particularly under apoptotic conditions remains lacking. The aim of this study was to uncover the changes in the expression of m6A RNA modifiers under apoptotic conditions across various cancer cell lines. Materials and methods: Initially, we quantified the abundance of m6A RNA modifiers in cervical (HeLa and ME180), breast (MCF7 and MDA-MB-231), lung (A549 and H1299), and colon (Caco-2 and HCT116) cancer cell lines using qPCR. Subsequently, we induced apoptosis using cisplatin and tumor necrosis factor-alpha (TNF-α) to activate intrinsic and extrinsic pathways, respectively, and assessed apoptosis rates via flow cytometry. Further, we examined the transcript abundance of m6A RNA modifiers under apoptotic conditions in cervical, breast, and lung cancer cell lines using qPCR. Results: Overall, treatment with cisplatin increased the abundance of m6A modifiers, whereas TNF-α treatment decreased their expression in cervical, breast, and lung cancer cell lines. Specifically, cisplatin-induced apoptosis, but not TNF-α-mediated apoptosis, resulted in decreased abundance of METTL14 and FTO transcripts. Additionally, cisplatin treatment drastically reduced the abundance of IGF2BP2 and IGF2BP3 readers. Conclusion: These results suggest that the differential response of cancer cells to apoptotic inducers may be partially attributed to the expression of m6A RNA modifiers.

An Overview of Current Detection Methods for RNA Methylation.

Epitranscriptomic mechanisms, which constitute an important layer in post-transcriptional gene regulation, are involved in numerous cellular processes under health and disease such as stem cell development or cancer. Among various such mechanisms, RNA methylation is considered to have vital roles in eukaryotes primarily due to its dynamic and reversible nature. There are numerous RNA methylations that include, but are not limited to, 2'-O-dimethyladenosine (m6Am), N7-methylguanosine (m7G), N6-methyladenosine (m6A) and N1-methyladenosine (m1A). These biochemical modifications modulate the fate of RNA by affecting the processes such as translation, target site determination, RNA processing, polyadenylation, splicing, structure, editing and stability. Thus, it is highly important to quantitatively measure the changes in RNA methylation marks to gain insight into cellular processes under health and disease. Although there are complicating challenges in identifying certain methylation marks genome wide, various methods have been developed recently to facilitate the quantitative measurement of methylated RNAs. To this end, the detection methods for RNA methylation can be classified in five categories such as antibody-based, digestion-based, ligation-based, hybridization-based or direct RNA-based methods. In this review, we have aimed to summarize our current understanding of the detection methods for RNA methylation, highlighting their advantages and disadvantages, along with the current challenges in the field.

Epitranscriptomics m6A analyses reveal distinct m6A marks under tumor necrosis factor α (TNF-α)-induced apoptotic conditions in HeLa cells.

Tumor necrosis factor-α (TNF-α) is a ligand that induces both intrinsic and extrinsic apoptotic pathways in HeLa cells by modulating complex gene regulatory mechanisms. However, the full spectrum of TNF-α-modulated epitranscriptomic m6A marks is unknown. We employed a genomewide approach to examine the extent of m6A RNA modifications under TNF-α-modulated apoptotic conditions in HeLa cells. miCLIP-seq analyses revealed a plethora of m6A marks on 632 target mRNAs with an enrichment on 99 mRNAs associated with apoptosis. Interestingly, the m6A RNA modification patterns were quite different under cisplatin- and TNF-α-mediated apoptotic conditions. We then examined the abundance and translational efficiencies of several mRNAs under METTL3 knockdown and/or TNF-α treatment conditions. Our analyses showed changes in the translational efficiency of TP53INP1 mRNA based on the polysome profile analyses. Additionally, TP53INP1 protein amount was modulated by METTL3 knockdown upon TNF-α treatment but not CP treatment, suggesting the existence of a pathway-specific METTL3-TP53INP1 axis. Congruently, METLL3 knockdown sensitized HeLa cells to TNF-α-mediated apoptosis, which was also validated in a zebrafish larval xenograft model. These results suggest that apoptotic pathway-specific m6A methylation marks exist in cells and TNF-α-METTL3-TP53INP1 axis modulates TNF-α-mediated apoptosis in HeLa cells.

Noncoding RNAs: A New Layer of Functional RNAs.

The conventional central dogma of molecular biology dictates that the genetic information contained within deoxyribonucleic acid (DNA) is passed onto messenger ribonucleic acids (mRNAs), which are then used as templates to synthesize proteins. Although these types of proteincoding genes have been historically prioritized in typical phenotype-genotype studies with a parallel disregard to the rest of the genome, the completion of genome projects has unveiled a surprising layer of genetic information that can play critical roles in cellular processes without coding for proteins. These types of genes are called noncoding genes as they do not code for proteins. Noncoding genes come in different sizes and shapes, and they are just as versatile in carrying out cellular biochemical processes as proteins. In this review, we cover a comprehensive review of housekeeping and regulatory noncoding genes and their mode of action.

Long Noncoding RNAs in Human Cancer and Apoptosis.

Genome annotations have uncovered the production of at least one transcript from nearly all loci in the genome at some given time throughout the development. Surprisingly, many of these transcripts do not code for proteins and are relatively long in size, thus called long noncoding RNAs (lncRNAs). Next- and third-generation sequencing technologies have amassed numerous lncRNAs expressed under different phenotypic conditions, yet many remain to be functionally characterized. LncRNAs regulate gene expression by functioning as scaffold, decoy, signaling, and guide molecules both at the transcriptional and post-transcriptional levels, interacting with different types of macromolecules, such as proteins, DNA, and RNA. Here, we review the potential regulatory role of lncRNAs in apoptosis and cancer as some of these lncRNAs may have the diagnostic and therapeutic potential in cancer.

A spectral approach on mineralogy and geochemistry of garnet skarns in Arc-Type granitoids.

Garnets that common constituent of skarn type iron deposits are wide ranges of chemical compositions, and they are also important as a semi-gemstone mineral. This study has been investigated garnets and inclusions of its formed in contacts of Pertek granitoid and Keban marble by using a combination of multiple techniques including Raman spectrum, electron microprobe, petrography and LA-ICP-MS. The main mineral assemblage observed in the skarn formation is diopside, garnet, quartz, magnetite, calcite and pyrite. The garnets are in size between 1 and 7 cm and have reddish, greenish and light-dark brown colour. The compositions of the garnets are mainly grossular-andradite, andradite-grossular, less grossular and andradite. Increased porosity and horizontal flow of hydrothermal fluids during metasomatism resulted in the formation of garnets of different sizes. Raman spectroscopy studies on garnets show to be that the idiomorphic and semi-idiomorphic garnets are predominantly grosular, andradite and uvarovite in the core. Raman spectroscopy studies on garnets showed to have predominantly grossular, andradite and uvarovite composition in the core of the idiomorphic and semi-idiomorphic garnets. As for the rare earth element (REE) analysis and distribution model, the chondrite-normalized REE patterns of all garnets in the study area have similar trends and generally in the right-slopping shape depleted in HREE and enriched in LREE, mostly controlled by adsorption. High Fe2O3, CaO and MgO contents may cause to high mobility during skarn formation associated with contact metamorphism and cation exchange via hydrothermal fluids (eg uvarovite in the core). All garnets have generally positive Ce and positive Eu anomalies. These results suggest that the garnets in the studya area formed in conditions showing increase in pH and high oxygen fugacity or decrease in temperature in the source of the hydrothermal fluids.

Genomewide m6A Mapping Uncovers Dynamic Changes in the m6A Epitranscriptome of Cisplatin-Treated Apoptotic HeLa Cells.

Cisplatin (CP), which is a conventional cancer chemotherapeutic drug, induces apoptosis by modulating a diverse array of gene regulatory mechanisms. However, cisplatin-mediated changes in the m6A methylome are unknown. We employed an m6A miCLIP-seq approach to investigate the effect of m6A methylation marks under cisplatin-mediated apoptotic conditions on HeLa cells. Our high-resolution approach revealed numerous m6A marks on 972 target mRNAs with an enrichment on 132 apoptotic mRNAs. We tracked the fate of differentially methylated candidate mRNAs under METTL3 knockdown and cisplatin treatment conditions. Polysome profile analyses revealed perturbations in the translational efficiency of PMAIP1 and PHLDA1 transcripts. Congruently, PMAIP1 amounts were dependent on METTL3. Additionally, cisplatin-mediated apoptosis was sensitized by METTL3 knockdown. These results suggest that apoptotic pathways are modulated by m6A methylation events and that the METTL3-PMAIP1 axis modulates cisplatin-mediated apoptosis in HeLa cells.

Development of AB3-Type Novel Phthalocyanine and Porphyrin Photosensitizers Conjugated with Triphenylphosphonium for Higher Photodynamic Efficacy.

There are a number of lipophilic cations that can be chosen; the triphenylphosphonium (TPP) ion is particularly unique for mitochondrion targeting, mainly due to its simplicity in structure and ease to be linked to the target molecules. In this work, mitochondrion-targeted AB3-type novel phthalocyanine and porphyrin photosensitizers (PSs) were synthesized and their photophysical photochemical properties were defined. Fluorescence quantum yields (ΦF) are 0.009, 0.14, 0.13, and 0.13, and the singlet-oxygen quantum yields (ΦΔ) are 0.27, 0.75, 0.57, and 0.58 for LuPcPox(OAc), AB 3 TPP-Pc, AB 3 TPP-Por-C4, and AB 3 TPP-Por-C6, respectively. To evaluate the photodynamic efficacy of the TPP-conjugated PS cell viabilities of A549 and BEAS-2B lung cells were comparatively measured and IC-50 values were determined. AB 3 TPP-Por-C4, AB 3 TPP-Por-C6, and AB 3 TPP-Pc compounds compared to the reference molecules ZnPc and H 2 TPP were found to be highly cytotoxic (sub-micromolar concentration) under the light. LuPcPox(OAc) is the most effective molecule regarding cell killing (the activity). The cell killing of the TPP-conjugated porphyrin derivatives exhibits a similar response compared to LuPcPox(OAc) when the light absorbing factor of the PS is normalized at 660 nm: TPP-conjugated porphyrins absorb less light (lower extinction coefficient) but produce more radical species (higher singlet-oxygen quantum yield) and therefore effectively kill the cells. The singlet oxygen-producing capacity of AB 3 TPP-Pc is almost 3 times higher compared to LuPcPox(OAc) and 50% more efficient with respect to ZnPc, suggesting that TPP-conjugated phthalocyanine may serve as a good photosensitizer for photodynamic therapy (PDT). The high singlet oxygen generation capacity of these novel TPP-conjugated porphyrin and phthalocyanine PS suggests that they might be useful for PDT requiring lower photosensitizer concentration and reduced energy deposited through less light exposure.

Epitranscriptomics Changes the Play: m6A RNA Modifications in Apoptosis.

Apoptosis is a form of programmed cell death that is essential for cellular and organismal homeostasis. Any irregularities that disturb the balance between apoptosis and cell survival have severe implications, such as improper development or life-threatening diseases. Thus, it is highly critical to maintain a proper rate of apoptosis throughout development. In fact, several complex transcriptional and posttranscriptional mechanisms exist in eukaryotes to critically regulate the rate of apoptotic processes. Recent studies suggest that not only RNA sequences but also their modifications, such as m6A methylation, play a fundamental role in these transcriptional and posttranscriptional processes. A specific set of proteins, called writer, eraser, and reader of m6A marks, modulate the rate of apoptosis by determining the m6A repertoire and the fate of certain transcripts associated with apoptosis. In this Review, we will cover the dynamic m6A RNA modifications and their impact on modulation of apoptosis.

Cytoplasmically localized tRNA-derived fragments inhibit translation in Drosophila S2 cells.

Transfer ribonucleic acids (tRNAs) serve not only as amino acid carriers during translation but also as a template for the biogenesis of short fragments that can regulate gene expression. Despite recent progress in the function of tRNA-derived fragments (tRFs), their intracellular localization, protein partners, and role in regulating translation are not well understood. We used synthetic tRFs to investigate their localization and function in Drosophila S2 cells. Under our experimental setting, all synthetic tRFs tested were localized at distinct sites within the cytoplasm in a similar manner in Drosophila S2 cells. Cytoplasmically-localized tRFs were positioned in close proximity to GW182 and XRN1 proteins. Functionally, tRFs, which slightly suppressed proliferation in S2 cells, inhibited translation without any major shift in the polysome profile. These results suggest that 5'-tRFs are cytoplasmically-localized and regulate gene expression through inhibition of translation in Drosophila.

Knockdown of death receptor 5 antisense long noncoding RNA and cisplatin treatment modulate similar macromolecular and metabolic changes in HeLa cells.

Background/aim: Despite great progress in complex gene regulatory mechanisms in the dynamic tumor microenvironment, the potential contribution of long noncoding RNAs (lncRNAs) to cancer cell metabolism is poorly understood. Death receptor 5 antisense (DR5-AS) is a cisplatin inducible lncRNA whose knockdown modulates cell morphology. However, its effect on cell metabolism is unknown. The aim of this study is to examine metabolic changes modulated by cisplatin and DR5-AS lncRNA in HeLa cells. Materials and methods: We used cisplatin as a universal cancer therapeutic drug to modulate metabolic changes in HeLa cervix cancer cells. We then examined the extent of metabolic changes by Fourier transform infrared spectroscopy (FTIR). We also performed transcriptomics analyses by generating new RNA-seq data with total RNAs isolated from cisplatin-treated HeLa cells. Then, we compared cisplatin-mediated transcriptomics and macromolecular changes with those mediated by DR5-AS knockdown. Results: Cisplatin treatment caused changes in the unsaturated fatty acid and lipid-to-protein ratios and the glycogen content. These observations in altered cellular metabolism were supported by transcriptomics analyses. FTIR spectroscopy analyses have revealed that DR5-AS knockdown causes a 20.9% elevation in the lipid/protein ratio and a 76.6% decrease in lipid peroxidation. Furthermore, we detected a 3.42% increase in the chain length of the aliphatic lipids, a higher content of RNA, and a lower amount of glycogen indicating relatively lower metabolic activity in the DR5-AS knockdown HeLa cells. Interestingly, we observed a similar gene expression pattern under cisplatin treatment and DR5-AS knockdown HeLa cells. Conclusion: These results suggest that DR5-AS lncRNA appears to account for a fraction of cisplatin-mediated macromolecular and metabolic changes in HeLa cervix cancer cells.

Noncoding RNAs in apoptosis: identification and function.

Apoptosis is a vital cellular process that is critical for the maintenance of homeostasis in health and disease. The derailment of apoptotic mechanisms has severe consequences such as abnormal development, cancer, and neurodegenerative diseases. Thus, there exist complex regulatory mechanisms in eukaryotes to preserve the balance between cell growth and cell death. Initially, protein-coding genes were prioritized in the search for such regulatory macromolecules involved in the regulation of apoptosis. However, recent genome annotations and transcriptomics studies have uncovered a plethora of regulatory noncoding RNAs that have the ability to modulate not only apoptosis but also many other biochemical processes in eukaryotes. In this review article, we will cover a brief summary of apoptosis and detection methods followed by an extensive discussion on microRNAs, circular RNAs, and long noncoding RNAs in apoptosis.

Experimental MicroRNA Detection Methods.

MicroRNAs (miRNAs) are considerably small yet highly important riboregulators involved in nearly all cellular processes. Due to their critical roles in posttranscriptional regulation of gene expression, they have the potential to be used as biomarkers in addition to their use as drug targets. Although computational approaches speed up the initial genomewide identification of putative miRNAs, experimental approaches are essential for further validation and functional analyses of differentially expressed miRNAs. Therefore, sensitive, specific, and cost-effective microRNA detection methods are imperative for both individual and multiplex analysis of miRNA expression in different tissues and during different developmental stages. There are a number of well-established miRNA detection methods that can be exploited depending on the comprehensiveness of the study (individual miRNA versus multiplex analysis), the availability of the sample and the location and intracellular concentration of miRNAs. This review aims to highlight not only traditional but also novel strategies that are widely used in experimental identification and quantification of microRNAs.

Endogenous miRNA Sponges.

MicroRNAs (miRNAs) are a class of noncoding RNAs of 17-22 nucleotides in length with a critical function in posttranscriptional gene regulation. These master regulators are themselves subject to regulation both transcriptionally and posttranscriptionally. Recently, miRNA function has been shown to be modulated by exogenous RNA molecules that function as miRNA sponges. Interestingly, endogenous transcripts such as transcribed pseudogenes, long noncoding RNAs (lncRNAs), circular RNAs (circRNAs) and mRNAs may serve as natural miRNA sponges. These transcripts, which bind to miRNAs and competitively sequester them away from their targets, are naturally existing endogenous miRNA sponges, called competing endogenous RNAs (ceRNAs). Here we present a historical background of miRNAs, exogenous and endogenous miRNA sponges as well as some examples of endogenous miRNA sponges involved in regulatory mechanisms associated with various diseases, developmental stages, and other cellular processes.

44 Current Challenges in miRNomics.

Mature microRNAs (miRNAs) are short RNA sequences about 18-24 nucleotide long, which provide the recognition key within RISC for the posttranscriptional regulation of target RNAs. Considering the canonical pathway, mature miRNAs are produced via a multistep process. Their transcription (pri-miRNAs) and first processing step via the microprocessor complex (pre-miRNAs) occur in the nucleus. Then they are exported into the cytosol, processed again by Dicer (dsRNA) and finally a single strand (mature miRNA) is incorporated into RISC (miRISC). The sequence of the incorporated miRNA provides the function of RNA target recognition via hybridization. Following binding of the target, the mRNA is either degraded or translation is inhibited, which ultimately leads to less protein production. Conversely, it has been shown that binding within the 5' UTR of the mRNA can lead to an increase in protein product. Regulation of homeostasis is very important for a cell; therefore, all steps in the miRNA-based regulation pathway, from transcription to the incorporation of the mature miRNA into RISC, are under tight control. While much research effort has been exerted in this area, the knowledgebase is not sufficient for accurately modelling miRNA regulation computationally. The computational prediction of miRNAs is, however, necessary because it is not feasible to investigate all possible pairs of a miRNA and its target, let alone miRNAs and their targets. We here point out open challenges important for computational modelling or for our general understanding of miRNA-based regulation and show how their investigation is beneficial. It is our hope that this collection of challenges will lead to their resolution in the near future.

Transcriptomics Profiling Identifies Cisplatin-Inducible Death Receptor 5 Antisense Long Non-coding RNA as a Modulator of Proliferation and Metastasis in HeLa Cells.

Cisplatin is a well-known cancer chemotherapeutic agent but how extensively long non-coding RNA (lncRNA) expression is modulated by cisplatin is unknown. It is imperative to employ a comprehensive approach to obtain a better account of cisplatin-mediated changes in the expression of lncRNAs. In this study, we used a transcriptomics approach to profile lncRNAs in cisplatin-treated HeLa cells, which resulted in identification of 10,214 differentially expressed lncRNAs, of which 2,500 were antisense lncRNAs. For functional analyses, we knocked down one of the cisplatin inducible lncRNAs, death receptor 5 antisense (DR5-AS) lncRNA, which resulted in a morphological change in HeLa cell shape without inducing any cell death. A second round of transcriptomics-based profiling revealed differential expression of genes associated with immune system, motility and cell cycle in DR5-AS knockdown HeLa cells. Cellular analyses showed that DR5-AS reduced cell proliferation and caused a cell cycle arrest at S and G2/M phases. Moreover, DR5-AS knockdown reduced the invasive capacity of HeLa cells in zebrafish xenograft model. These results suggest that cisplatin-mediated pleiotropic effects, such as reduction in cell proliferation, metastasis and cell cycle arrest, may be mediated by lncRNAs.

Transcriptomics Analysis of Circular RNAs Differentially Expressed in Apoptotic HeLa Cells.

Apoptosis is a form of regulated cell death that plays a critical role in survival and developmental homeostasis. There are numerous reports on regulation of apoptosis by protein-coding genes as well as small non-coding RNAs, such as microRNAs. However, there is no comprehensive investigation of circular RNAs (circRNA) that are differentially expressed under apoptotic conditions. We have performed a transcriptomics study in which we first triggered apoptosis in HeLa cells through treatment with four different agents, namely cisplatin, doxorubicin, TNF-α and anti-Fas mAb. Total RNAs isolated from control as well as treated cells were treated with RNAse R to eliminate the linear RNAs. The remaining RNAs were then subjected to deep-sequencing to identify differentially expressed circRNAs. Interestingly, some of the dys-regulated circRNAs were found to originate from protein-coding genes well-documented to regulate apoptosis. A number of candidate circRNAs were validated with qPCR with or without RNAse R treatment as well. We then took advantage of bioinformatics tools to investigate the coding potential of differentially expressed RNAs. Additionally, we examined the candidate circRNAs for the putative miRNA-binding sites and their putative target mRNAs. Our analyses point to a potential for circRNA-mediated sponging of miRNAs known to regulate apoptosis. In conclusion, this is the first transcriptomics study that provides a complete circRNA profile of apoptotic cells that might shed light onto the potential role of circRNAs in apoptosis.

Intracytoplasmic Re-localization of miRISC Complexes.

MicroRNAs (miRNAs) are a conserved class of non-coding RNAs of 22 nucleotides that post-transcriptionally regulate gene expression through translational repression and/or mRNA degradation. A great progress has been made regarding miRNA biogenesis and miRNA-mediated gene regulation. Additionally, an ample amount of information exists with respect to the regulation of miRNAs. However, the cytoplasmic localization of miRNAs and its effect on gene regulatory output is still in progress. We provide a current review of the cytoplasmic miRNA localization in metazoans. We then discuss the dynamic changes in the intracytoplasmic localization of miRNAs as a means to regulate their silencing activity. We then conclude our discussion with the potential molecules that could modulate miRNA localization.

Small RNA data set that includes tRNA-derived fragments from Jurkat cells treated with camptothecin.

In this article, we report a small RNA data set obtained from human T cell acute leukemia Jurkat cells, which were treated with the universal apoptotic agent camptothecin. Based on the Annexin-V labeling pattern, we sorted two Jurkat subpopulations in treated cells: one that is sensitive to the drug and the other being relatively more resistant. We report new original data that include the frequency of tRNA-derived fragments (tRF) in drug-sensitive and resistant cells. We also present partially analyzed data to show the origin of reads on tRNAs as well as the borders of the fragments. We believe that this data can benefit the science community working in the field of tRF and/or apoptosis.

Re-Arrangements in the Cytoplasmic Distribution of Small RNAs Following the Maternal-to-Zygotic Transition in Drosophila Embryos.

Small ribonucleic acids (RNAs) are known to regulate gene expression during early development. However, the dynamics of interaction between small RNAs and polysomes during this process is largely unknown. To investigate this phenomenon, 0-1 h and 7-8 h Drosophila melanogaster embryos were fractionated on sucrose density gradients into four fractions based on A254 reading (1) translationally inactive messenger ribonucleoprotein (mRNP), (2) 60S, (3) monosome, and (4) polysome. Comparative analysis of deep-sequencing reads from fractionated and un-fractionated 0-1 h and 7-8 h embryos revealed development-specific co-sedimentation pattern of small RNAs with the cellular translation machinery. Although most micro RNAs (miRNAs) did not have a specific preference for any state of the translational machinery, we detected fraction-specific enrichment of a few miRNAs such as dme-miR-1-3p, -184-3p, 5-5p and 263-5p. More interestingly, we observed changes in the subcellular location of a subset of miRNAs in fractionated embryos despite no measurable difference in their amount in unfractionated embryos. Transposon-derived endo small interfering RNAs (siRNAs) were over-expressed in 7-8 h embryos and associated mainly with the mRNP fraction. In contrast, transposon-derived PIWI-interacting RNAs (piRNA), which were more abundant in 0-1 h embryos, co-sedimented primarily with the polysome fractions. These results suggest that there appears to be a complex interplay among the small RNAs with respect to their polysome-cosedimentation pattern during early development in Drosophila melanogaster.