The Proteins of mRNA Modification: Writers, Readers, and Erasers.

Over the past decade, mRNA modifications have emerged as important regulators of gene expression control in cells. Fueled in large part by the development of tools for detecting RNA modifications transcriptome wide, researchers have uncovered a diverse epitranscriptome that serves as an additional layer of gene regulation beyond simple RNA sequence. Here, we review the proteins that write, read, and erase these marks, with a particular focus on the most abundant internal modification, N6-methyladenosine (m6A). We first describe the discovery of the key enzymes that deposit and remove m6A and other modifications and discuss how our understanding of these proteins has shaped our views of modification dynamics. We then review current models for the function of m6A reader proteins and how our knowledge of these proteins has evolved. Finally, we highlight important future directions for the field and discuss key questions that remain unanswered.

scDART-seq reveals distinct m6A signatures and mRNA methylation heterogeneity in single cells.

N6-methyladenosine (m6A) is an abundant RNA modification that plays critical roles in RNA regulation and cellular function. Global m6A profiling has revealed important aspects of m6A distribution and function, but to date such studies have been restricted to large populations of cells. Here, we develop a method to identify m6A sites transcriptome-wide in single cells. We uncover surprising heterogeneity in the presence and abundance of m6A sites across individual cells and identify differentially methylated mRNAs across the cell cycle. Additionally, we show that cellular subpopulations can be distinguished based on their RNA methylation signatures, independent from gene expression. These studies reveal fundamental features of m6A that have been missed by m6A profiling of bulk cells and suggest the presence of cell-intrinsic mechanisms for m6A deposition.

Thioridazine inhibits self-renewal in breast cancer cells via DRD2-dependent STAT3 inhibition, but induces a G1 arrest independent of DRD2.

Thioridazine is an antipsychotic that has been shown to induce cell death and inhibit self-renewal in a broad spectrum of cancer cells. The mechanisms by which these effects are mediated are currently unknown but are presumed to result from the inhibition of dopamine receptor 2 (DRD2). Here we show that the self-renewal of several, but not all, triple-negative breast cancer cell lines is inhibited by thioridazine. The inhibition of self-renewal by thioridazine in these cells is mediated by DRD2 inhibition. Further, we demonstrate that DRD2 promotes self-renewal in these cells via a STAT3- and IL-6-dependent mechanism. We also show that thioridazine induces a G1 arrest and a loss in cell viability in all tested cell lines. However, the reduction in proliferation and cell viability is independent of DRD2 and STAT3. Our results indicate that although there are cell types in which DRD2 inhibition results in inhibition of STAT3 and self-renewal, the dramatic block in cancer cell proliferation across many cell lines caused by thioridazine treatment is independent of DRD2 inhibition.

Studying m6A in the brain: a perspective on current methods, challenges, and future directions.

A major mechanism of post-transcriptional RNA regulation in cells is the addition of chemical modifications to RNA nucleosides, which contributes to nearly every aspect of the RNA life cycle. N6-methyladenosine (m6A) is a highly prevalent modification in cellular mRNAs and non-coding RNAs, and it plays important roles in the control of gene expression and cellular function. Within the brain, proper regulation of m6A is critical for neurodevelopment, learning and memory, and the response to injury, and m6A dysregulation has been implicated in a variety of neurological disorders. Thus, understanding m6A and how it is regulated in the brain is important for uncovering its roles in brain function and potentially identifying novel therapeutic pathways for human disease. Much of our knowledge of m6A has been driven by technical advances in the ability to map and quantify m6A sites. Here, we review current technologies for characterizing m6A and highlight emerging methods. We discuss the advantages and limitations of current tools as well as major challenges going forward, and we provide our perspective on how continued developments in this area can propel our understanding of m6A in the brain and its role in brain disease.

Single-cell m6A profiling in the mouse brain uncovers cell type-specific RNA methylomes and age-dependent differential methylation.

N6-methyladenosine (m6A) is an abundant mRNA modification in the brain that has important roles in neurodevelopment and brain function. However, because of technical limitations, global profiling of m6A sites within the individual cell types that make up the brain has not been possible. Here, we develop a mouse model that enables transcriptome-wide m6A detection in any tissue of interest at single-cell resolution. We use these mice to map m6A across different brain regions and within single cells of the mouse cortex and discover a high degree of shared methylation across brain regions and cell types. However, we also identify a small number of differentially methylated mRNAs in neurons that encode important regulators of neuronal signaling, and we discover that microglia have lower levels of m6A than other cell types. Finally, we perform single-cell m6A mapping in aged mice and identify many transcripts with age-dependent changes in m6A.

The Drosophila Nab2 RNA binding protein inhibits m6A methylation and male-specific splicing of Sex lethal transcript in female neuronal tissue.

The Drosophila polyadenosine RNA binding protein Nab2, which is orthologous to a human protein lost in a form of inherited intellectual disability, controls adult locomotion, axon projection, dendritic arborization, and memory through a largely undefined set of target RNAs. Here, we show a specific role for Nab2 in regulating splicing of ~150 exons/introns in the head transcriptome and focus on retention of a male-specific exon in the sex determination factor Sex-lethal (Sxl) that is enriched in female neurons. Previous studies have revealed that this splicing event is regulated in females by N6-methyladenosine (m6A) modification by the Mettl3 complex. At a molecular level, Nab2 associates with Sxl pre-mRNA in neurons and limits Sxl m6A methylation at specific sites. In parallel, reducing expression of the Mettl3, Mettl3 complex components, or the m6A reader Ythdc1 rescues mutant phenotypes in Nab2 flies. Overall, these data identify Nab2 as an inhibitor of m6A methylation and imply significant overlap between Nab2 and Mettl3 regulated RNAs in neuronal tissue.

Detection of m6A in single cultured cells using scDART-seq.

Most techniques for mapping m6A-methylated RNAs transcriptome-wide require large amounts of RNA and have been limited to bulk cells and tissues. Here, we provide a detailed protocol for the identification of m6A sites in single HEK293T cells using single-cell DART-seq (scDART-seq). The protocol details how to generate cell lines with inducible expression of the APOBEC1-YTH transgene and the use of important controls for minimizing false positives. We also describe the bioinformatic analysis to identify m6A sites. For complete details on the use and execution of this protocol, please refer to Tegowski et al. (2022).

Detecting m6A with In Vitro DART-Seq.

Recent studies have uncovered that cellular mRNAs contain a diverse epitranscriptome comprising chemically modified bases which play important roles in gene expression regulation. Among these is m6A, which is a highly prevalent modification that contributes to several aspects of RNA regulation and cellular function. Traditional methods for m6A profiling have used m6A antibodies to immunoprecipitate methylated RNAs. Although powerful, such methods require high amounts of input material. Recently, we developed DART-seq, an antibody-free method for m6A profiling from low-input RNA samples. DART-seq relies on deamination of cytidines that invariably follow m6A sites and can be performed using a simple in vitro assay with only 50 ng of total RNA. Here, we describe the in vitro DART method and present a detailed protocol for highly sensitive m6A profiling from any RNA sample of interest.

Selective Effects of Thioridazine on Self-Renewal of Basal-Like Breast Cancer Cells.

Several recent publications demonstrated that DRD2-targeting antipsychotics such as thioridazine induce proliferation arrest and apoptosis in diverse cancer cell types including those derived from brain, lung, colon, and breast. While most studies show that 10-20 µM thioridazine leads to reduced proliferation or increased apoptosis, here we show that lower doses of thioridazine (1-2 µM) target the self-renewal of basal-like breast cancer cells, but not breast cancer cells of other subtypes. We also show that all breast cancer cell lines tested express DRD2 mRNA and protein, regardless of thioridazine sensitivity. Further, DRD2 stimulation with quinpirole, a DRD2 agonist, promotes self-renewal, even in cell lines in which thioridazine does not inhibit self-renewal. This suggests that DRD2 is capable of promoting self-renewal in these cell lines, but that it is not active. Further, we show that dopamine can be detected in human and mouse breast tumor samples. This observation suggests that dopamine receptors may be activated in breast cancers, and is the first time to our knowledge that dopamine has been directly detected in human breast tumors, which could inform future investigation into DRD2 as a therapeutic target for breast cancer.

Noncanonical NF-κB in Cancer.

The NF-κB pathway is a critical regulator of immune responses and is often dysregulated in cancer. Two NF-κB pathways have been described to mediate these responses, the canonical and the noncanonical. While understudied compared to the canonical NF-κB pathway, noncanonical NF-κB and its components have been shown to have effects, usually protumorigenic, in many different cancer types. Here, we review noncanonical NF-κB pathways and discuss its important roles in promoting cancer. We also discuss alternative NF-κB-independent functions of some the components of noncanonical NF-κB signaling. Finally, we discuss important crosstalk between canonical and noncanonical signaling, which blurs the two pathways, indicating that understanding the full picture of NF-κB regulation is critical to deciphering how this broad pathway promotes oncogenesis.