The rise of epitranscriptomics: recent developments and future directions

Abstract

The epitranscriptomics field has undergone tremendous growth since the discovery that the RNA N⁶-methyladenosine (m⁶A) modification is reversible and is distributed throughout the transcriptome. Efforts to map RNA modifications transcriptome-wide and reshape the epitranscriptome in disease settings have facilitated mechanistic understanding and drug discovery in the field. In this review we discuss recent advancements in RNA modification detection methods and consider how these developments can be applied to gain novel insights into the epitranscriptome. We also highlight drug discovery efforts aimed at developing epitranscriptomic therapeutics for cancer and other diseases. Finally, we consider engineering of the epitranscriptome as an emerging direction to investigate RNA modifications and their causal effects on RNA processing at high specificity.

Keywords: N(6)-methyladenosine; RNA modifications; drug development; epitranscriptomics; machine learning; nanopore sequencing; pseudouridine.

Figure 1:. Methods for high-throughput detection of N⁶-methyladenosine (m⁶A).

(A) Protocols for m⁶A detection using m⁶A-specific antibodies (m⁶A-seq and MeRIP-seq) were first reported in 2012. Although m⁶A-seq and MeRIP-seq achieve 100–200 nucleotide resolution of mapping m⁶A, cross-linking of m⁶A-specific antibodies to their RNA targets using a modified protocol (miCLIP) enables m⁶A detection at higher resolution. (B) Antibody-free m⁶A detection using DART-seq is based on recruitment of a cytidine deaminase to m⁶A sites to induce a mutational signature that can later be used to infer the m⁶A position. Recently, DART-seq has also been combined with single-cell RNA sequencing (scRNA-seq) for detecting m⁶A in single cells (scDART-seq). (C) Chemical and metabolic labeling of m⁶A can be used to enrich m⁶A-decorated RNA fragments (m⁶A-SEAL) or induce a mutational signature around the m⁶A site during reverse transcription (m⁶A-label-seq and m⁶A-SAC-seq). (D) m⁶A-sensitive endoribonuclease MazF digests RNA at the ACA but not m⁶ACA motif and is used in m⁶A-REF-seq and MAZTER-seq protocols of m⁶A detection. (E) Recently developed protocols for selective adenosine but not m⁶A deamination enable transcriptome-wide stoichiometric m⁶A detection, reminiscent of bisulfite sequencing of DNA methylation. (F) Third-generation direct RNA sequencing can be used to detect m⁶A and other RNA modifications on native RNA by comparative analysis of modified and unmodified transcripts or by supervised learning.

Figure 2:. Therapeutic development focused on epitranscriptomics.

Dysregulation of the epitranscriptome in cancer and other diseases often stems from aberrant expression and function of associated RNA modification enzymes that can be targeted pharmacologically to reverse disease-associated phenotypes. (A) Therapeutic development begins at the stage of target identification, which has been greatly facilitated by tumor atlases that can be used to identify aberrantly expressed RNA modification machinery in cancer. RNA modification databases, such as MODOMICS, m⁶A-Atlas, and M6AREG, can be used to generate testable hypotheses regarding the mode of action of a particular RNA modification enzyme. (B) Having identified a target of interest, virtual drug screening is performed to uncover potential hit compounds. Drug-like compound libraries, such as the library curated by the National Cancer Institute Developmental Therapeutics Program (NCI-DTP) facilitate virtual screening and subsequent acquisition of selected compounds. (C) Identified hit compounds are tested using cell-free assays to characterize target-ligand interactions and evaluate drug inhibitory activity. (D) The hit compound can be further optimized by rational chemical optimization and synthesized for testing in vitro and in vivo. (E, F) Tumor cell growth assays can be used to assess the effects of the hit compound on cancer cell proliferation and cancer stem cell activity in vitro, whereas pharmacokinetics of the hit compound and its effects on tumor growth and survival can be assessed in vivo.

Figure 3:. Molecular tools for engineering RNA modifications.

(A) CRISPR/Cas technology enables sequence-specific targeting of RNA molecules by Cas endonucleases that recruit protein modalities for RNA modification. Catalytically inactive Cas13 (dCas13) or the RCas9 system that combines dCas9 and a single-stranded DNA monomer containing a protospacer adjacent motif (PAMmer) can be fused to catalytic domains of METTL3 or ALKBH5 to induce m⁶A methylation and demethylation of RNA targets, respectively. dCas endonucleases can also be used to induce other RNA modifications, such as A-to-I editing, highlighting the versatility of the RNA modification engineering technology. (B) In addition to the CRISPR/Cas-based tools, endogenous RNA modification machinery can be recruited to install pseudouridine (Ψ). Recruitment of the Ψ synthase dyskerin 1 (DKC1) complex (guide small nucleolar ribonucleoprotein, snoRNP) by supplying a specific guide RNA enables targeted pseudouridylation. (C) Temporal control of RNA modification engineering can be achieved by photoinducible dimerization of a dCas endonuclease and an RNA modification machinery component.

Figure 4:. Applications of molecular tools to study RNA modifications in various biological processes.

(A) RNA modification engineering facilitates the elucidation of causal relationships between individual RNA modification sites and their downstream effects on RNA processing and phenotypic outcomes. For example, targeted methylation of CDCP1 messenger RNA enhances its translation and bladder cancer progression. (B) Fluorescently-labeled dCas endonucleases fused to m⁶A reader proteins can be used to study how m⁶A reader proteins orchestrate RNA trafficking and localization in highly complex and elongated cells, such as neurons. (C) Molecular tools for RNA engineering can be packaged into adeno-associated virus (AAV) vectors for delivery in vivo to study the roles of RNA modifications in complex biological processes, such as learning and memory or tumor growth and metastasis.

Figure I:. The dynamics of N⁶-methyladenosine (m⁶A) RNA modification.

Adenosine moieties of RNA are methylated by m⁶A writer proteins, METTL3/14 and METTL16. The modified m⁶A can either be erased by oxygenases FTO and ALKBH5 or recognized by reader proteins of different protein families that in turn mediate downstream RNA processing. For example, YTHDF1 has been shown to promote m⁶A-decorated messenger RNA (mRNA) translation, whereas YTHDF2 facilitates mRNA degradation.