The Proteins of mRNA Modification: Writers, Readers, and Erasers

Abstract

Over the past decade, mRNA modifications have emerged as important regulators of gene expression control in cells. Fueled in large part by the development of tools for detecting RNA modifications transcriptome wide, researchers have uncovered a diverse epitranscriptome that serves as an additional layer of gene regulation beyond simple RNA sequence. Here, we review the proteins that write, read, and erase these marks, with a particular focus on the most abundant internal modification, N⁶-methyladenosine (m⁶A). We first describe the discovery of the key enzymes that deposit and remove m⁶A and other modifications and discuss how our understanding of these proteins has shaped our views of modification dynamics. We then review current models for the function of m⁶A reader proteins and how our knowledge of these proteins has evolved. Finally, we highlight important future directions for the field and discuss key questions that remain unanswered.

Keywords: RNA modifications; demethylase; epitranscriptome; m6A; methyltransferase; reader protein.

Figure 1.. Regulation and function of m⁶A writer proteins.

a) m⁶A is deposited co-transcriptionally by a methyltransferase complex which includes METTL3 as the catalytic subunit and additional proteins METTL14, WTAP, RBM15/15B, VIRMA, HAKAI, and ZC3H13. This complex is recruited to RNAs through interactions with H3K36me3 histone marks, transcription factors, and RNA polymerase II (RNA pol II), and its activity can be influenced by transcription rate. METTL3 deposits m⁶A within the DRACH consensus sequence, which is where the majority of m⁶A sites within mRNAs reside. b) In the cytoplasm, METTL3 and METTL16 interact with the translation initiation machinery to promote mRNA translation independently of their methyltransferase activity. c) A subset of mRNAs can be methylated by METTL16, which requires distinct sequence and structural elements. METTL16 also deposits m⁶A in snRNAs and lncRNAs. At the mRNA 5’ end, PCIF1 deposits m⁶A_m adjacent to the cap.

Figure 2.. Writers, readers, and erasers of other mRNA modifications.

Shown are the major regulatory proteins of pseudouridine, m¹A, m⁵C, ac⁴C, and Nm as well as the types of RNAs in which these modifications have been identified. Further characterization of many of these regulatory proteins will be important for understanding how these modifications are controlled in cells and their contribution to gene expression. In some cases, eraser and reader proteins have not yet been identified.

Figure 3.. m⁶A reader proteins have diverse functions and mechanisms of m⁶A recognition.

a) Schematic showing the domain structures of direct m⁶A binding proteins. YTHDF1/2/3, YTHDC1, and YTHDC2 recognize m⁶A through the highly conserved YTH domain. K-homology (KH) domains mediate m⁶A binding in FMRP and IGF2BP proteins. The KH domains required for m⁶A recognition are underlined. (b) Surface representation of the YTH domain of human YTHDF1 in complex with m⁶A (PDB: 4RCJ). m⁶A is nested in a hydrophobic pocket of the YTH domain. The three tryptophan residues forming the aromatic cage surrounding m⁶A are highlighted, as well as the position of the methyl moiety in m⁶A. c) m⁶A readers impact several aspects of RNA processing and function. In the nucleus, YTHDC1 regulates chromatin accessibility, promotes degradation of carRNAs, and binds to m⁶A sites within the lncRNA XIST to promote gene silencing. YTHDC1 also interacts with splicing factors and controls alternative splicing, polyadenylation, and nuclear export. FMRP recognition of methylated transcripts also promotes their export. In the cytoplasm, m⁶A is recognized by eIF3 to promote cap-independent translation of mRNAs with 5’-UTR m⁶A sites. YTHDF proteins can localize to stress granules and P-bodies, and all three proteins can promote mRNA decay. The role of YTHDF2 in mRNA destabilization is the most extensively studied and can include recruitment of various effector proteins to promote decapping, deadenylation, or endonucleolytic decay. Binding of YTHDF1 and YTHDF3 can also lead to increased translation of methylated mRNAs in some cell types.

Figure 4.. Targets of m⁶A eraser proteins.

a) In the nucleus, FTO and ALKBH5 demethylate m⁶A sites in mRNAs. FTO can also remove m⁶A and m⁶A_m in lncRNAs and snRNAs. b) In the cytoplasm, FTO can remove m⁶A and m⁶A_m from mRNAs and m¹A from tRNA. In the nervous system, FTO expression is regulated by activity and can promote m⁶A demethylation of specific transcripts in axons. ALKBH5 expression is increased in hypoxic conditions and in cancer cells, resulting in decreased m⁶A levels of specific mRNAs and enhanced cell proliferation and stem cell self-renewal.