Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2012 May 4;287(19):15635-47.
doi: 10.1074/jbc.M111.333450. Epub 2012 Mar 16.

TDP-43 and FUS RNA-binding proteins bind distinct sets of cytoplasmic messenger RNAs and differently regulate their post-transcriptional fate in motoneuron-like cells

Claudia Colombrita  1 , Elisa Onesto, Francesca Megiorni, Antonio Pizzuti, Francisco E Baralle, Emanuele Buratti, Vincenzo Silani, Antonia Ratti
Affiliations

TDP-43 and FUS RNA-binding proteins bind distinct sets of cytoplasmic messenger RNAs and differently regulate their post-transcriptional fate in motoneuron-like cells

Claudia Colombrita et al. J Biol Chem. .

Abstract

The RNA-binding proteins TDP-43 and FUS form abnormal cytoplasmic aggregates in affected tissues of patients with amyotrophic lateral sclerosis and frontotemporal lobar dementia. TDP-43 and FUS localize mainly in the nucleus where they regulate pre-mRNA splicing, but they are also involved in mRNA transport, stability, and translation. To better investigate their cytoplasmic activities, we applied an RNA immunoprecipitation and chip analysis to define the mRNAs associated to TDP-43 and FUS in the cytoplasmic ribonucleoprotein complexes from motoneuronal NSC-34 cells. We found that they bind different sets of mRNAs although converging on common cellular pathways. Bioinformatics analyses identified the (UG)(n) consensus motif in 80% of 3'-UTR sequences of TDP-43 targets, whereas for FUS the binding motif was less evident. By in vitro assays we validated binding to selected target 3'-UTRs, including Vegfa and Grn for TDP-43, and Vps54, Nvl, and Taf15 for FUS. We showed that TDP-43 has a destabilizing activity on Vegfa and Grn mRNAs and may ultimately affect progranulin protein content, whereas FUS does not affect mRNA stability/translation of its targets. We also demonstrated that three different point mutations in TDP-43 did not change the binding affinity for Vegfa and Grn mRNAs or their protein level. Our data indicate that TDP-43 and FUS recognize distinct sets of mRNAs and differently regulate their fate in the cytoplasm of motoneuron-like cells, therefore suggesting complementary roles in neuronal RNA metabolism and neurodegeneration.

PubMed Disclaimer

Figures

FIGURE 1.
FIGURE 1.
Isolation of the cytoplasmic RNP complexes containing TDP-43 and FUS. A, shown is immunoblot analysis with Histone H1 antibody as a nuclear marker. B, shown is a representative Western blot of RNP complexes immunopurified from cytoplasmic fraction with anti-TDP-43 (upper inset) and anti-FUS (lower inset) antibodies in NSC-34 cells. The irrelevant IgG antibody was used as negative control for IP assays. Total cell lysate was loaded as a positive control (Ctrl).
FIGURE 2.
FIGURE 2.
Identification of consensus sequence motifs in the 3′-UTR of TDP-43 and FUS mRNA targets. A, shown is the consensus sequence logo as predicted by MEME analysis of the 3′-UTR sequences of TDP-43 targets. The (TG)n motif (e value <1.0e−100; p value<1.0e−07) is present in 80% of RIP-identified targets. B, shown is the consensus sequence logo predicted for FUS targets (e value <1.0e−007; p value <1.0e−05) and identified in only 9.6% of 3′-UTR sequences. In each column, all letters with observed frequencies greater than 0.2 are shown. Single letters match that letter.
FIGURE 3.
FIGURE 3.
Validation of TDP-43 and FUS binding to selected mRNA targets by UV-CLIP assays. A, shown is a list of the RIP-chip-identified targets of TDP-43 and FUS selected for further validation. FC, fold change value corresponding to the enrichment value for TDP-43-IP or FUS-IP compared with control IgG-IP; SCA2, spinocerebellar ataxia 2. B, shown is SDS-PAGE of UV-CLIP experiment with TDP-43 antibody and radiolabeled Vegfa (first lane), Grn (third lane), Gemin7 (fifth lane), or Nrp (seventh lane) 3′-UTR riboprobes; the isotypic IgG antibody was used as a negative control (second, fourth, sixth, and eighth lanes). C, shown is SDS-PAGE of the UV-CLIP experiment with FUS antibody and radiolabeled Vps54 (first lane), Nvl (third lane), or Taf15 (fifth lane) 3′-UTRs; the isotypic IgG antibody was used as a negative control (second, fourth, and sixth lanes). The arrow indicates a FUS-containing RNP complex with an apparent molecular mass of 65 kDa. The 3′-UTR riboprobes in B and C were generated according to the GenBankTM Reference Sequences indicated in A.
FIGURE 4.
FIGURE 4.
TDP-43 specifically binds (UG)n motifs in Vegfa and Grn 3′-UTR sequences. A, shown is a representative Western blot of TDP-43 in cell lysates of mock, wt, or ΔRRM1 FLAG-hTDP-43-transfected NSC-34 cells (the arrowhead indicates FLAG-hTDP-43 protein). Tub, tubulin. B, shown is SDS-PAGE of UV-CLIP experiment with wt or ΔRRM1 FLAG-hTDP-43 and the radiolabeled Vegfa (left panel) and Grn (right panel) 3′-UTR riboprobes. The isotypic IgG antibody was used as a negative control. C, shown are SDS-PAGE of UV cross-linking assays using GST-hTDP-43 protein and radiolabeled Vegfa and Grn 3′-UTR riboprobes (first lanes) in the presence of increasing amounts (2.5, 10, and 100 ng) of unlabeled (UG)6 (second, third, and fourth lanes) or scrambled (N12) (fifth, sixth, and seventh lanes) oligoribonucleotides. D, shown is a schematic representation of the 3′ terminal portion of Vegfa 3′-UTR containing the (TG)n repeat. Different deletion fragments were generated (A–D) where only fragment C contained the TDP-43 consensus motif (TG)22. E, shown is SDS-PAGE of UV-CLIP with TDP-43 antibody and the four different radiolabeled Vegfa 3′-UTR fragments described in D. F, shown is a schematic representation of Grn 3′-UTR. A deletion fragment (del) without the TDP-43 consensus motif (TG)6 was generated. G, shown is SDS-PAGE of UV-CLIP with TDP-43 antibody and radiolabeled full length (FL) or deletion (del) Grn 3′-UTR fragments.
FIGURE 5.
FIGURE 5.
Effect of TDP-43 and FUS on the mRNA stability of their targets. A, shown is a representative Western blot of TDP-43 and FUS in cell lysates of parental (NT), mock-transfected (siCTRL), and FUS- and TDP-43-silenced (siFUS and siTDP-43, respectively) NSC-34 cells. α-Tubulin (α-Tub) was used for sample normalization. B, shown is densitometric analysis of normalized Western blot data for TDP-43 (upper inset) and FUS (lower inset) (mean ± S.E.; n = 5; one-way analysis of variance and Tukey post hoc test; ***, p < 0.001 versus NT and siCTRL). C, shown is real time PCR analysis of Tardbp (upper inset) and Fus (lower inset) mRNAs in parental (NT), siCTRL, and siTDP-43 or siFUS cells. Fold change values for each gene were calculated versus siCTRL cells (mean ± S.E.; n = 5; one-way analysis of variance and Tukey post hoc test; ***, p < 0.001 versus NT and siCTRL). D and E, mRNA content of the indicated gene was quantified by real time PCR in siCTRL and siTDP-43 or si-FUS NSC-34 cells after the indicated time of dichlorobenzamidazole riboside treatment. For each analyzed gene, the mRNA amount at each time point was compared with its initial mRNA level (100%) (n = 5; mean ± S.E.).
FIGURE 6.
FIGURE 6.
Effect of TDP-43 on the translatability of Vegfa and Grn mRNAs. A, shown is a representative Western blot of VEGF and PGRN in cell lysates of siCTRL and siTDP-43 NSC-34 cells (96-h silencing) (left). α-Tubulin (α-Tub) was used for sample normalization, and densitometric data are reported (right) (mean ± S.E.; n = 5; paired t test analysis; *, p < 0.05). B, real time PCR analysis of Vegfa and Grn mRNAs in siCTRL and siTDP-43 NSC-34 cells is shown. Fold change values for each gene were calculated versus siCTRL samples (mean ± S.E.; n = 5; paired t test analysis; *, p < 0.05). C, luciferase activity of Vegfa- and Grn-3′-UTR firefly constructs in siCTRL and siTDP-43 NSC-34 cells is shown. Normalized firefly luciferase activity was represented relative to siCTRL-transfected cells (mean ± S.E.; n = 8; paired t test analysis; *, p < 0.05; **, p < 0.01).
FIGURE 7.
FIGURE 7.
Modulation of TDP-43 influences PGRN protein content. A, shown is a representative Western blot of VEGF and PGRN in mock- and FLAG-hTDP-43-transfected NSC-34 cells (left) and densitometric analysis (mean ± S.E.; n = 4; paired t test analysis; *, p < 0.05) (right). The arrowhead indicates FLAG-hTDP-43 protein. B, real time PCR analysis of Vegfa and Grn mRNAs in mock- and FLAG-hTDP-43-transfected NSC-34 cells is shown. Fold change values for each gene were calculated versus mock-transfected samples (mean ± S.E.; n = 4). C, shown is a representative Western blot of rescue experiments with FLAG-hTDP-43 plasmid transfected for 24 h (siTDP-43 + FLAG-hTDP-43) after a 48-h TDP-43 gene silencing (siTDP-43) (left) and densitometric analysis (right) (mean ± S.E.; n = 4; one-way analysis of variance and Tukey post hoc test; *, p < 0.005). α-Tubulin (α-Tub) was used for sample normalization.
FIGURE 8.
FIGURE 8.
Effect of TDP-43 mutants on the post-transcriptional regulation of Vegfa and Grn target mRNAs. A, shown is a SDS-PAGE of UV-CLIP assay with wt or mutant (Q331K, M337V, A382T) FLAG-hTDP-43 and radiolabeled Vegfa (left) and Grn (right) 3′-UTR riboprobes. The isotypic IgG antibody was used as a negative control. B, shown is a representative Western blot of VEGF and PGRN in wt or mutant FLAG-hTDP-43-transfected NSC-34 cells (the arrowhead indicates FLAG-hTDP-43 protein). α-Tub, α-tubulin. C, shown is densitometric analysis of Western blot data (mean ± S.E.; n = 5; paired t test analysis).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Neumann M., Sampathu D. M., Kwong L. K., Truax A. C., Micsenyi M. C., Chou T. T., Bruce J., Schuck T., Grossman M., Clark C. M., McCluskey L. F., Miller B. L., Masliah E., Mackenzie I. R., Feldman H., Feiden W., Kretzschmar H. A., Trojanowski J. Q., Lee V. M. (2006) Ubiquitinated TDP-43 in frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Science 314, 130–133 - PubMed
    1. Arai T., Hasegawa M., Akiyama H., Ikeda K., Nonaka T., Mori H., Mann D., Tsuchiya K., Yoshida M., Hashizume Y., Oda T. (2006) TDP-43 is a component of ubiquitin-positive Tau-negative inclusions in frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Biochem. Biophys. Res. Commun. 351, 602–611 - PubMed
    1. Sreedharan J., Blair I. P., Tripathi V. B., Hu X., Vance C., Rogelj B., Ackerley S., Durnall J. C., Williams K. L., Buratti E., Baralle F., de Belleroche J., Mitchell J. D., Leigh P. N., Al-Chalabi A., Miller C. C., Nicholson G., Shaw C. E. (2008) TDP-43 mutations in familial and sporadic amyotrophic lateral sclerosis. Science 319, 1668–1672 - PMC - PubMed
    1. Mackenzie I. R., Rademakers R., Neumann M. (2010) TDP-43 and FUS in amyotrophic lateral sclerosis and frontotemporal dementia. Lancet Neurol. 9, 995–1007 - PubMed
    1. Lagier-Tourenne C., Polymenidou M., Cleveland D. W. (2010) TDP-43 and FUS/TLS. Emerging roles in RNA processing and neurodegeneration. Hum. Mol. Genet. 19, R46–R64 - PMC - PubMed

Publication types

MeSH terms

Associated data

LinkOut - more resources

-