Paternal methotrexate exposure affects sperm small RNA content and causes craniofacial defects in the offspring.

Folate is an essential vitamin for vertebrate embryo development. Methotrexate (MTX) is a folate antagonist that is widely prescribed for autoimmune diseases, blood and solid organ malignancies, and dermatologic diseases. Although it is highly contraindicated for pregnant women, because it is associated with an increased risk of multiple birth defects, the effect of paternal MTX exposure on their offspring has been largely unexplored. Here, we found MTX treatment of adult medaka male fish (Oryzias latipes) causes cranial cartilage defects in their offspring. Small non-coding RNA (sncRNAs) sequencing in the sperm of MTX treated males identify differential expression of a subset of tRNAs, with higher abundance for specific 5' tRNA halves. Sperm RNA methylation analysis on MTX treated males shows that m5C is the most abundant and differential modification found in RNAs ranging in size from 50 to 90 nucleotides, predominantly tRNAs, and that it correlates with greater testicular Dnmt2 methyltransferase expression. Injection of sperm small RNA fractions from MTX-treated males into normal fertilized eggs generated cranial cartilage defects in the offspring. Overall, our data suggest that paternal MTX exposure alters sperm sncRNAs expression and modifications that may contribute to developmental defects in their offspring.

miR-1 sustains muscle physiology by controlling V-ATPase complex assembly.

Muscle function requires unique structural and metabolic adaptations that can render muscle cells selectively vulnerable, with mutations in some ubiquitously expressed genes causing myopathies but sparing other tissues. We uncovered a muscle cell vulnerability by studying miR-1, a deeply conserved, muscle-specific microRNA whose ablation causes various muscle defects. Using Caenorhabditis elegans, we found that miR-1 represses multiple subunits of the ubiquitous vacuolar adenosine triphosphatase (V-ATPase) complex, which is essential for internal compartment acidification and metabolic signaling. V-ATPase subunits are predicted miR-1 targets in animals ranging from C. elegans to humans, and we experimentally validated this in Drosophila. Unexpectedly, up-regulation of V-ATPase subunits upon miR-1 deletion causes reduced V-ATPase function due to defects in complex assembly. These results reveal V-ATPase assembly as a conserved muscle cell vulnerability and support a previously unknown role for microRNAs in the regulation of protein complexes.