The Functions of N-Methyladenosine in Nuclear RNAs.

N-methyladenosine (mA) is one of the most common modifications in both eukaryotic and prokaryotic mRNAs. It has been experimentally confirmed that mA methylation is involved in the regulation of stability and translation of various mRNAs. Until recently, the majority of mA-related studies have been focused on the cytoplasmic functions of this modification.

Effect of Lactobacillus reuteri NCIMB 30351 drops on symptoms of infantile functional gastrointestinal disorders and gut microbiota in early infants: Results from a randomized, placebo-controlled clinical trial.

Infantile functional gastrointestinal disorders, such as colic, constipation, diarrhea, and gastroesophageal reflux (regurgitation), often occur in early infancy and, representing one of the causes of significant parental anxiety, lead to a significant strain on the healthcare resources. In this study, we aimed to evaluate the effects of Lactobacillus reuteri drops (L. reuteri NCIMB 30351) on the symptoms of infantile colic, constipation, diarrhea, and gastroesophageal reflux, as well as on the levels of intestinal microbiota in full-term newborns during the first months of life.

Analyzing the Effects of Single Nucleotide Polymorphisms on hnRNPA2/B1 Protein Stability and Function: Insights for Anticancer Therapeutic Design.

Heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNPA2/B1) is a pivotal player in m6A recognition, RNA metabolism, and antiviral responses. In the context of cancer, overexpression of hnRNPA2/B1, abnormal RNA levels, and m6A depositions are evident. This study focuses on two significant nonsynonymous single nucleotide polymorphisms (nsSNPs) within hnRNPA2/B1, namely, F66L and E92K.

Detecting and Mapping N6-Methyladenosine on RNA/DNA Hybrids.

N6-methyladenosine (mA) is an RNA modification essential for posttranscriptional regulation of gene expression in eukaryotes. We recently demonstrated that mA decorates the RNA components of R-loops, specific nucleic acid structures consisting of an RNA/DNA hybrid and a single strand of non-template DNA, that represent a major source of genetic instability and, at the same time, contribute to regulation of gene expression in mammalian cells. According to growing body of experimental evidence, adenosine methylation affects stability of these structures and potentially influences various aspects of their metabolism.

Detection and Quantification of RNA Modifications on RNA-DNA Hybrids Using SID-UPLC-MS/MS.

R-loops are three-stranded nucleic acid structures consisting of an RNA-DNA hybrid and an unpaired strand of nontemplate DNA that represent a major source of genomic instability and are involved in regulation of several important biological processes in eukaryotic cells. A growing body of experimental evidence suggests that RNA moieties of RNA-DNA hybrids may convey RNA modifications influencing various aspects of R-loop biology. Here we present a protocol for quantitative analysis of RNA modifications on RNA-DNA hybrids using stable-isotope dilution ultraperformance liquid chromatography coupled with tandem mass spectrometry (SID-UPLC-MS/MS).

LINE-1 transcription in round spermatids is associated with accretion of 5-carboxylcytosine in their open reading frames.

Chromatin of male and female gametes undergoes a number of reprogramming events during the transition from germ cell to embryonic developmental programs. Although the rearrangement of DNA methylation patterns occurring in the zygote has been extensively characterized, little is known about the dynamics of DNA modifications during spermatid maturation. Here, we demonstrate that the dynamics of 5-carboxylcytosine (5caC) correlate with active transcription of LINE-1 retroelements during murine spermiogenesis.

Analysis of 5-Carboxylcytosine Distribution Using DNA Immunoprecipitation.

DNA methylation (5-methylcytosine, 5mC) is involved in regulation of a wide range of biological processes. TET proteins can oxidize 5mC to 5-hydroxymethylcytosine, 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). Although both 5fC and 5caC serve as intermediates in active demethylation pathway, growing body of experimental evidence indicate that these DNA modifications may also interact with specific sets of reader proteins and therefore may represent bona fide epigenetic marks.

Detection of Low-Abundance DNA Modifications Using Signal Amplification-Based Immunocytochemistry.

Immunocytochemistry can be instrumental in assessing the spatial distribution and relative levels of epigenetic modifications. Although conventional immunostaining has been utilized for the detection of 5-methylcytosine (5mC) in animal cells and tissues for several decades, the sensitivity of techniques based on the use of fluorophore-conjugated secondary antibodies is not always sufficient for studying DNA modifications that are less abundant in DNA compared with 5mC. Here we describe a protocol for sensitive immunocytochemistry that utilizes peroxidase-conjugated secondary antibodies coupled with catalyzed reporter deposition and allows for detection of low-abundance noncanonical bases (e.

Evidence for Noncytosine Epigenetic DNA Modifications in Multicellular Eukaryotes: An Overview.

Cytosine DNA methylation (5-methylcytsone, 5mC) is the major DNA modification found in the genomes of animals and plants. Although the roles of 5mC and its oxidized derivatives in the regulation of gene expression are relatively well attested and extensively explored, a number of recent studies imply that noncytosine DNA modifications may also convey specific biological functions and act as "epigenetic" marks in multicellular organisms. Here we review experimental evidence for the presence of noncytosine epigenetic modifications in metazoans and plants focusing on two "unusual" DNA bases, 5-hydroxymethyluracil (5hmU) and N6-methyladenine (6mA), and suggest potential explanations for inconsistencies in the currently available data on abundance and potential biological roles of these DNA modifications in mammals.

Modified Forms of Cytosine in Eukaryotes: DNA (De)methylation and Beyond.

5-Methylcytosine (5mC) is an epigenetic mark known to contribute to the regulation of gene expression in a wide range of biological systems. Ten Eleven Translocation (TET) dioxygenases oxidize 5mC to 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine in metazoans and fungi. Moreover, two recent reports imply the existence of other species of modified cytosine in unicellular alga Chlamydomonas reinhardtii and malaria parasite Plasmodium falciparum.

Mass spectrometry reveals the presence of specific set of epigenetic DNA modifications in the Norway spruce genome.

5-Methylcytosine (5mC) is an epigenetic modification involved in regulation of gene expression in metazoans and plants. Iron-(II)/α-ketoglutarate-dependent dioxygenases can oxidize 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). Although these oxidized forms of 5mC may serve as demethylation intermediates or contribute to transcriptional regulation in animals and fungi, experimental evidence for their presence in plant genomes is ambiguous.

N-methyladenosine regulates the stability of RNA:DNA hybrids in human cells.

R-loops are nucleic acid structures formed by an RNA:DNA hybrid and unpaired single-stranded DNA that represent a source of genomic instability in mammalian cells. Here we show that N-methyladenosine (mA) modification, contributing to different aspects of messenger RNA metabolism, is detectable on the majority of RNA:DNA hybrids in human pluripotent stem cells. We demonstrate that mA-containing R-loops accumulate during G/M and are depleted at G/G phases of the cell cycle, and that the mA reader promoting mRNA degradation, YTHDF2 (ref.

Molecular Mechanisms Governing the Stem Cell's Fate in Brain Cancer: Factors of Stemness and Quiescence.

Cellular quiescence is a reversible, non-cycling state controlled by epigenetic, transcriptional and niche-associated molecular factors. Quiescence is a condition where molecular signaling pathways maintain the poised cell-cycle state whilst enabling rapid cell cycle re-entry. To achieve therapeutic breakthroughs in oncology it is crucial to decipher these molecular mechanisms employed by the cancerous milieu to control, maintain and gear stem cells towards re-activation.

Wilms' Tumor Protein 1 and Enzymatic Oxidation of 5-Methylcytosine in Brain Tumors: Potential Perspectives.

The patterns of 5-methylcytosine (5mC) and its oxidized derivatives, 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine (5caC) are reportedly altered in a range of cancers. Likewise, Wilms' Tumor protein 1 (WT1), a transcription factor essential for urogenital, epicardium, and kidney development exhibits aberrant expression in multiple tumors. Interestingly, WT1 directly interacts with TET proteins that catalyze the enzymatic oxidation of 5mC and exhibits high affinity for 5caC-containing DNA substrates .

Developmental Functions of the Dynamic DNA Methylome and Hydroxymethylome in the Mouse and Zebrafish: Similarities and Differences.

5-methylcytosine (5mC) is the best understood DNA modification and is generally believed to be associated with repression of gene expression. Over the last decade, sequentially oxidized forms of 5mC (oxi-mCs) have been discovered within the genomes of vertebrates. Their discovery was accompanied by that of the ten-eleven translocation (TET) methylcytosine dioxygenases, the enzymes that catalyze the formation of the oxi-mCs.

Immunostaining for DNA Modifications: Computational Analysis of Confocal Images.

For several decades, 5-methylcytosine (5mC) has been thought to be the only DNA modification with a functional significance in metazoans. The discovery of enzymatic oxidation of 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) as well as detection of N6-methyladenine (6mA) in the DNA of multicellular organisms provided additional degrees of complexity to the epigenetic research. According to a growing body of experimental evidence, these novel DNA modifications may play specific roles in different cellular and developmental processes.

Dynamics of 5-carboxylcytosine during hepatic differentiation: Potential general role for active demethylation by DNA repair in lineage specification.

Patterns of DNA methylation (5-methylcytosine, 5mC) are rearranged during differentiation contributing to the regulation of cell type-specific gene expression. TET proteins oxidize 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). Both 5fC and 5caC can be recognized and excised from DNA by thymine-DNA glycosylase (TDG) followed by the subsequent incorporation of unmodified cytosine into the abasic site via the base excision repair (BER) pathway.

Medulloblastoma and ependymoma cells display increased levels of 5-carboxylcytosine and elevated expression.

Background: Alteration of DNA methylation (5-methylcytosine, 5mC) patterns represents one of the causes of tumorigenesis and cancer progression. Tet proteins can oxidise 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine and 5-carboxylcytosine (5caC). Although the roles of these oxidised forms of 5mC (oxi-mCs) in cancer pathogenesis are still largely unknown, there are indications that they may be involved in the mechanisms of malignant transformation.

Detection of Modified Forms of Cytosine Using Sensitive Immunohistochemistry.

Methylation of cytosine bases (5-methylcytosine, 5mC) occurring in vertebrate genomes is usually associated with transcriptional silencing. 5-hydroxylmethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) are the recently discovered modified cytosine bases produced by enzymatic oxidation of 5mC, whose biological functions remain relatively obscure. A number of approaches ranging from biochemical to antibody based techniques have been employed to study the genomic distribution and global content of these modifications in various biological systems.

5-Carboxylcytosine levels are elevated in human breast cancers and gliomas.

Background: DNA methylation (5-methylcytosine (5mC)) patterns are often altered in cancers. Ten-eleven translocation (Tet) proteins oxidise 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). In addition to their presumptive specific biological roles, these oxidised forms of 5mC may serve as intermediates in demethylation process.

A B-cell targeting virus disrupts potentially protective genomic methylation patterns in lymphoid tissue by increasing global 5-hydroxymethylcytosine levels.

The mechanisms by which viruses modulate the immune system include changes in host genomic methylation. 5-hydroxymethylcytosine (5hmC) is the catalytic product of the Tet (Ten-11 translocation) family of enzymes and may serve as an intermediate of DNA demethylation. Recent reports suggest that 5hmC may confer consequences on cellular events including the pathogenesis of disease; in order to explore this possibility further we investigated both 5-methylcytosine (5mC) and 5hmC levels in healthy and diseased chicken bursas of Fabricius.