Cross-linking and immunoprecipitation coupled with high-throughput sequencing was used to identify binding sites within 6,304 genes as the brain RNA targets for TDP-43, an RNA binding protein which when mutated causes Amyotrophic Lateral Sclerosis (ALS). Use of massively parallel sequencing and splicing-sensitive junction arrays revealed that levels of 601 mRNAs are changed (including Fus/Tls, progranulin, and other transcripts encoding neurodegenerative disease-associated proteins) and 965 altered splicing events are detected (including in sortilin, the receptor for progranulin), following depletion of TDP-43 from mouse adult brain with antisense oligonucleotides. RNAs whose levels are most depleted by reduction in TDP-43 are derived from genes with very long introns and which encode proteins involved in synaptic activity. Lastly, TDP-43 was found to auto-regulate its synthesis, in part by directly binding and enhancing splicing of an intron within the 3′ untranslated region of its own transcript, thereby triggering nonsense mediated RNA degradation. (147 words)

Amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) are neurodegenerative diseases with clinical and pathological overlap. Landmark discoveries of mutations in the transactive response DNA-binding protein (TDP-43) and fused in sarcoma/translocated in liposarcoma (FUS/TLS) as causative of ALS and FTLD, combined with the abnormal aggregation of these proteins, have initiated a shifting paradigm for the underlying pathogenesis of multiple neurodegenerative diseases. TDP-43 and FUS/TLS are both RNA/DNA-binding proteins with striking structural and functional similarities. Their association with ALS and other neurodegenerative diseases is redirecting research efforts toward understanding the role of RNA processing regulation in neurodegeneration.

FUS/TLS (fused in sarcoma/translocated in liposarcoma) and TDP-43 are integrally involved in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. We found that FUS/TLS binds to RNAs from >5,500 genes in mouse and human brain, primarily through a GUGGU-binding motif. We identified a sawtooth-like binding pattern, consistent with co-transcriptional deposition of FUS/TLS. Depletion of FUS/TLS from the adult nervous system altered the levels or splicing of >950 mRNAs, most of which are distinct from RNAs dependent on TDP-43. Abundance of only 45 RNAs was reduced after depletion of either TDP-43 or FUS/TLS from mouse brain, but among these were mRNAs that were transcribed from genes with exceptionally long introns and that encode proteins that are essential for neuronal integrity. Expression levels of a subset of these were lowered after TDP-43 or FUS/TLS depletion in stem cell-derived human neurons and in TDP-43 aggregate–containing motor neurons in sporadic ALS, supporting a common loss-of-function pathway as one component underlying motor neuron death from misregulation of TDP-43 or FUS/TLS.