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

In vitro processing assay. Oligoribonucleotides P1 (5′ -GGAAACGUAUCCUUUGGGAG-3′ ) or P2 (5′ -GGAAACGUAUCCUUGGGAGG-3′ ) were 5′ -end labeled with [γ -32P]ATP (PerkinElmer Life Sciences) by T4 polynucleotide kinase (New England Biolabs) treatment. Processing reactions were carried out in the conditions already described 1 , by incubating 3 × 10 4 cpm of 32 P-labeled substrate together with 5-10 μ l of CG2145-or CG3303-expressing reticulocyte lysates, in the presence of 5 mM MnCl 2 (or other cations, for ion dependence determination experiments). Cleavage products were phenol-extracted and analysed on 20% polyacrylamide, 7 M urea gels. The RNA ladder derived from incubation of P1 oligo (200,000 cpm) in 500 mM NaHCO 3 at 90 °C 2-tailed unpaired t-tests. Tests were performed using Prism (GraphPad Software, Inc.). P value, ****p < 0.0001. (d) Diagram showing viability in D42-G4 > dendoU RNAi flies. Median lifespan in experimental flies (grey squares) was 21.53 ± 0.50d and it was 31.38 ± 0.26d in control flies (black circles). The log rank test showed that the two curves were significantly different (P < 0.001). (e) Locomotor deficit in 5-day-old flies interfered for dendoU in motor neurons. The climbing performance in D42-G4 > dendoU RNAi flies (grey bar) was significantly lower compared to control flies (black bar). Statistics as in panel c. P values, **p = 0.0015. (f) Western blot of dTDP-43 in head and thoracic-abdominal ganglia (H + TAG) dissected from pan-neuronal dendoU interfered pupae. Giotto was used as internal control. Result was expressed as means for at least three independent biological replicates. (g) qRT-PCR analysis of dendoU and dTDP-43 mRNAs (grey bars) in the same samples as in (f). Expression levels in control samples are set as 1 (black bar). Data are presented as mean ± SD in triplicates (***P < 0.001, Paired T-test). The full-length versions of the cropped blots are shown in Supplementary Figure 7b .