m6A governs length-dependent enrichment of mRNAs in stress granules.

Stress granules are biomolecular condensates composed of protein and mRNA. One feature of stress granule-enriched mRNAs is that they are often longer than average. Another feature of stress granule-enriched mRNAs is that they often contain multiple N6-methyladenosine (m6A) residues. m6A is bound by the YTHDF proteins, creating mRNA-protein complexes that partition into stress granules in mammalian cells. Here we show that length-dependent enrichment of mRNAs in stress granules is mediated by m6A. Long mRNAs often contain one or more long exons, which are preferential sites of m6A formation. In mammalian cells lacking m6A, long mRNAs no longer show preferential stress granule enrichment. Furthermore, we show that m6A abundance more strongly predicts which short or long mRNAs are enriched in stress granules, rather than length alone. Thus, mRNA length correlates with mRNA enrichment in stress granules owing to the high prevalence of m6A in long mRNAs.

miR-17 acts as a tumor suppressor by negatively regulating the miR-17-92 cluster.

Anaplastic thyroid cancer (ATC) is an aggressive, highly metastatic cancer that expresses high levels of the microRNA (miR)-17-92 cluster. We employ an miR inhibitor system to study the function of the different miRs within the miR-17-92 cluster based on seed sequence homology in the ATC SW579 cell line. While three of the four miR-17-92 families were oncogenic, we uncovered a novel role for miR-17 as a tumor suppressor in vitro and in vivo. Surprisingly, miR-17 inhibition increased expression of the miR-17-92 cluster and significantly increased the levels of the miR-18a and miR-19a mature miRs. miR-17 inhibition increased expression of the cell cycle activator CCND2, associated with increased cell proliferation and tumor growth in transplanted SW579 cells in xenograft mice. miR-17 regulates MYCN and c-MYC expression in SW579 cells, and the inhibition of miR-17 increased MYCN and c-MYC expression, which increased pri-miR-17-92 transcripts. Thus, inhibition of miR-17 activated the expression of the oncogenic miRs, miR-18a and miR-19a. While many cancers express high levels of miR-17, linking it with tumorigenesis, we demonstrate that miR-17 inhibition does not inhibit thyroid tumor growth in SW579 and MDA-T32 ATC cells but increases expression of the other miR-17-92 family members and genes to induce cancer progression.

Reading, writing and erasing mRNA methylation.

RNA methylation to form N6-methyladenosine (m6A) in mRNA accounts for the most abundant mRNA internal modification and has emerged as a widespread regulatory mechanism that controls gene expression in diverse physiological processes. Transcriptome-wide m6A mapping has revealed the distribution and pattern of m6A in cellular RNAs, referred to as the epitranscriptome. These maps have revealed the specific mRNAs that are regulated by m6A, providing mechanistic links connecting m6A to cellular differentiation, cancer progression and other processes. The effects of m6A on mRNA are mediated by an expanding list of m6A readers and m6A writer-complex components, as well as potential erasers that currently have unclear relevance to m6A prevalence in the transcriptome. Here we review new and emerging methods to characterize and quantify the epitranscriptome, and we discuss new concepts - in some cases, controversies - regarding our understanding of the mechanisms and functions of m6A readers, writers and erasers.

m6A enhances the phase separation potential of mRNA.

N6-methyladenosine (m6A) is the most prevalent modified nucleotide in mRNA1,2, with around 25% of mRNAs containing at least one m6A. Methylation of mRNA to form m6A is required for diverse cellular and physiological processes3. Although the presence of m6A in an mRNA can affect its fate in different ways, it is unclear how m6A directs this process and why the effects of m6A can vary in different cellular contexts. Here we show that the cytosolic m6A-binding proteins-YTHDF1, YTHDF2 and YTHDF3-undergo liquid-liquid phase separation in vitro and in cells. This phase separation is markedly enhanced by mRNAs that contain multiple, but not single, m6A residues. Polymethylated mRNAs act as a multivalent scaffold for the binding of YTHDF proteins, juxtaposing their low-complexity domains and thereby leading to phase separation. The resulting mRNA-YTHDF complexes then partition into different endogenous phase-separated compartments, such as P-bodies, stress granules or neuronal RNA granules. m6A-mRNA is subject to compartment-specific regulation, including a reduction in the stability and translation of mRNA. These studies reveal that the number and distribution of m6A sites in cellular mRNAs can regulate and influence the composition of the phase-separated transcriptome, and suggest that the cellular properties of m6A-modified mRNAs are governed by liquid-liquid phase separation principles.

Irx1 regulates dental outer enamel epithelial and lung alveolar type II epithelial differentiation.

The Iroquois genes (Irx) appear to regulate fundamental processes that lead to cell proliferation, differentiation, and maturation during development. In this report, the Iroquois homeobox 1 (Irx1) transcription factor was functionally disrupted using a LacZ insert and LacZ expression demonstrated stage-specific expression during embryogenesis. Irx1 is highly expressed in the brain, lung, digits, kidney, testis and developing teeth. Irx1 null mice are neonatal lethal and this lethality it due to pulmonary immaturity. Irx1-/- mice show delayed lung maturation characterized by defective surfactant protein secretion and Irx1 marks a population of SP-C expressing alveolar type II cells. Irx1 is specifically expressed in the outer enamel epithelium (OEE), stellate reticulum (SR) and stratum intermedium (SI) layers of the developing tooth. Irx1 mediates dental epithelial cell differentiation in the lower incisors resulting in delayed growth of the lower incisors. Irx1 is specifically and temporally expressed during developmental stages and we have focused on lung and dental development in this report. Irx1+ cells are unique to the development of the incisor outer enamel epithelium, patterning of Lef-1+ and Sox2+ cells as well as a new marker for lung alveolar type II cells. Mechanistically, Irx1 regulates Foxj1 and Sox9 to control cell differentiation during development.