SUMMARY Evidence from animal studies and human famines suggests that starvation may affect the health of the progeny of famished individuals. However, it is not clear whether starvation affects only immediate offspring or has lasting effects; it is also unclear how such epigenetic information is inherited. Small RNA-induced gene silencing can persist over several generations via transgenerationally inherited small RNA molecules in C. elegans, but all known transgenerational silencing responses are directed against foreign DNA introduced into the organism. We found that starvation-induced developmental arrest, a natural and drastic environmental change, leads to the generation of small RNAs that are inherited through at least three consecutive generations. These small, endogenous, transgenerationally transmitted RNAs target genes with roles in nutrition. We defined genes that are essential for this multigenerational effect. Moreover, we show that the F3 offspring of starved animals show an increased lifespan, corroborating the notion of a transgenerational memory of past conditions.

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Various methyltransferases and demethylases catalyse methylation and demethylation of N6-methyladenosine (m6A) and N6,2′-O-dimethyladenosine (m6Am) but precise methylomes uniquely mediated by each methyltransferase/demethylase are still lacking. Here, we develop m6A-Crosslinking-Exonuclease-sequencing (m6ACE-seq) to map transcriptome-wide m6A and m6Am at quantitative single-base-resolution. This allows for the generation of a comprehensive atlas of distinct methylomes uniquely mediated by every individual known methyltransferase or demethylase. Our atlas reveals METTL16 to indirectly impact manifold methylation targets beyond its consensus target motif and highlights the importance of precision in mapping PCIF1-dependent m6Am. Rather than reverse RNA methylation, we find that both ALKBH5 and FTO instead maintain their regulated sites in an unmethylated steady-state. In FTO’s absence, anomalous m6Am disrupts snRNA interaction with nuclear export machinery, potentially causing aberrant pre-mRNA splicing events.

MIWI catalytic activity is required for spermatogenesis, indicating that piRNA-guided cleavage is critical for germ cell development. To identify meiotic piRNA targets, we augmented the mouse piRNA repertoire by introducing a human meiotic piRNA cluster. This triggered a spermatogenesis defect by inappropriately targeting the piRNA machinery to mouse mRNAs essential for germ cell development. Analysis of such de novo targets revealed a signature for pachytene piRNA target recognition. This enabled identification of both transposable elements and meiotically expressed protein-coding genes as targets of native piRNAs. Cleavage of genic targets began at the pachytene stage and resulted in progressive repression through meiosis, driven at least in part via the ping-pong cycle. Our data support the idea that meiotic piRNA populations must be strongly selected to enable successful spermatogenesis, both driving the response away from essential genes and directing the pathway toward mRNA targets that are regulated by small RNAs in meiotic cells.

In animals, piRNAs and their associated Piwi proteins guard germ cell genomes against mobile genetic elements via an RNAi-like mechanism. In Caenorhabditis elegans, 21U-RNAs comprise the piRNA class, and these collaborate with 22G RNAs via unclear mechanisms to discriminate self from nonself and selectively and heritably silence the latter. Recent work indicates that 21U-RNAs are post-transcriptional processing products of individual transcription units that produce~26-nucleotide capped precursors. However, nothing is known of how the expression of precursors is controlled or how primary transcripts give rise to mature small RNAs. We conducted a genome-wide RNAi screen to identify components of the 21U biogenesis machinery. Screening by direct, quantitative PCR (qPCR)-based measurements of mature 21U-RNA levels, we identified 22 genes important for 21U-RNA production, termed TOFUs (Twenty-One-u Fouled Ups). We also identified seven genes that normally repress 21U production. By measuring mature 21U-RNA and precursor levels for the seven strongest hits from the screen, we assigned factors to discrete stages of 21U-RNA production. Our work identifies for the first time factors separately required for the transcription of 21U precursors and the processing of these precursors into mature 21U-RNAs, thereby providing a resource for studying the biogenesis of this important small RNA class.

N 6-methyldeoxyadenosine (6mA) is a well-characterized DNA modification in prokaryotes but reports on its presence and function in mammals have been controversial. To address this issue, we established the capacity of 6mA-Crosslinking-Exonuclease-sequencing (6mACE-seq) to detect genome-wide 6mA at single-nucleotide-resolution, demonstrating this by accurately mapping 6mA in synthesized DNA and bacterial genomes. Using 6mACE-seq, we generated a human-genome-wide 6mA map that accurately reproduced known 6mA enrichment at active retrotransposons and revealed mitochondrial chromosome-wide 6mA clusters asymmetrically enriched on the heavy-strand. We identified a novel putative 6mA-binding protein in single-stranded DNA-binding protein 1 (SSBP1), a mitochondrial DNA (mtDNA) replication factor known to coat the heavy-strand, linking 6mA with the regulation of mtDNA replication. Finally, we characterized AlkB homologue 1 (ALKBH1) as a mitochondrial protein with 6mA demethylase activity and showed that its loss decreases mitochondrial oxidative phosphorylation. Our results show that 6mA clusters play a previously unappreciated role in regulating human mitochondrial function, despite 6mA being an uncommon DNA modification in the human genome.

N 6-methylation of 2′-O-methyladenosine (Am) in RNA occurs in eukaryotic cells to generate N6,2′-O-dimethyladenosine (m6Am). Identification of the methyltransferase responsible for m6Am catalysis has accelerated studies on the function of m6Am in RNA processing. While m6Am is generally found in the first transcribed nucleotide of mRNAs, the modification is also found internally within U2 snRNA. However, the writer required for catalyzing internal m6Am formation had remained elusive. By sequencing transcriptome-wide RNA methylation at single-base-resolution, we identified human METTL4 as the writer that directly methylates Am at U2 snRNA position 30 into m6Am. We found that METTL4 localizes to the nucleus and its conserved methyltransferase catalytic site is required for U2 snRNA methylation. By sequencing human cells with overexpressed Mettl4, we determined METTL4’s in vivo target RNA motif specificity. In the absence of Mettl4 in human cells, U2 snRNA lacks m6Am thereby affecting a subset of splicing events that exhibit specific features such as 3′ splice-site weakness and an increase in exon inclusion. These findings suggest that METTL4 methylation of U2 snRNA regulates splicing of specific pre-mRNA transcripts.

Genome wide maps of nucleosome occupancy in yeast have recently been produced through deep sequencing of nuclease-protected DNA. These maps have been obtained from both crosslinked and uncrosslinked chromatin in vivo, and from chromatin assembled from genomic DNA and nucleosomes in vitro. Here, we analyze these maps in combination with existing ChIP-chip data, and with new ChIP-qPCR experiments reported here. We show that the apparent nucleosome density in crosslinked chromatin, when compared to uncrosslinked chromatin, is preferentially increased at transcription factor (TF) binding sites, suggesting a strategy for mapping generic transcription factor binding sites that would not require immunoprecipitation of a particular factor. We also confirm previous conclusions that the intrinsic, sequence dependent binding of nucleosomes helps determine the localization of TF binding sites. However, we find that the association between low nucleosome occupancy and TF binding is typically greater if occupancy at a site is averaged over a 600bp window, rather than using the occupancy at the binding site itself. We have also incorporated intrinsic nucleosome binding occupancies as weights in a computational model for TF binding, and by this measure as well we find better prediction if the high resolution nucleosome occupancy data is averaged over 600bp. We suggest that the intrinsic DNA binding specificity of nucleosomes plays a role in TF binding site selection not so much through the specification of precise nucleosome positions that permit or occlude binding, but rather through the creation of low occupancy regions that can accommodate competition from TFs through rearrangement of nucleosomes.