RNA Post-transcriptional Regulation Research Articles

DNA 5-hydroxymethylcytosine in pediatric central nervous system tumors may impact tumor classification and is a positive prognostic marker

BackgroundNucleotide-specific 5-hydroxymethylcytosine (5hmC) remains understudied in pediatric central nervous system (CNS) tumors. 5hmC is abundant in the brain, and alterations to 5hmC in adult CNS tumors have been reported. However, traditional approaches to measure DNA methylation do not distinguish between 5-methylcytosine (5mC) and its oxidized counterpart 5hmC, including those used to build CNS tumor DNA methylation classification systems. We measured 5hmC and 5mC epigenome-wide at nucleotide resolution in glioma, ependymoma, and embryonal tumors from children, as well as control pediatric brain tissues using tandem bisulfite and oxidative bisulfite treatments followed by hybridization to the Illumina Methylation EPIC Array that interrogates over 860,000 CpG loci.ResultsLinear mixed effects models adjusted for age and sex tested the CpG-specific differences in 5hmC between tumor and non-tumor samples, as well as between tumor subtypes. Results from model-based clustering of tumors was used to test the relation of cluster membership with patient survival through multivariable Cox proportional hazards regression. We also assessed the robustness of multiple epigenetic CNS tumor classification methods to 5mC-specific data in both pediatric and adult CNS tumors. Compared to non-tumor samples, tumors were hypohydroxymethylated across the epigenome and tumor 5hmC localized to regulatory elements crucial to cell identity, including transcription factor binding sites and super-enhancers. Differentially hydroxymethylated loci among tumor subtypes tended to be hypermethylated and disproportionally found in CTCF binding sites and genes related to posttranscriptional RNA regulation, such as DICER1. Model-based clustering results indicated that patients with low 5hmC patterns have poorer overall survival and increased risk of recurrence. Our results suggest 5mC-specific data from OxBS-treated samples impacts methylation-based tumor classification systems giving new opportunities for further refinement of classifiers for both pediatric and adult tumors.ConclusionsWe identified that 5hmC localizes to super-enhancers, and genes commonly implicated in pediatric CNS tumors were differentially hypohydroxymethylated. We demonstrated that distinguishing methylation and hydroxymethylation is critical in identifying tumor-related epigenetic changes. These results have implications for patient prognostication, considerations of epigenetic therapy in CNS tumors, and for emerging molecular neuropathology classification approaches.

Fucosylated Proteome Profiling Identifies a Fucosylated, Non-Ribosomal, Stress-Responsive Species of Ribosomal Protein S3.

Simple SummaryDysregulated fucosylation has been characterized as an underlying cause or a contributor to the pathogenesis of several disease states. However, to date, there is not a clear understanding of how and what proteins, signaling pathways, and cellular processes are impacted by fucosylation. Here, we characterized the proteins recognized by a fucose-binding lectin and unexpectedly discovered that many intracellular proteins are putatively subject to posttranslational fucosylation. We further found that fucosylation on intracellular ribosomal protein S3 responds to stimulus, and that it appears to be independent of the currently characterized fucosylation pathway. This work suggests a to-date-underappreciated role for fucosylation on intracellular proteins and supports the existence of fucosylation capabilities within cells that is not fully known.Alterations in genes encoding for proteins that control fucosylation are known to play causative roles in several developmental disorders, such as Dowling-Degos disease 2 and congenital disorder of glycosylation type IIc (CDGIIc). Recent studies have provided evidence that changes in fucosylation can contribute to the development and progression of several different types of cancers. It is therefore important to gain a detailed understanding of how fucosylation is altered in disease states so that interventions may be developed for therapeutic purposes. In this report, we find that fucosylation occurs on many intracellular proteins. This is an interesting finding, as the fucosylation machinery is restricted to the secretory pathway and is thought to predominately affect cell-membrane-bound and secreted proteins. We find that Ribosomal protein S3 (RPS3) is fucosylated in normal tissues and in cancer cells, and that the extent of its fucosylation appears to respond to stress, including MAPK inhibitors, suggesting a new role in posttranslational protein function. Our data identify a new ribosome-independent species of fucosylated RPS3 that interacts with proteins involved in posttranscriptional regulation of RNA, such as Heterogeneous nuclear ribonucleoprotein U (HNRNPU), as well as with a predominance of non-coding RNAs. These data highlight a novel role for RPS3, which, given previously reported oncogenic roles for RPS3, might represent functions that are perturbed in pathologies such as cancer. Together, our findings suggest a previously unrecognized role for fucosylation in directly influencing intracellular protein functions.

Mitochondrial DNA Methylation and Human Diseases

Epigenetic modifications of the nuclear genome, including DNA methylation, histone modifications and non-coding RNA post-transcriptional regulation, are increasingly being involved in the pathogenesis of several human diseases. Recent evidence suggests that also epigenetic modifications of the mitochondrial genome could contribute to the etiology of human diseases. In particular, altered methylation and hydroxymethylation levels of mitochondrial DNA (mtDNA) have been found in animal models and in human tissues from patients affected by cancer, obesity, diabetes and cardiovascular and neurodegenerative diseases. Moreover, environmental factors, as well as nuclear DNA genetic variants, have been found to impair mtDNA methylation patterns. Some authors failed to find DNA methylation marks in the mitochondrial genome, suggesting that it is unlikely that this epigenetic modification plays any role in the control of the mitochondrial function. On the other hand, several other studies successfully identified the presence of mtDNA methylation, particularly in the mitochondrial displacement loop (D-loop) region, relating it to changes in both mtDNA gene transcription and mitochondrial replication. Overall, investigations performed until now suggest that methylation and hydroxymethylation marks are present in the mtDNA genome, albeit at lower levels compared to those detectable in nuclear DNA, potentially contributing to the mitochondria impairment underlying several human diseases.

Massively Parallel Analysis of Regulatory RNA Sequences.

The stability of RNA transcripts is regulated by signals within their sequences, but theidentity ofthose signals still remain elusive in many biological systems. Recently introduced massively parallel tools for the analysis of regulatory RNA sequences provide the ability to detect functional cis-regulatory sequences of post-transcriptional RNA regulation at a much larger scale and resolution than before. Their application formulates theunderlying sequence-based rules and predictsthe impact of genetic variations. Here, we describe the application of UTR-Seq, as a strategy to uncover cis-regulatory signals of RNA stability during early zebrafish embryogenesis. The method combines massively parallel reporter assays (MPRA) with computational regression models. It surveys the effect of tens of thousands of regulatory sequences on RNA stability andanalyzes the results via regression models to identify sequence signals that impact RNA stability and to predict the in vivo effect of sequence variations.

Identification of RNA-Binding Protein Landscapes Across Zebrafish Embryonic Transcriptome via iCLIP Approach.

In metazoans, fertilization initiates vast remodeling of the embryonic proteome and transcriptome. This is accomplished via complex post-transcriptional regulation of maternal and zygotic RNA. RNA-binding proteins (RBPs) are one of the major mediators of embryonic post-transcriptional RNA regulation. Thus, elucidation of the molecular mechanisms by which maternal and zygotic transcripts change their translational capacities and expression levels requires thorough and precise determination of the targets and binding sites of individual RBPs in embryonic transcriptomes. Here, I provide a detailed protocol for the UV crosslinking-based method, named iCLIP, to study RBP functions during early zebrafish embryogenesis.

Loss of Pseudouridine Synthases in the RluA Family Causes Hypersensitive Nociception in Drosophila.

Nociceptive neurons of Drosophila melanogaster larvae are characterized by highly branched dendritic processes whose proper morphogenesis relies on a large number of RNA-binding proteins. Post-transcriptional regulation of RNA in these dendrites has been found to play an important role in their function. Here, we investigate the neuronal functions of two putative RNA modification genes, RluA-1 and RluA-2, which are predicted to encode pseudouridine synthases. RluA-1 is specifically expressed in larval sensory neurons while RluA-2 expression is ubiquitous. Nociceptor-specific RNAi knockdown of RluA-1 caused hypersensitive nociception phenotypes, which were recapitulated with genetic null alleles. These were rescued with genomic duplication and nociceptor-specific expression of UAS-RluA-1-cDNA. As with RluA-1, RluA-2 loss of function mutants also displayed hyperalgesia. Interestingly, nociceptor neuron dendrites showed a hyperbranched morphology in the RluA-1 mutants. The latter may be a cause or a consequence of heightened sensitivity in mutant nociception behaviors.

Altered Expression of the m6A Methyltransferase METTL3 in Alzheimer's Disease.

Cognitive impairment in Alzheimer’s disease (AD) is associated with dysregulation of the RNA and protein expression profiles in the brain. Recent studies have highlighted the importance of RNA post-transcriptional regulation (epitranscriptomics) in higher order brain functions. Specifically, N6-methyladenosine (m6A), which controls RNA stability, splicing, translation and trafficking, plays an important role in learning and memory. This raises the question of whether m6A signaling is perturbed in AD. To address this, we investigated the expression profile of known m6A-regulatory genes using a public RNA-seq dataset and identified a subset of genes which were significantly dysregulated in the human AD brain. Among these, genes encoding the m6A methyltransferase, METTL3, and a member of the m6A methyltransferase complex (MACOM), RBM15B, were downregulated and upregulated in the hippocampus, respectively. These findings were validated at the protein level using an independent cohort of postmortem human brain samples. Unexpectedly, we observed an accumulation of methyltransferase-like 3 (METTL3), but not RBM15B, in the insoluble fractions, which positively correlated with the levels of insoluble Tau protein in the postmortem human AD samples. Aberrant expression and distribution of METTL3 in the hippocampus of the AD brain may therefore represent an epitranscriptomic mechanism underlying the altered gene expression patterns associated with disease pathogenesis.

Dynamic RNA 3' Uridylation and Guanylation during Mitosis.

SummarySuccessful cell division involves highly regulated transcriptional and post-transcriptional control. The RNA poly(A) tail represents an important layer of RNA post-transcriptional regulation. Previous TAIL-seq analysis of S phase and M phase poly(A) tail information showed that only a small number of genes showed more than 2-fold change in their poly(A) tail length between the two cell cycle stages. In addition, the changes in poly(A) tail length between these two stages showed minimal impact on the translation of the genes as long as the poly(A) tails were longer than 20 nt. Therefore, the significance of poly(A) tail dynamics during the cell cycle remains largely unknown. Here, by re-analyzing the S phase and M phase TAIL-seq data, we uncovered an interesting global dynamics of RNA poly(A) tails in terms of their terminal modifications, implying global RNA regulation between mitotic cell cycles through poly(A) tail terminal modifications.

Abundance of miRNA during muscle growth is not influenced by dietary protein inclusion levels in neonatal pigs

Postnatal muscle growth requires satellite cell (SC) activation and fusion with existing myofibers. Delay or absence of SC proliferation and differentiation leads to a reduction in muscle hypertrophy. Nutrition during neonatal life impacts developmental growth programing by altering SC activity. Growth as a percentage of body weight is highest during the neonatal period and restricting neonatal nutrition can lead to permanent muscle growth deficits. Reducing dietary intake of protein can improve gut function during weaning, however, this may limit amino acid availability for muscle growth. The feed‐induced protein synthesis response in muscle is highest in the neonatal period and subsequently progressively declines throughout life. The heightened responsiveness of protein synthesis during this period is mediated mainly by the activation of translation initiation factors. Post‐transcriptional regulation of RNA is mediated in part by micro‐RNAs (miRNA) by preventing translation and/or promoting degradation and are known to be important modulators of stem cell activity. Proliferation of SCs is promoted by miR‐133a, while differentiation is promoted by miR‐1, miR‐133b and miR‐206 through repression of Pax7. The objective of this study was to determine miRNA abundance during neonatal muscle growth in response to a protein deficient, adequate, and excess diets in pigs. Twenty‐eight piglets obtained at 1d of age received isocaloric dietary treatments of either an adequate protein diet (A), a 25% deficient protein diet (D), or a 25% excess protein diet (E) over a 17‐day trial. Piglet starting weights were 1.47kg±0.1 kg for D and 1.46±0.1 kg for E and A treatments. Final body weight and ADG were increased in E compared to D pigs (5.60±0.27kg vs 4.75±0.27kg and 0.27±0.01kg/d vs 0.21±0.01kg/d) despite an equal feed intake across treatments. Gene expression in the longissimus dorsi was evaluated by qPCR. There were no differences IGF1, or in myogenic genes MyoD and MyoG despite the increased growth rates of E treatment pigs. The SC marker Pax‐7 did not differ between treatments. Genes associated with lipid oxidation, PPARd and CPT1, were not different between treatments. There were no differences in degradation pathway genes muRF‐1, atrogin‐1, and FOXO despite the reduction in growth of D pigs. The differences in growth response without alterations in gene expression may be due to post‐transcriptional regulation, however, no differences were observed in miRNA expression. The differentiation promoting miRs‐1, 133b and 206 and proliferation promoting miR‐133a were not different between treatments. These data indicate that diets excess in protein increased growth rates which may lead a sustained increase in muscle mass throughout life. While miRNA expression was not different between treatments, miRNAs may still play a role in the differences observed in growth between D and E treatments through their activity and rate of binding to their target mRNAs.

A novel epigenetic mechanism of FXR inhibiting GLP-1 secretion via miR-33 and its downstream targets

Type II diabetes is a complex, chronic, and progressive disease. Previously, we demonstrate that FXR inhibits GLP-1 secretion via interacting with CREB to inhibit the transcriptional activity of CREB, thus promoting the development of type II diabetes. Epigenetic modifications, such as DNA methylation, histone acetylation, and post-transcriptional RNA regulation, are essential mediators contributing to diabetes-associated morbidity and mortality. Thus, we attempted to investigate the epigenetic mechanisms of FXR modulating GLP-1 secretion. Firstly, the involvement of histone acetylation, DNA methylation, and post-transcriptional regulation in FXR inhibiting GLP-1 secretion was verified. As FXR overexpression significantly inhibited the activity of GCG 3′-UTR, we hypothesize that miRNA might participate in the mechanism. Two online tools and real-time PCR revealed that FXR promoted miR-33 expression. Moreover, miR-33 inhibited the expression of GCG and CREB1 through direct targeting in STC-1 cells. FXR overexpression in STC-1 cells significantly reduced the mRNA expression and protein levels of both GCG and CREB1, as well as the secretion of GLP-1; miR-33 inhibition exerted opposing effects. More importantly, the effects of FXR overexpression were significantly reversed by miR-33 inhibition, indicating that FXR inhibited GLP-1 secretion through promoting miR-33 expression, therefore inhibiting the expression of miR-33 targets, GCG and CREB1. In conclusion, we provide a novel epigenetic mechanism by which FXR inhibits the secretion of GLP-1 through miR-33 and its two downstream targets, GCG and CREB1. These findings might provide innovative strategies for improving type II diabetes, which needs further in vivo and clinical investigation.

Targeted m6A reader proteins to study the epitranscriptome.

Posttranscriptional regulation of RNA has emerged as an important regulator of genetic information flow in eukaryotic systems. In particular, chemical modifications of RNA have recently been established as key regulatory marks that affect the lifetime, location, trafficking, and function of messenger RNA (mRNA). In mammalian systems, N6-methyladenosine (m6A) is the most prevalent mRNA modification, and the writer, eraser, and reader proteins that install, remove, or recognize m6A have been rapidly uncovered and studied at the whole cell level. Understanding the effects of specific m6A modifications and their regulation at the single transcript level is the key next step to understanding the mechanism and consequences of epitranscriptomic regulation. We recently developed programmable m6A reader proteins to study the effects of epitranscriptomic regulatory factors at individual RNA transcripts. In this chapter, we discuss the application of targeted m6A readers to study RNA regulation at single endogenous sites. We briefly introduce what is currently known about the N6-methyltranscriptome and the Cas13 RNA-targeting family of proteins before detailing our protocol to study RNA modifications with targeted reader proteins.

Non-canonical post-transcriptional RNA regulation of neural stem cell potential.

Adult brain structures and complexity emerge from a single layer of neuroepithelial cells that early during the development give rise to neural stem cells (NSCs). NSCs persist in restricted regions of the postnatal brain where they support neurogenesis throughout life thus allowing brain plasticity and adaptation. NSC regulation involves a precise coordination of intrinsic and extrinsic mechanisms that finely modulate the neurogenic process. Here we will discuss new mechanisms of post-transcriptional gene regulation that act in the embryonic and adult brain to regulate NSC maintenance and differentiation. In our recent work we found that the RNAaseIII Drosha not only regulates microRNA production, but also directly affects the stability of mRNAs and thereby controls proteome composition. This non-canonical (miRNA-independent) function of Drosha is central in the maintenance and fate choices made by adult hippocampal NSCs in the healthy brain. We found that Drosha targets the mRNA of the gliogenic transcription factor Nuclear Factor I/B and thereby blocks its expression in the NSCs. In the absence of Drosha, NSCs aberrantly differentiate into oligodendrocytes and are lost leading to an impairment of neurogenesis. Overall these findings reveal an unprecedented Drosha-mediated post-transcriptional mechanism for the regulation of hippocampal NSC potential.

Emerging Evidence of Epigenetic Modifications in Vascular Complication of Diabetes.

Genes, dietary, and lifestyle factors have been shown to be important in the pathophysiology of diabetes and associated microvascular complications. Epigenetic modifications, such as DNA methylation, histone acetylation, and post-transcriptional RNA regulation, are being increasingly recognized as important mediators of the complex interplay between genes and the environment. Recent studies suggest that diabetes-induced dysregulation of epigenetic mechanisms resulting in altered gene expression in target cells can lead to diabetes-associated complications, such as diabetic cardiomyopathy, diabetic nephropathy, retinopathy, and so on, which are the major contributors to diabetes-associated morbidity and mortality. Thus, knowledge of dysregulated epigenetic pathways involved in diabetes can provide much needed new drug targets for these diseases. In this review, we constructed our search strategy to highlight the role of DNA methylation, modifications of histones and role of non-coding RNAs (microRNAs and long non-coding RNAs) in vascular complications of diabetes, including cardiomyopathy, nephropathy, and retinopathy.

SsHMM: extracting intuitive sequence-structure motifs from high-throughput RNA-binding protein data.

RNA-binding proteins (RBPs) play an important role in RNA post-transcriptional regulation and recognize target RNAs via sequence-structure motifs. The extent to which RNA structure influences protein binding in the presence or absence of a sequence motif is still poorly understood. Existing RNA motif finders either take the structure of the RNA only partially into account, or employ models which are not directly interpretable as sequence-structure motifs. We developed ssHMM, an RNA motif finder based on a hidden Markov model (HMM) and Gibbs sampling which fully captures the relationship between RNA sequence and secondary structure preference of a given RBP. Compared to previous methods which output separate logos for sequence and structure, it directly produces a combined sequence-structure motif when trained on a large set of sequences. ssHMM’s model is visualized intuitively as a graph and facilitates biological interpretation. ssHMM can be used to find novel bona fide sequence-structure motifs of uncharacterized RBPs, such as the one presented here for the YY1 protein. ssHMM reaches a high motif recovery rate on synthetic data, it recovers known RBP motifs from CLIP-Seq data, and scales linearly on the input size, being considerably faster than MEMERIS and RNAcontext on large datasets while being on par with GraphProt. It is freely available on Github and as a Docker image.

Moving messages in the developing brain—emerging roles for mRNA transport and local translation in neural stem cells

The mammalian cerebral cortex is a complex brain structure integral to our higher cognition. During embryonic cortical development, radial glial progenitors (RGCs) produce neurons and serve as physical structures for migrating neurons. Recent discoveries highlight new roles for RNA localization and local translation in RGCs, both at the cell body and at distal structures called basal endfeet. By implementing technologies from the field of RNA research to brain development, investigators can manipulate RNA-binding proteins as well as visualize single-molecule RNAs, live movement of mRNAs and their binding proteins, and translation. Going forward, these studies establish a framework for investigating how post-transcriptional RNA regulation helps shape RGC function and triggers neurodevelopmental diseases.

RNA-binding motif protein 47 inhibits Nrf2 activity to suppress tumor growth in lung adenocarcinoma.

RNA-binding proteins provide a new layer of posttranscriptional regulation of RNA during cancer progression. We identified RNA-binding motif protein 47 (RBM47) as a target gene of transforming growth factor (TGF)-β in mammary gland epithelial cells (NMuMG cells) that have undergone the epithelial-to-mesenchymal transition. TGF-β repressed RBM47 expression in NMuMG cells and lung cancer cell lines. Expression of RBM47 correlated with good prognosis in patients with lung, breast and gastric cancer. RBM47 suppressed the expression of cell metabolism-related genes, which were the direct targets of nuclear factor erythroid 2-related factor 2 (Nrf2; also known as NFE2L2). RBM47 bound to KEAP1 and Cullin 3 mRNAs, and knockdown of RBM47 inhibited their protein expression, which led to enhanced binding of Nrf2 to target genomic regions. Knockdown of RBM47 also enhanced the expression of some Nrf2 activators, p21/CDKN1A and MafK induced by TGF-β. Both mitochondrial respiration rates and the side population cells in lung cancer cells increased in the absence of RBM47. Our findings, together with the enhanced tumor formation and metastasis of xenografted mice by knockdown of the RBM47 expression, suggested tumor-suppressive roles for RBM47 through the inhibition of Nrf2 activity.

Dissecting the target specificity of RNase H recruiting oligonucleotides using massively parallel reporter analysis of short RNA motifs.

Processing and post-transcriptional regulation of RNA often depend on binding of regulatory molecules to short motifs in RNA. The effects of such interactions are difficult to study, because most regulatory molecules recognize partially degenerate RNA motifs, embedded in a sequence context specific for each RNA. Here, we describe Library Sequencing (LibSeq), an accurate massively parallel reporter method for completely characterizing the regulatory potential of thousands of short RNA sequences in a specific context. By sequencing cDNA derived from a plasmid library expressing identical reporter genes except for a degenerate 7mer subsequence in the 3′UTR, the regulatory effects of each 7mer can be determined. We show that LibSeq identifies regulatory motifs used by RNA-binding proteins and microRNAs. We furthermore apply the method to cells transfected with RNase H recruiting oligonucleotides to obtain quantitative information for >15000 potential target sequences in parallel. These comprehensive datasets provide insights into the specificity requirements of RNase H and allow a specificity measure to be calculated for each tested oligonucleotide. Moreover, we show that inclusion of chemical modifications in the central part of an RNase H recruiting oligonucleotide can increase its sequence-specificity.

Memory acquisition and retrieval impact different epigenetic processes that regulate gene expression.

BackgroundA fundamental question in neuroscience is how memories are stored and retrieved in the brain. Long-term memory formation requires transcription, translation and epigenetic processes that control gene expression. Thus, characterizing genome-wide the transcriptional changes that occur after memory acquisition and retrieval is of broad interest and importance. Genome-wide technologies are commonly used to interrogate transcriptional changes in discovery-based approaches. Their ability to increase scientific insight beyond traditional candidate gene approaches, however, is usually hindered by batch effects and other sources of unwanted variation, which are particularly hard to control in the study of brain and behavior.ResultsWe examined genome-wide gene expression after contextual conditioning in the mouse hippocampus, a brain region essential for learning and memory, at all the time-points in which inhibiting transcription has been shown to impair memory formation. We show that most of the variance in gene expression is not due to conditioning and that by removing unwanted variance through additional normalization we are able provide novel biological insights. In particular, we show that genes downregulated by memory acquisition and retrieval impact different functions: chromatin assembly and RNA processing, respectively. Levels of histone 2A variant H2AB are reduced only following acquisition, a finding we confirmed using quantitative proteomics. On the other hand, splicing factor Rbfox1 and NMDA receptor-dependent microRNA miR-219 are only downregulated after retrieval, accompanied by an increase in protein levels of miR-219 target CAMKIIγ.ConclusionsWe provide a thorough characterization of coding and non-coding gene expression during long-term memory formation. We demonstrate that unwanted variance dominates the signal in transcriptional studies of learning and memory and introduce the removal of unwanted variance through normalization as a necessary step for the analysis of genome-wide transcriptional studies in the context of brain and behavior. We show for the first time that histone variants are downregulated after memory acquisition, and splicing factors and microRNAs after memory retrieval. Our results provide mechanistic insights into the molecular basis of cognition by highlighting the differential involvement of epigenetic mechanisms, such as histone variants and post-transcriptional RNA regulation, after acquisition and retrieval of memory.

RNA and lessons from viruses.

RNA and lessons from viruses.

The Role of Extracellular Vesicles: An Epigenetic View of the Cancer Microenvironment

Exosomes, microvesicles, and other extracellular vesicles are released by many cell types, including cancer cells and cancer-related immune cells. Extracellular vesicles can directly or indirectly facilitate the transfer of bioinformation to recipient cells or to the extracellular environment. In cancer, exosomes have been implicated in tumor initiation, proliferation, and metastasis. Extracellular vesicles can transmit proteins and nucleic acids that participate in DNA methylation, histone modification, and posttranscriptional regulation of RNA. Factors transmitted by extracellular vesicles reflect the donor cell status, and extracellular vesicles derived from tumor cells may be also responsible for altering expression of tumor promoting and tumor suppressing genes in recipient cells. Thus, circulating extracellular vesicles may act as biomarkers of cancer, and detection of these biomarkers may be applied to diagnosis or assessment of prognosis in patients with cancer.