Novel roles of Drosophila FUS and Aub responsible for piRNA biogenesis in neuronal disorders.

piRNAs, small non-coding RNAs, were considered to be restricted to germline cells. Although they have recently been detected in somatic cells including neurons, it remains unclear how piRNA biogenesis is involved in neuronal diseases. We herein examined the possible roles of Aubergine (Aub), a Piwi-family protein (PIWI) responsible for piRNA biogenesis, in the neuronal disorders, using the Cabeza (Caz) knockdown Drosophila. Caz is a Drosophila homologue of FUS, which is one of the genes causing amyotrophic lateral sclerosis (ALS). Aub overexpression enhanced the mobility defects accompanied by anatomical defects in motoneurons at neuromuscular junctions induced by the neuron-specific knockdown of Caz. In order to elucidate the underlying mechanisms, we examined pre-piRNA and mature-size piRNA levels under these conditions. qRT-PCR and RNA-seq analyses revealed that the Caz knockdown increased pre-piRNA levels, but reduced mature-size piRNA levels in the central nervous system (CNS), suggesting a role in the pre-piRNAs production. Aub overexpression did not increase mature-size piRNA levels. These results suggest that the accumulated pre-piRNAs are abnormal abortive pre-piRNAs that cannot be further processed by slicers, including Aub. We also demonstrated a relationship between Caz and pre-piRNAs in the CNS by RNA immunoprecipitation. Aub overexpression induced the abnormal cytoplasmic localization of Caz. Based on these results, we propose a model in which Caz knockdown-induced abnormal pre-piRNAs associate with Caz, then translocate and accumulate in the cytoplasm, a process that may be mediated by Aub. The novel roles for Caz and Aub demonstrated herein using the Caz-knockdown fly will contribute to a deeper understanding of the pathogenesis of ALS.

The ABD on the nascent polypeptide and PH domain are required for the precise Anillin localization in Drosophila syncytial blastoderm.

Targeting proteins to regions where they are required is essential for proper development of organisms. For achievement of this, subcellular mRNA localization is one of the critical mechanisms. Subcellular mRNA localization is an evolutionarily conserved phenomenon from E. coli to human and contributes to limiting the regions at which its products function and efficiently supplies substrates for protein translation. During early Drosophila embryogenesis, while 71% of the 3370 mRNAs analyzed have shown prominent subcellular localization, the underlying molecular mechanisms have not been elucidated. Here, we reveal that anillin mRNA, one of the localized mRNAs in early Drosophila embryo, localizes to the tip of the pseudo-cleavage furrow in the Drosophila syncytial blastoderm using in situ hybridization combined with immunohistochemistry. Localization analyses with transgenic fly lines carrying a series of deletion mRNAs indicate that this localization is dependent on its own nascent polypeptides including the actin binding domain (ABD). In addition to the mRNA localization, it is revealed that the pleckstrin homology (PH) domain of Anillin protein is also required for its proper localization. Thus, we indicate that the precise localization of Anillin protein is tightly regulated by the ABD on the nascent polypeptide and PH domain in the Drosophila syncytial blastoderm.