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

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

Predictions of protein features such as occurrence of signal peptides, disordered segments, trans-membrane regions, and secondary structure elements were obtained from several available servers including: Pfam 28 ; SMART 29 ; PSIPRED 30 ; TMHMM 31 ; SignalP 32 and GeneSilico MetaDisorder Service 33 . In vitro translation of CG2145 and CG3303. Total RNA was purified from whole larvae and adults by tissue homogenization and Qiazol reagent extraction. CG2145 and CG3303 ORFs were amplified, through the oligonucleotides A (5′ -ATGCGCTGCCTGGCATTGAGC-3′ ), B (5′ -TCAAATCTCCGGATAGGCGCTGCC-3′ ) and C (5′ -ATGGACGCAGTGCAGCAGCAAAC-3′ ), D (5′ -CTAGGAGCTGTCCGGATAGAC-3′ ), respectively, and cloned into the pGEX-4T1 vector, between the EcoRI and NotI sites. CG2145 and CG3303 [ 35 S] methionine-labeled proteins were produced by in vitro transcription and translation reactions using the TNT-coupled Reticulocyte Lysate System kit (Promega), according to manifacturer's instructions.