The epitranscriptome in stem cell biology and neural development

Abstract

The blossoming field of epitranscriptomics has recently garnered attention across many fields by findings that chemical modifications on RNA have immense biological consequences. Methylation of nucleotides in RNA, including N6-methyladenosine (m⁶A), 2-O-dimethyladenosine (m⁶A_m), N1-methyladenosine (m¹A), 5-methylcytosine (m⁵C), and isomerization of uracil to pseudouridine (Ψ), have the potential to alter RNA processing events and contribute to developmental processes and different diseases. Though the abundance and roles of some RNA modifications remain contentious, the epitranscriptome is thought to be especially relevant in stem cell biology and neurobiology. In particular, m⁶A occurs at the highest levels in the brain and plays major roles in embryonic stem cell differentiation, brain development, and neurodevelopmental disorders. However, studies in these areas have reported conflicting results on epitranscriptomic regulation of stem cell pluripotency and mechanisms in neural development. In this review we provide an overview of the current understanding of several RNA modifications and disentangle the various findings on epitranscriptomic regulation of stem cell biology and neural development.

Keywords: Brain development; Brain disorders; Epitranscriptome; Stem cells; m(6)A.

Figure 1.. Overview of common epitranscriptomic marks.

This pinwheel shows the current knowledge for m⁶A, m⁷G, m¹A, Ψ, m⁶A_m, and m⁵C (from top, clockwise). Each slice shows the known writer proteins, known eraser enzymes, known reader proteins and downstream functions of the epitranscriptomic mark.

Figure 2.. The epitranscriptome in embryonic development and ESCs.

Top: Marks that have been studied in the developing embryo include m⁶A on mRNA to promote mRNA degradation, m⁷G on tRNA to promote translation, METTL16-mediated addition of m⁶A to ncRNA like Mat2a, m⁵C on mRNA to promote RNA Binding Protein (RBP) mRNA binding, and m⁵C on mitochondrial tRNA to regulate germ layer specification. Bottom: The most-studied modification in embryonic stem cells (ESCs) is m⁶A. While several conflicting studies have reported different phenotypes after Mettl3 knockdown, the current consensus is that m⁶A functions to destabilize whichever gene transcripts are modified at the time. In naïve ESCs purified from preimplantation blastocysts, m⁶A is primarily added to pluripotency-promoting gen transcripts. Therefore loss of m⁶A improves self-renewal and impairs differentiation. In contrast, in primed ESCs derived from post-implantation blastocysts, m⁶A is primarily added to lineage-commitment gene transcripts, so loss of m⁶A promotes differentiation and impairs self-renewal.

Figure 3.. m⁶A in neural development.

Top: In the cortex, loss of m⁶A elongates the timeframe of cortical neurogenesis such that the postnatal brain is still generating upper-layer neurons. This is accomplished through altered mRNA degradation rates of key pluripotency and fate-determining gene transcripts. Middle: In the cerebellum, loss of m⁶A causes disorganization of the Purkinje cell layer (PCL) into the inner granule cell layer (IGL). IGL cells also exhibit a higher rate of apoptosis, resulting in fewer total IGL cells. Mechanistically, m⁶A has been shown to promote alternative splicing and mRNA degradation in the cerebellum. Bottom: In hippocampal adult neurogenesis, in vitro studies found that loss of m⁶A impairs self-renewal and neurogenesis, but the mechanism of action remains unclear.

Figure 4.. The epitranscriptome in neural disorders.

Top left: Fragile X Syndrome is correlated with m⁶A in that the central protein involved in Fragile X, FMRP, can bind m⁶A to promote nuclear export of modified mRNAs through interaction with CRM1, a component of the nuclear pore complex. Loss of FMRP in mice impairs this export and causes decreased levels of embryonic neurogenesis and NSC proliferation. Top right: PUS3 mutations in humans significantly correlate with intellectual disability and microcephaly. Though the exact mechanism is unknown, mutations in PUS3 cause significantly lower levels of pseudouridine addition on tRNA relative to wildtype controls. Bottom left: PUS7 mutations identified in humans and validated in drosophila cause increased aggression, speech delay, intellectual disability, and microcephaly through decreased levels of pseudouridine on both tRNA and mRNA. Bottom right: METTL5 mutation in humans and validated in zebrafish and mice cause intellectual disability and microcephaly through decreased levels of m⁶A on 18S ribosomal RNA.