No abstract

The N6-methyladenosine (m6A) modification is the most prevalent post-transcriptional mRNA modification, regulating mRNA decay and splicing. It plays a major role during normal development, differentiation, and disease progression. The modification is regulated by a set of writer, eraser, and reader proteins. The YTH domain family of proteins consists of three homologous m6A-binding proteins, Ythdf1, Ythdf2, and Ythdf3, which were suggested to have different cellular functions. However, their sequence similarity and their tendency to bind the same targets suggest that they may have overlapping roles. We systematically knocked out (KO) the Mettl3 writer, each of the Ythdf readers, and the three readers together (triple-KO). We then estimated the effect in vivo in mouse gametogenesis, postnatal viability, and in vitro in mouse embryonic stem cells (mESCs). In gametogenesis, Mettl3-KO severity is increased as the deletion occurs earlier in the process, and Ythdf2 has a dominant role that cannot be compensated by Ythdf1 or Ythdf3, due to differences in readers’ expression pattern across different cell types, both in quantity and in spatial location. Knocking out the three readers together and systematically testing viable offspring genotypes revealed a redundancy in the readers’ role during early development that is Ythdf1/2/3 gene dosage-dependent. Finally, in mESCs there is compensation between the three Ythdf reader proteins, since the resistance to differentiate and the significant effect on mRNA decay occur only in the triple-KO cells and not in the single KOs. Thus, we suggest a new model for the Ythdf readers function, in which there is profound dosage-dependent redundancy when all three readers are equivalently coexpressed in the same cell types.

Summary Mutations in superoxide dismutase (SOD1) cause amyotrophic lateral sclerosis (ALS), a neurodegenerative disease characterized by loss of motor neurons and accompanied by accumulation of misfolded SOD1 onto the cytoplasmic faces of intracellular organelles, including mitochondria and endoplasmic reticulum (ER). Using inhibition of misfolded SOD1 deposition onto mitochondria as an assay, a chaperone activity abundant in non-neuronal tissues is now purified and identified to be the multifunctional macrophage migration inhibitory factor (MIF), whose activities include an ATP-independent protein folding chaperone. Purified MIF is shown to directly inhibit mutant SOD1 misfolding. Elevating MIF in neuronal cells suppresses accumulation of misfolded SOD1 and its association with mitochondria and ER and extends survival of mutant SOD1-expressing motor neurons. Accumulated MIF protein is identified to be low in motor neurons, implicating correspondingly low chaperone activity as a component of vulnerability to mutant SOD1 misfolding and supporting therapies to enhance intracellular MIF chaperone activity.

Summary Isolating human MEK/ERK signaling-independent pluripotent stem cells (PSCs) with naive pluripotency characteristics while maintaining differentiation competence and (epi)genetic integrity remains challenging. Here, we engineer reporter systems that allow the screening for defined conditions that induce molecular and functional features of human naive pluripotency. Synergistic inhibition of WNT/β-CATENIN, protein kinase C (PKC), and SRC signaling consolidates the induction of teratoma-competent naive human PSCs, with the capacity to differentiate into trophoblast stem cells (TSCs) and extraembryonic naive endodermal (nEND) cells in vitro . Divergent signaling and transcriptional requirements for boosting naive pluripotency were found between mouse and human. P53 depletion in naive hPSCs increased their contribution to mouse-human cross-species chimeric embryos upon priming and differentiation. Finally, MEK/ERK inhibition can be substituted with the inhibition of NOTCH/RBPj, which induces alternative naive-like hPSCs with a diminished risk for deleterious global DNA hypomethylation. Our findings set a framework for defining the signaling foundations of human naive pluripotency.

Mutations in superoxide dismutase (SOD1) cause amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease caused by the progressive loss of motor neurons in the brain and spinal cord. It has been suggested that toxicity of mutant SOD1 results from its misfolding, however, it is yet unclear why misfolded SOD1 accumulates specifically within motor neurons. We recently demonstrated that macrophage migration inhibitory factor (MIF)—a multifunctional protein with cytokine/chemokine activity and cytosolic chaperone-like properties—inhibits the accumulation of misfolded SOD1. Here, we show that MIF inhibits mutant SOD1 nuclear clearance when overexpressed in motor neuron-like NSC-34 cells. In addition, MIF alters the typical SOD1 amyloid aggregation pathway in vitro, and, instead, promotes the formation of disordered aggregates, as measured by Thioflavin T (ThT) assay and transmission electron microscopy (TEM) imaging. Moreover, we report that MIF reduces the toxicity of misfolded SOD1 by directly interacting with it, and that the chaperone function and protective effect of MIF in neuronal cultures do not require its intrinsic catalytic activities. Importantly, we report that the locked-trimeric MIFN110C mutant, which exhibits strongly impaired CD74-mediated cytokine functions, has strong chaperone activity, dissociating, for the first time, these two cellular functions. Altogether, our study implicates MIF as a potential therapeutic candidate in the treatment of ALS.