Identification of Neuronal RNA Targets of TDP-43-containing Ribonucleoprotein Complexes

Abstract

TAR DNA-binding protein 43 (TDP-43) is associated with a spectrum of neurodegenerative diseases. Although TDP-43 resembles heterogeneous nuclear ribonucleoproteins, its RNA targets and physiological protein partners remain unknown. Here we identify RNA targets of TDP-43 from cortical neurons by RNA immunoprecipitation followed by deep sequencing (RIP-seq). The canonical TDP-43 binding site (TG)n is 55.1-fold enriched, and moreover, a variant with adenine in the middle, (TG)nTA(TG)m, is highly abundant among reads in our TDP-43 RIP-seq library. TDP-43 RNA targets can be divided into three different groups: those primarily binding in introns, in exons, and across both introns and exons. TDP-43 RNA targets are particularly enriched for Gene Ontology terms related to synaptic function, RNA metabolism, and neuronal development. Furthermore, TDP-43 binds to a number of RNAs encoding for proteins implicated in neurodegeneration, including TDP-43 itself, FUS/TLS, progranulin, Tau, and ataxin 1 and -2. We also identify 25 proteins that co-purify with TDP-43 from rodent brain nuclear extracts. Prominent among them are nuclear proteins involved in pre-mRNA splicing and RNA stability and transport. Also notable are two neuron-enriched proteins, methyl CpG-binding protein 2 and polypyrimidine tract-binding protein 2 (PTBP2). A PTBP2 consensus RNA binding motif is enriched in the TDP-43 RIP-seq library, suggesting that PTBP2 may co-regulate TDP-43 RNA targets. This work thus reveals the protein and RNA components of the TDP-43-containing ribonucleoprotein complexes and provides a framework for understanding how dysregulation of TDP-43 in RNA metabolism contributes to neurodegeneration.

Linking RNA Dysfunction and Neurodegeneration in Amyotrophic Lateral Sclerosis

“…Consistent with this hypothesis, disease-associated mutations in TARDBP and FUS promote cytoplasmic mislocalization of TDP43 and FUS, and enhance neurotoxicity [61,141]. TDP43 also regulates the expression of Ran, a master mediator of nucleocytoplasmic transport [37,142], and Ran expression was reduced to 60 % of normal levels in FTLD-TDP cortex [143]. Furthermore, dominant-negative Ran mutations induce cytoplasmic TDP43 deposition and neurotoxicity, while overexpression of WT Ran in a neuron model of FTLD-TDP prevented TDP43 mislocalization and improved survival [143].…”

“…TDP43 binds to and regulates thousands of RNA targets [13,14,[36][37][38], and is thus in a unique position to dramatically affect gene expression on a global scale. The RNA-binding domains of TDP43 and FUS are essential for toxicity in ALS model systems, testifying to the importance of RNA dysregulation in ALS pathogenesis [34,[39][40][41].…”

“…The RNA-binding domains of TDP43 and FUS are essential for toxicity in ALS model systems, testifying to the importance of RNA dysregulation in ALS pathogenesis [34,[39][40][41]. Immunoprecipitation studies in murine brain [14], dissociated primary cortical neurons [37], and human brain [13] and cell lines [13,38] showed that TDP43 recognizes nearly one-thrid of all transcribed genes by binding to redundant GU-rich sequences [42]. While nuclear TDP43 binds preferentially to distal intronic sequences, cytoplasmic TDP43 recognizes more 3' untranslated region (UTR) binding sites [13].…”

“…However, GRN mutations rarely cause ALS [43][44][45][46], indicating that mutations in different genes may result in convergent molecular pathologies but distinct clinical phenotypes. TDP43 also binds several transcripts involved in RNA splicing, nucleocytoplasmic protein trafficking, synapse formation and function, neurotransmitter metabolism, and neurite development [13,14,37]. TDP43 dysfunction, therefore, can disrupt fundamental and ubiquitous processes, as well as neuron-specific pathways.…”

“…Third, ADAR2 recognizes the expanded C9orf72 G 4 C 2 hexanucleotide repeat and is incorporated into RNA-rich nuclear foci characteristic of C9orf72-linked fALS [68]. Fourth, TDP43 binds directly to ADAR1 RNA and protein [37,77]. The ADAR 1 and 2 isoforms share considerable overlap in substrate specificity [78], such that either or both may mediate GluA2 RNA editing.…”