The microRNA processor DROSHA is a candidate gene for a severe progressive neurological disorder.

DROSHA encodes a ribonuclease that is a subunit of the Microprocessor complex and is involved in the first step of microRNA (miRNA) biogenesis. To date, DROSHA has not yet been associated with a Mendelian disease. Here, we describe two individuals with profound intellectual disability, epilepsy, white matter atrophy, microcephaly and dysmorphic features, who carry damaging de novo heterozygous variants in DROSHA. DROSHA is constrained for missense variants and moderately intolerant to loss-of-function (o/e = 0.24). The loss of the fruit fly ortholog drosha causes developmental arrest and death in third instar larvae, a severe reduction in brain size and loss of imaginal discs in the larva. Loss of drosha in eye clones causes small and rough eyes in adult flies. One of the identified DROSHA variants (p.Asp1219Gly) behaves as a strong loss-of-function allele in flies, while another variant (p.Arg1342Trp) is less damaging in our assays. In worms, a knock-in that mimics the p.Asp1219Gly variant at a worm equivalent residue causes loss of miRNA expression and heterochronicity, a phenotype characteristic of the loss of miRNA. Together, our data show that the DROSHA variants found in the individuals presented here are damaging based on functional studies in model organisms and likely underlie the severe phenotype involving the nervous system.

In Situ Hybridization for Detecting Mature MicroRNAs In Vivo at Single-Cell Resolution.

MicroRNAs (miRNAs) are key regulators of cell and tissue development. However, spatial resolution of miRNA heterogeneity and accumulation patterns in vivo remains uncharted. Next-generation sequencing methods assay miRNA abundance in tissues, yet these analyses do not provide spatial resolution. A method to assay miRNA expression at single-cell resolution in vivo should clarify the cell-autonomous functions of miRNAs, their roles in influencing the cellular microenvironment, and their perdurance and turnover rate. We present an in situ hybridization protocol to map miRNA subcellular expression in single cells in vivo in four days. Using this protocol, we mapped distinct miRNAs that accumulate in the cytoplasm of one sibling oocyte but not another, dependent on the oocyte developmental stage. Thus, this method provides spatial and temporal resolution of the heterogeneity in expression of miRNAs during Caenorhabditis elegans oogenesis. This protocol can generally be adapted to any tissue amenable to dissection and fixation. © 2019 by John Wiley & Sons, Inc.

Functional genomic analysis identifies miRNA repertoire regulating C. elegans oocyte development.

Oocyte-specific miRNA function remains unclear in mice and worms because loss of Dgcr8 and Dicer from mouse and worm oocytes, respectively, does not yield oogenic defects. These data lead to several models: (a) miRNAs are not generated in oocytes; (b) miRNAs are generated but do not perform an oogenic function; (c) functional oocyte miRNAs are generated in a manner independent of these enzymes. Here, we test these models using a combination of genomic, expression and functional analyses on the C. elegans germline. We identify a repertoire of at least twenty-three miRNAs that accumulate in four spatial domains in oocytes. Genetic tests demonstrate that oocyte-expressed miRNAs regulate key oogenic processes within their respective expression domains. Unexpectedly, we find that over half of the oocyte-expressed miRNAs are generated through an unknown Drosha independent mechanism. Thus, a functional miRNA repertoire generated via Drosha dependent and independent pathways regulates C. elegans oocyte development.