mRNA ageing shapes the Cap2 methylome in mammalian mRNA.

The mRNA cap structure is a major site of dynamic mRNA methylation. mRNA caps exist in either the Cap1 or Cap2 form, depending on the presence of 2'-O-methylation on the first transcribed nucleotide or both the first and second transcribed nucleotides, respectively1,2. However, the identity of Cap2-containing mRNAs and the function of Cap2 are unclear. Here we describe CLAM-Cap-seq, a method for transcriptome-wide mapping and quantification of Cap2. We find that unlike other epitranscriptomic modifications, Cap2 can occur on all mRNAs. Cap2 is formed through a slow continuous conversion of mRNAs from Cap1 to Cap2 as mRNAs age in the cytosol. As a result, Cap2 is enriched on long-lived mRNAs. Large increases in the abundance of Cap1 leads to activation of RIG-I, especially in conditions in which expression of RIG-I is increased. The methylation of Cap1 to Cap2 markedly reduces the ability of RNAs to bind to and activate RIG-I. The slow methylation rate of Cap2 allows Cap2 to accumulate on host mRNAs, yet ensures that low levels of Cap2 occur on newly expressed viral RNAs. Overall, these results reveal an immunostimulatory role for Cap1, and that Cap2 functions to reduce activation of the innate immune response.

Identification of RNA-Binding Protein Landscapes Across Zebrafish Embryonic Transcriptome via iCLIP Approach.

In metazoans, fertilization initiates vast remodeling of the embryonic proteome and transcriptome. This is accomplished via complex post-transcriptional regulation of maternal and zygotic RNA. RNA-binding proteins (RBPs) are one of the major mediators of embryonic post-transcriptional RNA regulation. Thus, elucidation of the molecular mechanisms by which maternal and zygotic transcripts change their translational capacities and expression levels requires thorough and precise determination of the targets and binding sites of individual RBPs in embryonic transcriptomes. Here, I provide a detailed protocol for the UV crosslinking-based method, named iCLIP, to study RBP functions during early zebrafish embryogenesis.

FTO controls reversible m6Am RNA methylation during snRNA biogenesis.

Small nuclear RNAs (snRNAs) are core spliceosome components and mediate pre-mRNA splicing. Here we show that snRNAs contain a regulated and reversible nucleotide modification causing them to exist as two different methyl isoforms, m1 and m2, reflecting the methylation state of the adenosine adjacent to the snRNA cap. We find that snRNA biogenesis involves the formation of an initial m1 isoform with a single-methylated adenosine (2'-O-methyladenosine, Am), which is then converted to a dimethylated m2 isoform (N6,2'-O-dimethyladenosine, m6Am). The relative m1 and m2 isoform levels are determined by the RNA demethylase FTO, which selectively demethylates the m2 isoform. We show FTO is inhibited by the oncometabolite D-2-hydroxyglutarate, resulting in increased m2-snRNA levels. Furthermore, cells that exhibit high m2-snRNA levels show altered patterns of alternative splicing. Together, these data reveal that FTO controls a previously unknown central step of snRNA processing involving reversible methylation, and suggest that epitranscriptomic information in snRNA may influence mRNA splicing.

RNA tales - how embryos read and discard messages from mom.

Following fertilization, embryos develop for a substantial amount of time with a transcriptionally silent genome. Thus, early development is maternally programmed, as it solely relies on RNAs and proteins that are provided by the female gamete. However, these maternal instructions are not sufficient to support later steps of embryogenesis and are therefore gradually replaced by novel products synthesized from the zygotic genome. This switch in the origin of molecular players that drive early development is known as the maternal-to-zygotic transition (MZT). MZT is a universal phenomenon among all metazoans and comprises two interconnected processes: maternal mRNA degradation and the transcriptional awakening of the zygotic genome. The recent adaptation of high-throughput methods for use in embryos has deepened our knowledge of the molecular principles underlying MZT. These mechanisms comprise conserved strategies for RNA regulation that operate in many well-studied cellular contexts but that have adapted differently to early development. In this Review, we will discuss advances in our understanding of post-transcriptional regulatory pathways that drive maternal mRNA clearance during MZT, with an emphasis on recent data in zebrafish embryos on codon-mediated mRNA decay, the contributions of microRNAs (miRNAs) and RNA-binding proteins to this process, and the roles of RNA modifications in the stability control of maternal mRNAs.

Analysis of RNA-protein interactions in vertebrate embryos using UV crosslinking approaches.

A decade ago, we believed that at least 300 RNA binding proteins (RBPs) were encoded in our genomes based on annotations of known or predicted RNA binding domains. Deciphering the roles of those RBPs in regulated gene expression was a vast frontier awaiting exploration. Since then, the field has developed a number of key tools that navigate the landscape of cellular RNA. These rely principally on UV crosslinking to create covalent bonds between RBPs and target RNAs in vivo, revealing not only target identities but also local binding sites upon RNA-Seq. More recently, a reverse protocol - mRNA interactome capture - has enabled the identification of the proteins that interact with mRNA. Astonishingly, the number of RBPs has grown to more than 1000, and we must now understand what they do. Here, we discuss the application of these methods to model organisms, focusing on the zebrafish Danio rerio, which provide unique biological contexts for the analysis of RBPs and their functions.

The m6A pathway facilitates sex determination in Drosophila.

The conserved modification N6-methyladenosine (m6A) modulates mRNA processing and activity. Here, we establish the Drosophila system to study the m6A pathway. We first apply miCLIP to map m6A across embryogenesis, characterize its m6A 'writer' complex, validate its YTH 'readers' CG6422 and YT521-B, and generate mutants in five m6A factors. While m6A factors with additional roles in splicing are lethal, m6A-specific mutants are viable but present certain developmental and behavioural defects. Notably, m6A facilitates the master female determinant Sxl, since multiple m6A components enhance female lethality in Sxl sensitized backgrounds. The m6A pathway regulates Sxl processing directly, since miCLIP data reveal Sxl as a major intronic m6A target, and female-specific Sxl splicing is compromised in multiple m6A pathway mutants. YT521-B is a dominant m6A effector for Sxl regulation, and YT521-B overexpression can induce female-specific Sxl splicing. Overall, our transcriptomic and genetic toolkit reveals in vivo biologic function for the Drosophila m6A pathway.

Dynamic RNA-protein interactions underlie the zebrafish maternal-to-zygotic transition.

During the maternal-to-zygotic transition (MZT), transcriptionally silent embryos rely on post-transcriptional regulation of maternal mRNAs until zygotic genome activation (ZGA). RNA-binding proteins (RBPs) are important regulators of post-transcriptional RNA processing events, yet their identities and functions during developmental transitions in vertebrates remain largely unexplored. Using mRNA interactome capture, we identified 227 RBPs in zebrafish embryos before and during ZGA, hereby named the zebrafish MZT mRNA-bound proteome. This protein constellation consists of many conserved RBPs, some of which are potential stage-specific mRNA interactors that likely reflect the dynamics of RNA-protein interactions during MZT. The enrichment of numerous splicing factors like hnRNP proteins before ZGA was surprising, because maternal mRNAs were found to be fully spliced. To address potentially unique roles of these RBPs in embryogenesis, we focused on Hnrnpa1. iCLIP and subsequent mRNA reporter assays revealed a function for Hnrnpa1 in the regulation of poly(A) tail length and translation of maternal mRNAs through sequence-specific association with 3' UTRs before ZGA. Comparison of iCLIP data from two developmental stages revealed that Hnrnpa1 dissociates from maternal mRNAs at ZGA and instead regulates the nuclear processing of pri-mir-430 transcripts, which we validated experimentally. The shift from cytoplasmic to nuclear RNA targets was accompanied by a dramatic translocation of Hnrnpa1 and other pre-mRNA splicing factors to the nucleus in a transcription-dependent manner. Thus, our study identifies global changes in RNA-protein interactions during vertebrate MZT and shows that Hnrnpa1 RNA-binding activities are spatially and temporally coordinated to regulate RNA metabolism during early development.