C19ORF84 connects piRNA and DNA methylation machineries to defend the mammalian germ line.

In the male mouse germ line, PIWI-interacting RNAs (piRNAs), bound by the PIWI protein MIWI2 (PIWIL4), guide DNA methylation of young active transposons through SPOCD1. However, the underlying mechanisms of SPOCD1-mediated piRNA-directed transposon methylation and whether this pathway functions to protect the human germ line remain unknown. We identified loss-of-function variants in human SPOCD1 that cause defective transposon silencing and male infertility. Through the analysis of these pathogenic alleles, we discovered that the uncharacterized protein C19ORF84 interacts with SPOCD1. DNMT3C, the DNA methyltransferase responsible for transposon methylation, associates with SPOCD1 and C19ORF84 in fetal gonocytes. Furthermore, C19ORF84 is essential for piRNA-directed DNA methylation and male mouse fertility. Finally, C19ORF84 mediates the in vivo association of SPOCD1 with the de novo methylation machinery. In summary, we have discovered a conserved role for the human piRNA pathway in transposon silencing and C19ORF84, an uncharacterized protein essential for orchestrating piRNA-directed DNA methylation.

SPOCD1 is an essential executor of piRNA-directed de novo DNA methylation.

In mammals, the acquisition of the germline from the soma provides the germline with an essential challenge: the need to erase and reset genomic methylation1. In the male germline, RNA-directed DNA methylation silences young, active transposable elements2-4. The PIWI protein MIWI2 (PIWIL4) and its associated PIWI-interacting RNAs (piRNAs) instruct DNA methylation of transposable elements3,5. piRNAs are proposed to tether MIWI2 to nascent transposable element transcripts; however, the mechanism by which MIWI2 directs the de novo methylation of transposable elements is poorly understood, although central to the immortality of the germline. Here we define the interactome of MIWI2 in mouse fetal gonocytes undergoing de novo genome methylation and identify a previously unknown MIWI2-associated factor, SPOCD1, that is essential for the methylation and silencing of young transposable elements. The loss of Spocd1 in mice results in male-specific infertility but does not affect either piRNA biogenesis or the localization of MIWI2 to the nucleus. SPOCD1 is a nuclear protein whose expression is restricted to the period of de novo genome methylation. It co-purifies in vivo with DNMT3L and DNMT3A, components of the de novo methylation machinery, as well as with constituents of the NURD and BAF chromatin remodelling complexes. We propose a model whereby tethering of MIWI2 to a nascent transposable element transcript recruits repressive chromatin remodelling activities and the de novo methylation apparatus through SPOCD1. In summary, we have identified a previously unrecognized and essential executor of mammalian piRNA-directed DNA methylation.

Applying Human ADAR1p110 and ADAR1p150 for Site-Directed RNA Editing-G/C Substitution Stabilizes GuideRNAs against Editing.

Site-directed RNA editing is an approach to reprogram genetic information at the RNA level. We recently introduced a novel guideRNA that allows for the recruitment of human ADAR2 to manipulate genetic information. Here, we show that the current guideRNA design is already able to recruit another human deaminase, ADAR1, in both isoforms, p110 and p150. However, further optimization seems necessary as the current design is less efficient for ADAR1 isoforms. Furthermore, we describe hotspots at which the guideRNA itself is edited and show a way to circumvent this auto-editing without losing editing efficiency at the target. Both findings are important for the advancement of site-directed RNA editing as a tool in basic biology or as a platform for therapeutic editing.