Thioguanosine Conversion Enables mRNA-Lifetime Evaluation by RNA Sequencing Using Double Metabolic Labeling (TUC-seq DUAL).

Temporal information about cellular RNA populations is essential to understand the functional roles of RNA. We have developed the hydrazine/NH4 Cl/OsO4 -based conversion of 6-thioguanosine (6sG) into A', where A' constitutes a 6-hydrazino purine derivative. A' retains the Watson-Crick base-pair mode and is efficiently decoded as adenosine in primer extension assays and in RNA sequencing. Because 6sG is applicable to metabolic labeling of freshly synthesized RNA and because the conversion chemistry is fully compatible with the conversion of the frequently used metabolic label 4-thiouridine (4sU) into C, the combination of both modified nucleosides in dual-labeling setups enables high accuracy measurements of RNA decay. This approach, termed TUC-seq DUAL, uses the two modified nucleosides in subsequent pulses and their simultaneous detection, enabling mRNA-lifetime evaluation with unprecedented precision.

RNA cytosine methyltransferase Nsun3 regulates embryonic stem cell differentiation by promoting mitochondrial activity.

Chemical modifications of RNA have been attracting increasing interest because of their impact on RNA fate and function. Therefore, the characterization of enzymes catalyzing such modifications is of great importance. The RNA cytosine methyltransferase NSUN3 was recently shown to generate 5-methylcytosine in the anticodon loop of mitochondrial tRNAMet. Further oxidation of this position is required for normal mitochondrial translation and function in human somatic cells. Because embryonic stem cells (ESCs) are less dependent on oxidative phosphorylation than somatic cells, we examined the effects of catalytic inactivation of Nsun3 on self-renewal and differentiation potential of murine ESCs. We demonstrate that Nsun3-mutant cells show strongly reduced mt-tRNAMet methylation and formylation as well as reduced mitochondrial translation and respiration. Despite the lower dependence of ESCs on mitochondrial activity, proliferation of mutant cells was reduced, while pluripotency marker gene expression was not affected. By contrast, ESC differentiation was skewed towards the meso- and endoderm lineages at the expense of neuroectoderm. Wnt3 was overexpressed in early differentiating mutant embryoid bodies and in ESCs, suggesting that impaired mitochondrial function disturbs normal differentiation programs by interfering with cellular signalling pathways. Interestingly, basal levels of reactive oxygen species (ROS) were not altered in ESCs, but Nsun3 inactivation attenuated induction of mitochondrial ROS upon stress, which may affect gene expression programs upon differentiation. Our findings not only characterize Nsun3 as an important regulator of stem cell fate but also provide a model system to study the still incompletely understood interplay of mitochondrial function with stem cell pluripotency and differentiation.

A novel role for the histone acetyltransferase Hat1 in the CENP-A/CID assembly pathway in Drosophila melanogaster.

The incorporation of CENP-A into centromeric chromatin is an essential prerequisite for kinetochore formation. Yet, the molecular mechanisms governing this process are surprisingly divergent in different organisms. While CENP-A loading mechanisms have been studied in some detail in mammals, there are still large gaps to our understanding of CENP-A/Cid loading pathways in Drosophila. Here, we report on the characterization and delineation of at least three different CENP-A preloading complexes in Drosophila. Two complexes contain the CENP-A chaperones CAL1, FACT and/or Caf1/Rbap48. Notably, we identified a novel complex consisting of the histone acetyltransferase Hat1, Caf1 and CENP-A/H4. We show that Hat1 is required for proper CENP-A loading into chromatin, since knock-down in S2 cells leads to reduced incorporation of newly synthesized CENP-A. In addition, we demonstrate that CENP-A/Cid interacts with the HAT1 complex via an N-terminal region, which is acetylated in cytoplasmic but not in nuclear CENP-A. Since Hat1 is not responsible for acetylation of CENP-A/Cid, these results suggest a histone acetyltransferase activity-independent escort function for Hat1. Thus, our results point toward intriguing analogies between the complex processing pathways of newly synthesized CENP-A and canonical histones.

Thiouridine-to-Cytidine Conversion Sequencing (TUC-Seq) to Measure mRNA Transcription and Degradation Rates.

The study of RNA dynamics, specifically RNA transcription and decay rates, has gained increasing attention in recent years because various mechanisms have been discovered that affect mRNA half-life, thereby ultimately controlling protein output. Therefore, there is a need for methods enabling minimally invasive, simple and high-throughput determination of RNA stability that can be applied to determine RNA transcription and decay rates in cells and organisms. We have recently developed a protocol which we named TUC-seq to directly distinguish newly synthesized transcripts from the preexisting pool of transcripts by metabolic labeling of nascent RNAs with 4-thiouridine (4sU) followed by osmium tetroxide-mediated conversion of 4sU to cytidine (C) and direct sequencing. In contrast to other related methods (SLAM-seq, TimeLapse-seq), TUC-seq converts 4sU to a native C instead of an alkylated or otherwise modified nucleoside derivative. TUC-seq can be applied to any cell type that is amenable to 4sU labeling. By employing different labeling strategies (pulse or pulse-chase labeling), it is suitable for a broad field of applications and provides a fast and highly efficient means to determine mRNA transcription and decay rates.

Publisher Correction: RNA modification: Getting a hold on cytosine methylation in mRNA.

In the version of this article initially published, the author order (Liu, J., Huang, T., Chen, W., Gu, N. & Zhang, R.) for the linked Resource (ref. 7) was incorrect, and all named citations of that reference in the text ("Liu et al.") were incorrect. The correct author order is "Huang, T., Chen, W., Liu, J., Gu, N. & Zhang, R." and all named citations should reflect that ("Huang et al."). The error has been corrected in the HTML and PDF versions of the article.

The dynamic RNA modification 5-methylcytosine and its emerging role as an epitranscriptomic mark.

It is a well-known fact that RNA is the target of a plethora of modifications which currently amount to over a hundred. The vast majority of these modifications was observed in the two most abundant classes of RNA, rRNA and tRNA. With the recent advance in mapping technologies, modifications have been discovered also in mRNA and in less abundant non-coding RNA species. These developments have sparked renewed interest in elucidating the nature and functions of those "epitransciptomic" modifications in RNA. N6-methyladenosine (m6 A) is the best understood and most frequent mark of mRNA with demonstrated functions ranging from pre-mRNA processing, translation, miRNA biogenesis to mRNA decay. By contrast, much less research has been conducted on 5-methylcytosine (m5C), which was detected in tRNAs and rRNAs and more recently in poly(A)RNAs. In this review, we discuss recent developments in the discovery of m5C RNA methylomes, the functions of m5C as well as the proteins installing, translating and manipulating this modification. Although our knowledge about m5C in RNA transcripts is just beginning to consolidate, it has become clear that cytosine methylation represents a powerful mechanistic strategy to regulate cellular processes on an epitranscriptomic level. This article is categorized under: RNA Processing > RNA Editing and Modification RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications RNA Processing > tRNA Processing RNA Turnover and Surveillance > Regulation of RNA Stability.

Bisulfite Sequencing of RNA for Transcriptome-Wide Detection of 5-Methylcytosine.

A powerful method to determine the methylation status of specific cytosine residues within RNA is bisulfite sequencing. In combination with high-throughput sequencing methods cytosine methylation can be determined at nucleotide resolution on a transcriptome-wide level. Nevertheless, several critical aspects need to be considered before starting such a project. Below we describe a detailed step-by-step protocol for planning and performing a transcriptome-wide bisulfite sequencing experiment and subsequent data analysis to determine methyl-cytosine in poly(A)RNA from cells and tissues.

Distinct 5-methylcytosine profiles in poly(A) RNA from mouse embryonic stem cells and brain.

BACKGROUND: Recent work has identified and mapped a range of posttranscriptional modifications in mRNA, including methylation of the N6 and N1 positions in adenine, pseudouridylation, and methylation of carbon 5 in cytosine (m5C). However, knowledge about the prevalence and transcriptome-wide distribution of m5C is still extremely limited; thus, studies in different cell types, tissues, and organisms are needed to gain insight into possible functions of this modification and implications for other regulatory processes. RESULTS: We have carried out an unbiased global analysis of m5C in total and nuclear poly(A) RNA of mouse embryonic stem cells and murine brain. We show that there are intriguing differences in these samples and cell compartments with respect to the degree of methylation, functional classification of methylated transcripts, and position bias within the transcript. Specifically, we observe a pronounced accumulation of m5C sites in the vicinity of the translational start codon, depletion in coding sequences, and mixed patterns of enrichment in the 3' UTR. Degree and pattern of methylation distinguish transcripts modified in both embryonic stem cells and brain from those methylated in either one of the samples. We also analyze potential correlations between m5C and micro RNA target sites, binding sites of RNA binding proteins, and N6-methyladenosine. CONCLUSION: Our study presents the first comprehensive picture of cytosine methylation in the epitranscriptome of pluripotent and differentiated stages in the mouse. These data provide an invaluable resource for future studies of function and biological significance of m5C in mRNA in mammals.