TDP-43 is an RNA-binding protein that, when mutated, causes Amyotrophic Lateral Sclerosis (ALS) and other neurodegenerative diseases. This study shows that the loss of TDP-43 leads to the depletion of long pre-mRNAs with long introns and altered splicing patterns, contributing to neuronal vulnerability. Using CLIP-seq and RNA sequencing, the researchers identified TDP-43 binding sites in 6,304 genes, revealing that TDP-43 is crucial for maintaining normal levels and splicing of over 1,000 mRNAs. The most affected RNAs are those with long introns and encode proteins involved in synaptic activity. TDP-43 also auto-regulates its synthesis by enhancing splicing of an intron in its own 3' untranslated region, triggering nonsense-mediated RNA degradation. The study further shows that TDP-43 regulates the expression of disease-related transcripts, including those encoding FUS/TLS and progranulin, which are associated with ALS and FTLD-U. TDP-43 depletion leads to the reduction of Fus/Tls and progranulin mRNA levels, while progranulin levels are increased. TDP-43 also regulates alternative splicing of its mRNA targets, affecting the splicing of genes such as sortilin. The findings highlight the critical role of TDP-43 in maintaining RNA levels and splicing patterns, and its loss contributes to neuronal dysfunction and neurodegeneration. The study provides insights into the molecular mechanisms underlying TDP-43-related neurodegenerative diseases and the importance of TDP-43 in RNA processing and neuronal survival.TDP-43 is an RNA-binding protein that, when mutated, causes Amyotrophic Lateral Sclerosis (ALS) and other neurodegenerative diseases. This study shows that the loss of TDP-43 leads to the depletion of long pre-mRNAs with long introns and altered splicing patterns, contributing to neuronal vulnerability. Using CLIP-seq and RNA sequencing, the researchers identified TDP-43 binding sites in 6,304 genes, revealing that TDP-43 is crucial for maintaining normal levels and splicing of over 1,000 mRNAs. The most affected RNAs are those with long introns and encode proteins involved in synaptic activity. TDP-43 also auto-regulates its synthesis by enhancing splicing of an intron in its own 3' untranslated region, triggering nonsense-mediated RNA degradation. The study further shows that TDP-43 regulates the expression of disease-related transcripts, including those encoding FUS/TLS and progranulin, which are associated with ALS and FTLD-U. TDP-43 depletion leads to the reduction of Fus/Tls and progranulin mRNA levels, while progranulin levels are increased. TDP-43 also regulates alternative splicing of its mRNA targets, affecting the splicing of genes such as sortilin. The findings highlight the critical role of TDP-43 in maintaining RNA levels and splicing patterns, and its loss contributes to neuronal dysfunction and neurodegeneration. The study provides insights into the molecular mechanisms underlying TDP-43-related neurodegenerative diseases and the importance of TDP-43 in RNA processing and neuronal survival.