Reversible methylation of m6Am in the 5' cap controls mRNA stability.

Internal bases in mRNA can be subjected to modifications that influence the fate of mRNA in cells. One of the most prevalent modified bases is found at the 5' end of mRNA, at the first encoded nucleotide adjacent to the 7-methylguanosine cap. Here we show that this nucleotide, N6,2'-O-dimethyladenosine (m6Am), is a reversible modification that influences cellular mRNA fate. Using a transcriptome-wide map of m6Am we find that m6Am-initiated transcripts are markedly more stable than mRNAs that begin with other nucleotides. We show that the enhanced stability of m6Am-initiated transcripts is due to resistance to the mRNA-decapping enzyme DCP2. Moreover, we find that m6Am is selectively demethylated by fat mass and obesity-associated protein (FTO). FTO preferentially demethylates m6Am rather than N6-methyladenosine (m6A), and reduces the stability of m6Am mRNAs. Together, these findings show that the methylation status of m6Am in the 5' cap is a dynamic and reversible epitranscriptomic modification that determines mRNA stability.

Distinguishing RNA modifications from noise in epitranscriptome maps.

Messenger RNA (mRNA) and long noncoding RNA (lncRNA) can be subjected to a variety of post-transcriptional modifications that markedly influence their fate and function. This concept of 'epitranscriptomic' modifications and the understanding of their function has been driven by new technologies for transcriptome-wide mapping of modified nucleotides using next-generation sequencing. Mapping technologies have successfully documented the location and prevalence of several modified nucleotides in the transcriptome. However, some mapping methods have led to proposals of pervasive novel RNA modifications that have subsequently been shown to be exceptionally rare. These controversies have resulted in confusion about the identity of the modified nucleotides comprising the epitranscriptome in mRNA and lncRNA. Here we discuss the different transcriptome-wide technologies for mapping modified nucleotides. We describe why these methods can have poor accuracy and specificity. Finally, we describe emerging strategies that minimize false positives and other pitfalls associated with mapping and measuring epitranscriptomic modifications.

Single-nucleotide-resolution mapping of m6A and m6Am throughout the transcriptome.

N(6)-methyladenosine (m6A) is the most abundant modified base in eukaryotic mRNA and has been linked to diverse effects on mRNA fate. Current mapping approaches localize m6A residues to transcript regions 100-200 nt long but cannot identify precise m6A positions on a transcriptome-wide level. Here we developed m6A individual-nucleotide-resolution cross-linking and immunoprecipitation (miCLIP) and used it to demonstrate that antibodies to m6A can induce specific mutational signatures at m6A residues after ultraviolet light-induced antibody-RNA cross-linking and reverse transcription. We found that these antibodies similarly induced mutational signatures at N(6),2'-O-dimethyladenosine (m6Am), a modification found at the first nucleotide of certain mRNAs. Using these signatures, we mapped m6A and m6Am at single-nucleotide resolution in human and mouse mRNA and identified small nucleolar RNAs (snoRNAs) as a new class of m6A-containing non-coding RNAs (ncRNAs).

Hormonal regulation of vasotocin receptor mRNA in a seasonally breeding songbird.

Behaviors associated with breeding are seasonally modulated in a variety of species. These changes in behavior are mediated by sex steroids, levels of which likewise vary with season. The effects of androgens on behaviors associated with breeding may in turn be partly mediated by the nonapeptides vasopressin (VP) and oxytocin (OT) in mammals, and vasotocin (VT) in birds. The effects of testosterone (T) on production of these neuropeptides have been well-studied; however, the regulation of VT receptors by T is not well understood. In this study, we investigated steroid-dependent regulation of VT receptor (VTR) mRNA in a seasonally breeding songbird, the white-throated sparrow (Zonotrichia albicollis). We focused on VTR subtypes that have been most strongly implicated in social behavior: V1a and oxytocin-like receptor (OTR). Using in situ hybridization, we show that T-treatment of non-breeding males altered V1a and OTR mRNA expression in several regions associated with seasonal reproductive behaviors. For example, T-treatment increased V1a mRNA expression in the medial preoptic area, bed nucleus of the stria terminalis, and ventromedial hypothalamus. T-treatment also affected both V1a and OTR mRNA expression in nuclei of the song system; some of these effects depended on the presence or absence of a chromosomal rearrangement that affects singing behavior, plasma T, and VT immunolabeling in this species. Overall, our results strengthen evidence that VT helps mediate the behavioral effects of T in songbirds, and suggest that the chromosomal rearrangement in this species may affect the sensitivity of the VT system to seasonal changes in T.

Antibody cross-reactivity accounts for widespread appearance of m1A in 5'UTRs.

N1-methyladenosine (m1A) was proposed to be a highly prevalent modification in mRNA 5'UTRs based on mapping studies using an m1A-binding antibody. We developed a bioinformatic approach to discover m1A and other modifications in mRNA throughout the transcriptome by analyzing preexisting ultra-deep RNA-Seq data for modification-induced misincorporations. Using this approach, we detected appreciable levels of m1A only in one mRNA: the mitochondrial MT-ND5 transcript. As an alternative approach, we also developed an antibody-based m1A-mapping approach to detect m1A at single-nucleotide resolution, and confirmed that the commonly used m1A antibody maps sites to the transcription-start site in mRNA 5'UTRs. However, further analysis revealed that these were false-positives caused by binding of the antibody to the m7G-cap. A different m1A antibody that lacks cap-binding cross-reactivity does not show enriched binding in 5'UTRs. These results demonstrate that high-stoichiometry m1A sites are exceedingly rare in mRNAs and that previous mappings of m1A to 5'UTRs were the result of antibody cross-reactivity to the 5' cap.

Mapping m6A at Individual-Nucleotide Resolution Using Crosslinking and Immunoprecipitation (miCLIP).

N 6 -methyladenosine (m6A) is the most abundant modified base in eukaryotic mRNA and has been linked to diverse effects on mRNA fate. Current m6A mapping approaches localize m6A residues to 100-200 nt-long regions of transcripts. The precise position of m6A in mRNAs cannot be identified on a transcriptome-wide level because there are no chemical methods to distinguish between m6A and adenosine. Here, we describe a method for using anti-m6A antibodies to induce specific mutational signatures at m6A residues after ultraviolet light-induced antibody-RNA crosslinking and reverse transcription. Then, we describe how to use these mutational signatures to map m6A residues at nucleotide resolution. Taken together, our protocol allows for high-throughput detection of individual m6A residues throughout the transcriptome.

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.