TDP-43 regulates miRNA biogenesis and cytotoxicity by obstructing Dicer activity

Description

The cytoplasmic Dicer proteins play a critical role in miRNA biogenesis. Deficiency of TDP-43 fly homologs, TBPH, and expression of its A315T pathogenic mutations reduces the levels of Drosophila dicer proteins (DCR-1 and DCR-2) in ALS fly models. Figure 1A and Figure 5A contain the original image files of Western Blot that were used to evaluate the expression levels of miRNA biogenesis-related proteins (including DCR-1 and DCR-2) in TBPH KO (compare to W- and heterozygous, Figure 1A) and TBPH A315T mutant (compare to TBPH WT and LacZ control, Figure 5A), flies. The files "W-vs-TBPHdelta23-mRNA.filter.annot.xls.zip" and "W-vs-TBPHdelta23-miRNA.filter.xls.zip" contain the miRNA and RNA reference sequencing data, including annotations and expression levels, from TBPH KO and W- control flies.

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Western Blot The whole lysate of adult flies or cultured cells was homogenated with 40 μl of 4x SDS loading buffer (200 mM Tris-HCl pH 6.8, 8% (w/v) SDS, 0.4% (w/v) BPB, 40% (v/v) glycerin, 4% (v/v) 2-ME). Pieces were added to the SDS-PAGE GEL for electrophoresis with the Electrophoresis system. Running buffers were composed of 0.125 M Tris-base and 1.25 M Glycine. Proteins were transferred to 0.45 μm PVDF membrane (Merck Millipore) using semi-dry overnight or wet transfer (Tenon) for 60 min. Unspecific binding was blocked using 5% non-fat skim milk or BAS in TBST for 15-30 min. Primary antibodies were diluted and incubated overnight at 4℃. According to the primary antibody, HRP-conjugated secondary antibodies were incubated at room temperature for two hours. Signals were developed using ECL LuminataTM Western HRP Substrates (Merck Millipore) and the ChemiDoc system (Bio-Rad). RNA and miRNA sequencing Total RNA was extracted from the cells or tissues using a Trizol reagent kit (Invitrogen, Carlsbad, CA, USA) per the manufacturer’s protocol. RNA quality was assessed on an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA) and verified through RNase-free agarose gel electrophoresis. After total RNA was extracted, eukaryotic mRNA was enriched by Oligo(dT) beads. Then, the increased mRNA was fragmented into short fragments using a fragmentation buffer. Next, the details were reverse transcribed into cDNA using NEBNext Ultra RNA Library Prep Kit for Illumina (NEB #7530, New England Biolabs, Ipswich, MA, USA). The double-stranded cDNA fragments were purified and end repaired. An A base was added, and the pieces were ligated to Illumina sequencing adapters. The ligation reaction was filtered with the AMPure XP Beads (1.0X). Ligated fragments were selected by size using agarose gel electrophoresis and polymerase chain reaction (PCR). The resulting cDNA library was sequenced using Illumina Novaseq6000 by Gene Denovo Biotechnology Co. After total RNA was extracted by Trizol reagent kit (Invitrogen, Carlsbad, CA, USA), the RNA molecules in a size range of 18–30 nt were enriched by polyacrylamide gel electrophoresis (PAGE). The 3’ adapters were added, increasing the 36-48 nt RNAs. The 5’ adapters were then ligated to the RNAs as well. The ligation products were reverse transcribed and purified using PCR. The 140-160bp size PCR products were enriched to generate a cDNA library and sequenced using Illumina HiSeq Xten by Gene Denovo (Biotechnology Co).

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