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)(n)TA(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.

FIGURE 1.

Genomic distribution of reads from TDP-43 RNA library. A, Western blot (IB) of fractions from HeLa nuclear extracts (± RNase A) applied to a size exclusion column, blotted for TDP-43, hnRNPA1, and lamin A/C. Fraction 6 = blue dextran (2000 kDa), fraction 30 = apoferritin (443 kDa), fraction 40 = alcohol dehydrogenase (150 kDa), and fraction 54 = bovine serum albumin (54 kDa) (data not shown). B, Western blot of fractions from rat brain nuclear extracts (± micrococcal nuclease (± MNase)) blotted for TDP-43. Fraction 5 = blue dextran (2,000 kDa), fraction 45 = bovine serum albumin (54 kDa), and fraction 54 = carbonic anhydrase (29 kDa) (data not shown). Note that different size exclusion columns were used in A and B. C, panel i, diagram of TDP-43 RIP method. C, panel ii, representative Western blot of TDP-43 RIP. IP:CTL, control immunoprecipitation. D, distribution of raw reads from the TDP-43 library mapped to exonic and intronic genes regions. CDS, coding sequence. E, read density, number of reads per 1,000 mappable nucleotides per million reads (mRPKM) of gene regions from the TDP-43 library.

FIGURE 2.

Identification of short nucleotide sequences enriched in TDP-43 library. A, short nucleotide sequences of type (TG)_nTA(TG)_m are highly enriched in the TDP-43 library when compared with the control library. The number of 36-nt reads with at least one occurrence of each variant is shown. The graph depicts sequences where adenine replaces guanine in positions 1, 3, 5, 7, 9, or 11. The shape of the distribution reveals that adenine tends to appear in the middle of the sequence. B, same as panel A, except that the adenine is in positions 2, 4, 6, 8, 10, or 12.

FIGURE 3.

Distribution of the read density for the top 25% of TDP-43 RNA targets. The scatter plot depicts exonic (A) and intronic (B) read density of TDP-43 RNA targets and -fold enrichment of reads in the TDP-43 library relative to the control library. C, summary of the number of genes in each of the TDP-43 RNA target categories.

FIGURE 4.

Functional categorization of top TDP-43 RNA targets. A–C, summary of the top 30 most enriched Gene Ontology terms in TDP-43 RNA targets in exonic (A, panel i); intronic (B, panel i); and dual sets (C, panel i). For the complete functional listing, see supplemental Tables S3–S5. A–C, panel ii, snapshots of genes representing each category of binding. A, panel ii, exonic-TDP-43; B, panel ii, intronic-Slit3; and C, panel ii, dual Notch1. The number of uniquely mapped reads to the gene were shown for both the TDP-43 library and the control (CTL) library. The asterisk indicates TG-rich regions. Note the adenine (bolded) in the (TG)_n motifs shown for Slit3 and Notch1.

FIGURE 5.

TDP-43 nuclear interactome. A, 25 proteins co-purified with TDP-43 from mouse brain in two independent immunoprecipitation experiments, IP (1) and IP (2). B, functional classification of TDP-43 nuclear interactome using Gene Ontology terms (m.p., metabolic process; N, number of proteins from the TDP-43 IP that are in the functional category; M, total proteins involved in that functional category, LOD, logarithm (base 10) of odds ratio; P-adj, p-value adjusted for multiple hypothesis testing). C, Western blot of TDP-43 co-immunoprecipitation products from mouse brain nuclear extracts showing co-precipitated proteins hnRNPA1 and MECP2. FT, flow through; W, wash; E, elution (1% of total elution was loaded). Arrows indicate TDP-43-specific bands. IB, immunoblot.