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Identification of neuronal RNA targets of TDP-43-containing ribonucleoprotein complexes.

Sephton CF, Cenik C, Kucukural A, Dammer EB, Cenik B, Han Y, Dewey CM, Roth FP, Herz J, Peng J, Moore MJ, Yu G - J. Biol. Chem. (2010)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience, University of Texas Southwestern Medical Center,Dallas, Texas 75390-9111, USA.

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.

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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.
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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.

Mentions: We immunoprecipitated endogenous TDP-43 from rodent brain nuclear extracts and analyzed the resultant precipitation products with semiquantitative mass spectrometry. Taking into consideration the abundance index (>3.33) and consistency of spectral count trend (Fig. 5A, under spectral counts) of two independent experiments, we reliably identified 25 co-precipitating proteins highly enriched in the TDP-43 precipitate relative to control (Fig. 5A). There were 34 co-purified proteins that did not meet our criteria (supplemental Table S8), which nevertheless may represent transient interacting proteins of TDP-43.


Identification of neuronal RNA targets of TDP-43-containing ribonucleoprotein complexes.

Sephton CF, Cenik C, Kucukural A, Dammer EB, Cenik B, Han Y, Dewey CM, Roth FP, Herz J, Peng J, Moore MJ, Yu G - J. Biol. Chem. (2010)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3020728&req=5

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.
Mentions: We immunoprecipitated endogenous TDP-43 from rodent brain nuclear extracts and analyzed the resultant precipitation products with semiquantitative mass spectrometry. Taking into consideration the abundance index (>3.33) and consistency of spectral count trend (Fig. 5A, under spectral counts) of two independent experiments, we reliably identified 25 co-precipitating proteins highly enriched in the TDP-43 precipitate relative to control (Fig. 5A). There were 34 co-purified proteins that did not meet our criteria (supplemental Table S8), which nevertheless may represent transient interacting proteins of TDP-43.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience, University of Texas Southwestern Medical Center,Dallas, Texas 75390-9111, USA.

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.

Show MeSH
Related in: MedlinePlus