To elucidate the intracellular molecular mechanism of TAR DNA-binding protein of 43 kDa(TDP-43)and its role in the pathogenesis of amyotrophic lateral sclerosis (ALS).

TDP-43 has been implicated in diverse aspects of RNA metabolism, including transcriptional regulation, mRNA transport and degradation. Recently, it has been shown that TDP-43 is involved in regulation of non-coding RNA (ncRNA) metabolism. Nevertheless, the molecular details of how TDP-43 contributes to RNA surveillance remain unclear. We performed several omics analysis to address the molecular mechanism of TDP-43 in regulation of ncRNA metabolism.

To identify RNAs bound to TDP-43, we performed ultraviolet RNA immunoprecipitation (UV-RIP) followed by RNA-seq using doxycycline-inducible FLAG-TDP-43 TREx293 cells. To identify TDP-43-associated proteins, we conducted immunoprecipitation–mass spectrometry analysis under the same conditions. To investigate functional consequences, we established TDP-43-FKBP12F36V knock-in cells using the dTAG system to induce rapid protein degradation, followed by RNA-seq analysis. In addition, single and double knockdowns of TDP-43 and NEXT components were performed, and RNA levels were measured by RT-qPCR. We used ChatGPT (GPT-5; OpenAI) to assist with R code for figure preparation. Analyses and data interpretation were conducted by the authors.