Characterizing the RNA targets and position-dependent splicing regulation by TDP-43

Abstract

TDP-43 is a predominantly nuclear RNA-binding protein that forms inclusion bodies in frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). The mRNA targets of TDP-43 in the human brain and its role in RNA processing are largely unknown. Using individual-nucleotide resolution UV-crosslinking and immunoprecipitation (iCLIP), we demonstrated that TDP-43 preferentially binds long clusters of UG-rich sequences in vivo. Analysis of TDP-43 RNA binding in FTLD-TDP brains revealed the greatest increases in binding to MALAT1 and NEAT1 non-coding RNAs. We also showed that TDP-43 binding on pre-mRNAs influences alternative splicing in a similar position-dependent manner to Nova proteins. In addition, we identified unusually long clusters of TDP-43 binding at deep intronic positions downstream of silenced exons. A significant proportion of alternative mRNA isoforms regulated by TDP-43 encode proteins that regulate neuronal development or are implicated in neurological diseases, highlighting the importance of TDP-43 for splicing regulation in the brain.

Mice with endogenous TDP ‐43 mutations exhibit gain of splicing function and characteristics of amyotrophic lateral sclerosis

“…To address whether the reduced RNA binding capacity in the RRM2mut was associated with changes in RNA binding specificity, we performed TDP‐43 iCLIP (Tollervey et al , 2011) on CNS tissue. As RRM2mut homozygotes are not viable, we performed iCLIP in embryonic brains for that strain.…”

Linking RNA Dysfunction and Neurodegeneration in Amyotrophic Lateral Sclerosis

“…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].…”

“…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]. TDP43 regulates its own pre-mRNA, as well as several others involved in neurodegenerative diseases, including those encoding FUS and progranulin [14].…”

“…Mounting genetic evidence suggests that motor neuron degeneration in ALS represents a final common pathway initiated by ≥1 conserved upstream mechanisms, including protein misfolding/aggregation [5][6][7][8][9][10][11][12], RNA misprocessing [10,[13][14][15][16][17][18][19], and disruptions in axonal transport [12,[20][21][22][23][24]. These mechanisms are not mutually exclusive, and instead may enhance one another, thereby accentuating neurodegeneration in ALS.…”