Drosophila CG3303 is an essential endoribonuclease linked to TDP-43-mediated neurodegeneration

Pietro Laneve,Assunta Maria Casale,Ubaldo Gioia,Irene Bozzoni,Lucia Piacentini,Ugo Cappucci,Veronica Morea,Carmela Antonia Di Franco,Davide Capauto,Elisa Caffarelli,Valerio Di Carlo,Patrizio Di Micco

doi:10.1038/srep41559

Abstract

Endoribonucleases participate in almost every step of eukaryotic RNA metabolism, acting either as degradative or biosynthetic enzymes. We previously identified the founding member of the Eukaryotic EndoU ribonuclease family, whose components display unique biochemical features and are flexibly involved in important biological processes, such as ribosome biogenesis, tumorigenesis and viral replication. Here we report the discovery of the CG3303 gene product, which we named DendoU, as a novel family member in Drosophila. Functional characterisation revealed that DendoU is essential for Drosophila viability and nervous system activity. Pan-neuronal silencing of dendoU resulted in fly immature phenotypes, highly reduced lifespan and dramatic motor performance defects. Neuron-subtype selective silencing showed that DendoU is particularly important in cholinergic circuits. At the molecular level, we unveiled that DendoU is a positive regulator of the neurodegeneration-associated protein dTDP-43, whose downregulation recapitulates the ensemble of dendoU-dependent phenotypes. This interdisciplinary work, which comprehends in silico, in vitro and in vivo studies, unveils a relevant role for DendoU in Drosophila nervous system physio-pathology and highlights that DendoU-mediated neurotoxicity is, at least in part, contributed by dTDP-43 loss-of-function.

Highlights

We previously identified and characterised the amphibian XendoU protein[1,2,3]
Sequence analysis highlighted the occurrence of two Drosophila proteins homologous to X. laevis XendoU, encoded by CG2145 and CG3303 genes
The multiple sequence alignment of CG3303 and CG2145 with XendoU and human Placental Protein 11 (PP11) homologues, shown in Fig. 1b, highlighted that the 1–250 region of XendoU, comprising most of the residues previously reported to be involved in catalytic activity[2], was well conserved in both Drosophila isoforms and PP11

Summary

Introduction

We previously identified and characterised the amphibian XendoU protein[1,2,3]. XendoU is an endoribonuclease involved in the biogenesis of small nucleolar RNAs, which participate in ribosome function[4,5]. Three-dimensional structure determination revealed that XendoU had a novel fold[3], which led it to be defined the founding member of the Eukaryotic EndoU ribonuclease family by the Structure Classification of Proteins Database (SCOP)[6]. We later characterised another member of the same family, closely related to XendoU, namely human Placental Protein 11 (PP11)[7], which is highly tissue-specific and represents a tumour-marker in several cancers. Deep biochemical analyses revealed that CG3303 product alone shares the aforementioned biochemical features We named this protein, which is annotated by the major databases as serine protease, DendoU (Drosophila endoribonuclease U-specific). These phenotypes overlap with those caused by loss- and gain-of-function of the neurodegeneration-associated protein dTDP-43, which we revealed to be regulated by DendoU

Methods

Results

Conclusion