N6-methyladenosine promotes induction of ADAR1-mediated A-to-I RNA editing to suppress aberrant antiviral innate immune responses.

Among over 150 distinct RNA modifications, N6-methyladenosine (m6A) and adenosine-to-inosine (A-to-I) RNA editing represent 2 of the most studied modifications on mammalian mRNAs. Although both modifications occur on adenosine residues, knowledge on potential functional crosstalk between these 2 modifications is still limited. Here, we show that the m6A modification promotes expression levels of the ADAR1, which encodes an A-to-I RNA editing enzyme, in response to interferon (IFN) stimulation. We reveal that YTH N6-methyladenosine RNA binding protein 1 (YTHDF1) mediates up-regulation of ADAR1; YTHDF1 is a reader protein that can preferentially bind m6A-modified transcripts and promote translation. Knockdown of YTHDF1 reduces the overall levels of IFN-induced A-to-I RNA editing, which consequently activates dsRNA-sensing pathway and increases expression of various IFN-stimulated genes. Physiologically, YTHDF1 deficiency inhibits virus replication in cells through regulating IFN responses. The A-to-I RNA editing activity of ADAR1 plays important roles in the YTHDF1-dependent IFN responses. Therefore, we uncover that m6A and YTHDF1 affect innate immune responses through modulating the ADAR1-mediated A-to-I RNA editing.

Post-transcriptional repression of circadian component CLOCK regulates cancer-stemness in murine breast cancer cells.

Disruption of the circadian clock machinery in cancer cells is implicated in tumor malignancy. Studies on cancer therapy reveal the presence of heterogeneous cells, including breast cancer stem-like cells (BCSCs), in breast tumors. BCSCs are often characterized by high aldehyde dehydrogenase (ALDH) activity, associated with the malignancy of cancers. In this study, we demonstrated the negative regulation of ALDH activity by the major circadian component CLOCK in murine breast cancer 4T1 cells. The expression of CLOCK was repressed in high-ALDH-activity 4T1, and enhancement of CLOCK expression abrogated their stemness properties, such as tumorigenicity and invasive potential. Furthermore, reduced expression of CLOCK in high-ALDH-activity 4T1 was post-transcriptionally regulated by microRNA: miR-182. Knockout of miR-182 restored the expression of CLOCK, resulted in preventing tumor growth. Our findings suggest that increased expression of CLOCK in BCSCs by targeting post-transcriptional regulation overcame stemness-related malignancy and may be a novel strategy for breast cancer treatments.

Stabilization of ERK-Phosphorylated METTL3 by USP5 Increases m6A Methylation.

N6-methyladenosine (m6A) is the most abundant mRNA modification and is installed by the METTL3-METTL14-WTAP methyltransferase complex. Although the importance of m6A methylation in mRNA metabolism has been well documented recently, regulation of the m6A machinery remains obscure. Through a genome-wide CRISPR screen, we identify the ERK pathway and USP5 as positive regulators of the m6A deposition. We find that ERK phosphorylates METTL3 at S43/S50/S525 and WTAP at S306/S341, followed by deubiquitination by USP5, resulting in stabilization of the m6A methyltransferase complex. Lack of METTL3/WTAP phosphorylation reduces decay of m6A-labeled pluripotent factor transcripts and traps mouse embryonic stem cells in the pluripotent state. The same phosphorylation can also be found in ERK-activated human cancer cells and contribute to tumorigenesis. Our study reveals an unrecognized function of ERK in regulating m6A methylation.

A-to-I RNA editing enzyme ADAR2 regulates light-induced circadian phase-shift.

In mammals, the central circadian clock is located in the suprachiasmatic nucleus (SCN) of the hypothalamus and it orchestrates peripheral clocks in the whole body to organize physiological and behavioral rhythms. Light-induced phase-shift of the SCN clock enables synchronization of the circadian clock system with 24-h environmental light/dark cycle. We previously found that adenosine deaminase acting on RNA 2 (Adar2), an A-to-I RNA editing enzyme catalyzing rhythmic A-to-I RNA editing, governs a wide range of mRNA rhythms in the mouse liver and regulates the circadian behavior. In brain, ADAR2-mediated A-to-I RNA editing was reported to occur in various transcripts encoding ion channels and neurotransmitter receptors, which could influence neuronal function of the SCN. Here we show that ADAR2 plays a crucial role for light-induced phase-shift of the circadian clock. Intriguingly, exposure of Adar2-knockout mice to a light pulse at late night caused an aberrant phase-advance of the locomotor rhythms. By monitoring the bioluminescence rhythms of the mutant SCN slices, we found that a phase-advance induced by treatment with pituitary adenylyl cyclase-activating polypeptide (PACAP) was markedly attenuated. The present study suggests that A-to-I RNA editing in the SCN regulates a proper phase response to light in the mouse circadian system.

ADARB1 catalyzes circadian A-to-I editing and regulates RNA rhythm.

It has been proposed that the CLOCK-ARNTL (BMAL1) complex drives circadian transcription of thousands of genes, including Per and Cry family genes that encode suppressors of CLOCK-ARNTL-dependent transcription. However, recent studies demonstrated that 70-80% of circadian-oscillating mRNAs have no obvious rhythms in their de novo transcription, indicating the potential importance of post-transcriptional regulation. Our CLOCK-ChIP-seq analysis identified rhythmic expression of adenosine deaminase, RNA-specific, B1 (Adarb1, also known as Adar2), an adenosine-to-inosine (A-to-I) RNA-editing enzyme. RNA-seq showed circadian rhythms of ADARB1-mediated A-to-I editing in a variety of transcripts. In Adarb1-knockout mice, rhythms of large populations of mRNA were attenuated, indicating a profound impact of ADARB1-mediated A-to-I editing on RNA rhythms. Furthermore, Adarb1-knockout mice exhibited short-period rhythms in locomotor activity and gene expression. These phenotypes were associated with abnormal accumulation of CRY2. The present study identifies A-to-I RNA editing as a key mechanism of post-transcriptional regulation in the circadian clockwork.

CLOCK-controlled polyphonic regulation of circadian rhythms through canonical and noncanonical E-boxes.

In mammalian circadian clockwork, the CLOCK-BMAL1 complex binds to DNA enhancers of target genes and drives circadian oscillation of transcription. Here we identified 7,978 CLOCK-binding sites in mouse liver by chromatin immunoprecipitation-sequencing (ChIP-Seq), and a newly developed bioinformatics method, motif centrality analysis of ChIP-Seq (MOCCS), revealed a genome-wide distribution of previously unappreciated noncanonical E-boxes targeted by CLOCK. In vitro promoter assays showed that CACGNG, CACGTT, and CATG(T/C)G are functional CLOCK-binding motifs. Furthermore, we extensively revealed rhythmically expressed genes by poly(A)-tailed RNA-Seq and identified 1,629 CLOCK target genes within 11,926 genes expressed in the liver. Our analysis also revealed rhythmically expressed genes that have no apparent CLOCK-binding site, indicating the importance of indirect transcriptional and posttranscriptional regulations. Indirect transcriptional regulation is represented by rhythmic expression of CLOCK-regulated transcription factors, such as Krüppel-like factors (KLFs). Indirect posttranscriptional regulation involves rhythmic microRNAs that were identified by small-RNA-Seq. Collectively, CLOCK-dependent direct transactivation through multiple E-boxes and indirect regulations polyphonically orchestrate dynamic circadian outputs.

A biochemical landscape of A-to-I RNA editing in the human brain transcriptome.

Inosine is an abundant RNA modification in the human transcriptome and is essential for many biological processes in modulating gene expression at the post-transcriptional level. Adenosine deaminases acting on RNA (ADARs) catalyze the hydrolytic deamination of adenosines to inosines (A-to-I editing) in double-stranded regions. We previously established a biochemical method called "inosine chemical erasing" (ICE) to directly identify inosines on RNA strands with high reliability. Here, we have applied the ICE method combined with deep sequencing (ICE-seq) to conduct an unbiased genome-wide screening of A-to-I editing sites in the transcriptome of human adult brain. Taken together with the sites identified by the conventional ICE method, we mapped 19,791 novel sites and newly found 1258 edited mRNAs, including 66 novel sites in coding regions, 41 of which cause altered amino acid assignment. ICE-seq detected novel editing sites in various repeat elements as well as in short hairpins. Gene ontology analysis revealed that these edited mRNAs are associated with transcription, energy metabolism, and neurological disorders, providing new insights into various aspects of human brain functions.

Single-particle tracking of quantum dot-conjugated prion proteins inside yeast cells.

Yeast is a model eukaryote with a variety of biological resources. Here we developed a method to track a quantum dot (QD)-conjugated protein in the budding yeast Saccharomyces cerevisiae. We chemically conjugated QDs with the yeast prion Sup35, incorporated them into yeast spheroplasts, and tracked the motions by conventional two-dimensional or three-dimensional tracking microscopy. The method paves the way toward the individual tracking of proteins of interest inside living yeast cells.