The epitranscriptome landscape of small noncoding RNAs in stem cells

Abstract

Stem cells (SCs) are unique cells that have an inherent ability to self-renew or differentiate. Both fate decisions are strongly regulated at the molecular level via intricate signaling pathways. The regulation of signaling networks promoting self-renewal or differentiation was thought to be largely governed by the action of transcription factors. However, small noncoding RNAs (ncRNAs), such as vault RNAs, and their post-transcriptional modifications (the epitranscriptome) have emerged as additional regulatory layers with essential roles in SC fate decisions. RNA post-transcriptional modifications often modulate RNA stability, splicing, processing, recognition, and translation. Furthermore, modifications on small ncRNAs allow for dual regulation of RNA activity, at both the level of biogenesis and RNA-mediated actions. RNA post-transcriptional modifications act through structural alterations and specialized RNA-binding proteins (RBPs) called writers, readers, and erasers. It is through SC-context RBPs that the epitranscriptome coordinates specific functional roles. Small ncRNA post-transcriptional modifications are today exploited by different mechanisms to facilitate SC translational studies. One mechanism readily being studied is identifying how SC-specific RBPs of small ncRNAs regulate fate decisions. Another common practice of using the epitranscriptome for regenerative applications is using naturally occurring post-transcriptional modifications on synthetic RNA to generate induced pluripotent SCs. Here, we review exciting insights into how small ncRNA post-transcriptional modifications control SC fate decisions in development and disease. We hope, by illustrating how essential the epitranscriptome and their associated proteome are in SCs, they would be considered as novel tools to propagate SCs for regenerative medicine.

Keywords: adult stem cells; cancer stem cells; epigenetics; epitranscriptome; microRNAs; noncoding RNAs.

FIGURE 1

Novel features of the small ncRNA transcriptomes. Here, we show major small ncRNA species alongside their typical abundance in cells, primary localization, major functions, and known epitranscriptome modifications. The positions of major modifications are denoted (colored circles) on representative secondary structures (colored by modification type, according to the side legend). miRNA, micro RNA; ncRNA, noncoding RNA; piRNA, PIWI‐interacting RNA; pRNA, promoter‐associated RNA; snRNA, small nuclear RNA; snoRNA, small nucleolar RNA; tRNA, transfer RNA; VT‐RNA, vault RNA. A to I editing indicates adenosine to inosine editing. ψ, pseudouridylation; D, dihydrouridine; terminal uridylation, addition of one or more uridyl nucleotides at the RNA terminal; 5′Pme2, 5′ terminal phosphate methylation, Nm: 2′‐O ribose methylation; m6A, 6‐methyladenosine; m5C, 5‐methylcytosine; m2G, 2‐methylguanidine; m1A, 1‐methyladenosine; m7G, 7‐methylguanidine; hm5C, 5‐hydroxymethylcytosine and 5‐formylcytosine

FIGURE 2

Major RNA post‐transcriptional modifications of eukaryotic small ncRNAs. The structures of RNA bases, 5′ phosphate, and ribose moieties with major chemical modifications are highlighted. Where relevant, these color conventions are continued throughout subsequent figures. ncRNA, noncoding RNA

FIGURE 3

How the epitranscriptome of small ncRNAs controls RNAi and translation. A, The occurrence and position of RNA modifications placed by writers such as the pseudouridine synthases CBF5 and PUS7 or methyltransferases NSUN2 and DNMT2 in several small ncRNAs (such as snoRNA, tRNA, and VT‐RNA) dictate endonuclease (eg, angiogenin [ANG], Ro‐associated 1 [Rny1], Dicer, or Drosha) processing. In this way, small ncRNA modifications modulate the production of regulatory RNA fragments (eg, snoRNA‐derived sdRNAs, tRNA‐derived tRFs and VT‐RNA‐derived svRNAs), which coordinate stem cell decisions by silencing mRNAs or altering the translational machinery (as observed for certain tRFs). ¹⁰⁶ B, In piRNAs, 3′ terminal ribose methylation by HEN1 orthologues (HENMET in humans) protect against 3′‐5′ endonuclease activity (Nibbler in Drosophila, possibly PARN in humans ¹⁰⁷ ). Conversely, terminal uridylation of Let‐7 miRNAs by TUTases are carefully coordinated as oligouridylation labels pre‐Let‐7 miRNAs for degradation by the endonuclease DIS3L2. C, The METTL3‐mediated deposition of m6A at the 5′ end of pri‐miRNAs promotes engagement with Drosha, thereby facilitating pre‐miRNA synthesis. Conversely, the m6A eraser, FTO (a dioxygenase) removes m6A from pri‐miRNAs, thereby reducing Drosha‐DGCR8 recognition. Such differential processing alters cell's miRNA pool hence adjusting RNAi and, ultimately, stem cell decisions through suppression of transcripts involved in self‐renewal, proliferation, commitment, and differentiation. The delta symbol (Δ) has been used to represent “a difference in.” Annotations for proteins and modifications are colored according to their associated graphic. ? indicates currently unknowns. iPSC, induced pluripotent stem cell; NSC, neuronal stem cell; ncRNA, noncoding RNA; piRNA, PIWI‐interacting RNAs; RNAi, RNA interference; SC, stem cell; snoRNA, small nucleolar RNAs; tRNA, transfer RNA; VT‐RNA, vault RNA

FIGURE 4

The epitranscriptome signatures of stem cell miRNAs in homeostasis and disease. A, Modifications edited by ADAR1 or ADAR2 (adenosine deaminases, which convert adenosine to inosine) on pri‐miRNAs and pre‐miRNAs provide additional layers of control over miRNA biogenesis. A to I editing of pri‐miRNA and pre‐miRNAs flag them for degradation by endonuclease SND1, as has been observed for Let‐7 miRNAs. B, The activity of Let‐7 miRNAs is suppressed by Lin28B (a Let‐7 miRNA‐binding protein). In normal HSC development, Let‐7 miRNA expression is elevated in line with Lin28B reduction. However, in some cancer low levels of Let‐7 caused by ADAR‐editing coincides with high levels of Lin28B (a dysregulation seen in LSCs), which promotes the continuance (or establishment) of proliferation and self‐renewal. C, miR‐21 and miR‐145 comprise an antagonistic regulatory axis (wherein the expression of each miR ultimately results in the suppression of the other). As certain stem cells (eg, HSCs and ESCs) undergo differentiation, miR‐21 levels are decrease in favor of increasing the expression of miR‐145 and, in doing so, lose their self‐renewal capacity (indicated by circular arrows). Both miR‐21 and miR‐145 coordinate self‐renewal decisions by modulating K‐Ras activities (K‐Ras activation is promoted by miR‐21, whereas inhibited by miR‐145). The BCDIN3D‐dependent installation of 5′ phosphate methyl groups on pre‐miR‐21 and pre‐miR‐145 prevents their Dicer‐dependent maturation. By preventing miR‐21 or miR‐145 maturation, BCDIN3D also blocks subsequent suppression of their antagonistic counterparts (which for miR‐21, is miR‐145 and vice versa), hence forming an epitranscriptome regulatory circuit governing differentiation, the dysregulation of which appears to be linked to the regression of normal cells into CSC like cells with self‐renewal capacity. D, Concept of contextual effects of miRNA modifications in tumorigenesis. Here, we demonstrate how identical modifications in different miRNAs (marked as red circles for modifications suppressing miRNA activities [eg, tagging the miRNA for degradation] or green circles for modifications enhancing miRNA activities [eg, RNA‐stabilizing modifications]) can give rise to distinct fate decisions (ie, whether it is an oncomiR or tumor suppressive miR). Arrows marked with a Red “X” indicate a lost interaction as a result of the epitranscriptome. Annotations for proteins and modifications are colored according to their associated graphic. CSC, cancer stem cell; HSC, hematopoietic stem cell; LSC, leukemic stem cell; miRNAs, micro RNAs

FIGURE 5

An emerging view of the epitranscriptome by specialized RBPs. (1) Writers modify naked RNA. Modifiers process post‐transcriptional modifications into novel modifications. Readers recognize and bind to modified RNA. Repellers lose binding to modified RNA. Erasers remove RNA modifications (2). The interactions of RBPs with modifications determine RNA stability, processing, localization, and translation (3) enabling additional stem cell regulatory layers. Examples for each class of epitranscriptome‐associated RBPs are given and color‐coded according to their respective modifications in previous figures. RBP, RNA‐binding proteins