Methods for isolation of messenger RNA from biological samples.

RNA molecules contain many chemical modifications that can regulate a variety of biological processes. Messenger RNA (mRNA) molecules are critical components in the central dogma of molecular biology. The discovery of reversible chemical modifications in eukaryotic mRNA brings forward a new research field in RNA modification-mediated regulation of gene expression. The modifications in mRNA generally exist in low abundance. The use of highly pure mRNA is critical for the confident identification of new modifications as well as for the accurate quantification of existing modifications in mRNA. In addition, isolation of highly pure mRNA is the first step in many biological research studies. Therefore, the methods for isolating highly pure mRNA are important for mRNA-based downstream studies. A variety of methods for isolating mRNA have been developed in the past few decades and new methods continuously emerge. This review focuses on the methodologies and protocols for isolating mRNA populations. In addition, we discuss the advantages and limitations of these methods. We hope this paper will provide a general view of mRNA isolation strategies and facilitate studies that involve mRNA modifications and functions.

Direct decarboxylation of ten-eleven translocation-produced 5-carboxylcytosine in mammalian genomes forms a new mechanism for active DNA demethylation.

DNA cytosine methylation (5-methylcytosine, 5mC) is the most important epigenetic mark in higher eukaryotes. 5mC in genomes is dynamically controlled by writers and erasers. DNA (cytosine-5)-methyltransferases (DNMTs) are responsible for the generation and maintenance of 5mC in genomes. Active demethylation of 5-methylcytosine (5mC) is achieved by ten-eleven translocation (TET) dioxygenase-mediated oxidation of 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). 5fC and 5caC are further processed by thymine DNA glycosylase (TDG)-initiated base excision repair (BER) to restore unmodified cytosines. The TET-TDG-BER pathway could cause the production of DNA strand breaks and therefore jeopardize the integrity of genomes. Here, we investigated the direct decarboxylation of 5caC in mammalian genomes by using metabolic labeling with 2'-fluorinated 5caC (F-5caC) and mass spectrometry analysis. Our results clearly demonstrated the decarboxylation of 5caC occurring in mammalian genomes, which unveiled that, in addition to the TET-TDG-BER pathway, the direct decarboxylation of TET-produced 5caC constituted a new pathway for active demethylation of 5mC in mammalian genomes.