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Spliceosome integrity is defective in the motor neuron diseases ALS and SMA.

Tsuiji H, Iguchi Y, Furuya A, Kataoka A, Hatsuta H, Atsuta N, Tanaka F, Hashizume Y, Akatsu H, Murayama S, Sobue G, Yamanaka K - EMBO Mol Med (2013)

Bottom Line: We also show that in ALS, Gems are lost, U snRNA levels are up-regulated and spliceosomal U snRNPs abnormally and extensively accumulate in motor neuron nuclei, but not in the temporal lobe of FTLD with TDP-43 pathology.This aberrant accumulation of U snRNAs in ALS motor neurons is in direct contrast to SMA motor neurons, which show reduced amounts of U snRNAs, while both have defects in the spliceosome.These findings indicate that a profound loss of spliceosome integrity is a critical mechanism common to neurodegeneration in ALS and SMA, and may explain cell-type specific vulnerability of motor neurons.

View Article: PubMed Central - PubMed

Affiliation: Laboratory for Motor Neuron Disease, RIKEN Brain Science Institute, Wako, Saitama, Japan. hitomitsuiji@brain.riken.jp

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TDP-43 is associated with U snRNPs and is required for maintaining proper expression levels of U snRNAsA. TDP-43 colocalized with U snRNPs that were marked with an anti-dimethylated Sm proteins antibody (Y12) in the nuclei of Hela cells and primary cultured mouse hippocampal neurons. Note that U snRNPs and TDP-43 were concentrated in the same nuclear bodies (arrows). Bar: 10 µm.B,C. U snRNA levels in Hela cells (B) or SH-SY5Y cells (C) treated with siRNAs for TDP-43 or control were determined by quantitative RT-PCR. Average from three independent experiments with transfections performed in triplicate were plotted (Bars: standard errors, *p < 0.05, **p < 0.01, Student's t-test).D. Mature U snRNP-associated U snRNA levels in Hela cells treated with siRNAs for TDP-43 or control. U snRNA levels were determined by quantitative RT-PCR from the RNAs isolated from mature U snRNP complex which was immunopurified using anti-Sm proteins antibody (Y12) as described in Materials and Methods.
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fig04: TDP-43 is associated with U snRNPs and is required for maintaining proper expression levels of U snRNAsA. TDP-43 colocalized with U snRNPs that were marked with an anti-dimethylated Sm proteins antibody (Y12) in the nuclei of Hela cells and primary cultured mouse hippocampal neurons. Note that U snRNPs and TDP-43 were concentrated in the same nuclear bodies (arrows). Bar: 10 µm.B,C. U snRNA levels in Hela cells (B) or SH-SY5Y cells (C) treated with siRNAs for TDP-43 or control were determined by quantitative RT-PCR. Average from three independent experiments with transfections performed in triplicate were plotted (Bars: standard errors, *p < 0.05, **p < 0.01, Student's t-test).D. Mature U snRNP-associated U snRNA levels in Hela cells treated with siRNAs for TDP-43 or control. U snRNA levels were determined by quantitative RT-PCR from the RNAs isolated from mature U snRNP complex which was immunopurified using anti-Sm proteins antibody (Y12) as described in Materials and Methods.

Mentions: The association and localization of TDP-43, FUS/TLS and SMN in Gems imply a functional convergence of three proteins. SMN is well known to assist in assembly of U snRNPs, which is central to splicing, in the cytoplasm and to recruit U snRNPs into the nucleus (Pellizzoni et al, 2002). In SMA mice and SMN-depleted cells, the levels of U snRNAs and components of U snRNPs are unbalanced, resulting in aberrant splicing (Gabanella et al, 2007; Zhang et al, 2008). Therefore, we hypothesized that TDP-43 might have a function in U snRNP biogenesis and alterations in U snRNPs may be also responsible for ALS. To test this hypothesis, we first analysed if TDP-43 associated with U snRNPs. TDP-43 distribution was similar to U snRNPs, which were marked by the anti-dimethylated Sm proteins antibody (Y12), and both TDP-43 and U snRNPs were concentrated to same nuclear bodies in Hela cells and primary cultured mouse hippocampal neurons (Fig 4A, arrows), suggesting the interaction of TDP-43 and snRNPs. Since C-terminus of TDP-43 is required for TDP-43-containing foci in nuclei (Fig 2B, Supporting Information Fig S3C), we identified proteins interacting with C-terminus of TDP-43. Comparison of proteins immunoprecipitated with wild type FLAG-tagged TDP-43 or deletion mutant of C-terminal domain, followed by LC–MS/MS, revealed many proteins associated with a TDP-43 C-terminus including U snRNP components such as PRPF3 (Supporting Information Fig S4A and B). The association of TDP-43 with U snRNP components PRPF3 and U1-70K was confirmed by IP-Western blotting (Supporting Information Fig S4C). To investigate the association between U snRNPs and TDP-43, U snRNPs were immunoprecipitated with the anti-Sm proteins antibody (Y12) (Supporting Information Fig S4D). Subsequent immunoblotting confirmed that TDP-43 was co-immunoprecipitated with U snRNPs although at a relatively low level. These results suggest a possible involvement of TDP-43 in maintaining the integrity of U snRNPs.


Spliceosome integrity is defective in the motor neuron diseases ALS and SMA.

Tsuiji H, Iguchi Y, Furuya A, Kataoka A, Hatsuta H, Atsuta N, Tanaka F, Hashizume Y, Akatsu H, Murayama S, Sobue G, Yamanaka K - EMBO Mol Med (2013)

TDP-43 is associated with U snRNPs and is required for maintaining proper expression levels of U snRNAsA. TDP-43 colocalized with U snRNPs that were marked with an anti-dimethylated Sm proteins antibody (Y12) in the nuclei of Hela cells and primary cultured mouse hippocampal neurons. Note that U snRNPs and TDP-43 were concentrated in the same nuclear bodies (arrows). Bar: 10 µm.B,C. U snRNA levels in Hela cells (B) or SH-SY5Y cells (C) treated with siRNAs for TDP-43 or control were determined by quantitative RT-PCR. Average from three independent experiments with transfections performed in triplicate were plotted (Bars: standard errors, *p < 0.05, **p < 0.01, Student's t-test).D. Mature U snRNP-associated U snRNA levels in Hela cells treated with siRNAs for TDP-43 or control. U snRNA levels were determined by quantitative RT-PCR from the RNAs isolated from mature U snRNP complex which was immunopurified using anti-Sm proteins antibody (Y12) as described in Materials and Methods.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig04: TDP-43 is associated with U snRNPs and is required for maintaining proper expression levels of U snRNAsA. TDP-43 colocalized with U snRNPs that were marked with an anti-dimethylated Sm proteins antibody (Y12) in the nuclei of Hela cells and primary cultured mouse hippocampal neurons. Note that U snRNPs and TDP-43 were concentrated in the same nuclear bodies (arrows). Bar: 10 µm.B,C. U snRNA levels in Hela cells (B) or SH-SY5Y cells (C) treated with siRNAs for TDP-43 or control were determined by quantitative RT-PCR. Average from three independent experiments with transfections performed in triplicate were plotted (Bars: standard errors, *p < 0.05, **p < 0.01, Student's t-test).D. Mature U snRNP-associated U snRNA levels in Hela cells treated with siRNAs for TDP-43 or control. U snRNA levels were determined by quantitative RT-PCR from the RNAs isolated from mature U snRNP complex which was immunopurified using anti-Sm proteins antibody (Y12) as described in Materials and Methods.
Mentions: The association and localization of TDP-43, FUS/TLS and SMN in Gems imply a functional convergence of three proteins. SMN is well known to assist in assembly of U snRNPs, which is central to splicing, in the cytoplasm and to recruit U snRNPs into the nucleus (Pellizzoni et al, 2002). In SMA mice and SMN-depleted cells, the levels of U snRNAs and components of U snRNPs are unbalanced, resulting in aberrant splicing (Gabanella et al, 2007; Zhang et al, 2008). Therefore, we hypothesized that TDP-43 might have a function in U snRNP biogenesis and alterations in U snRNPs may be also responsible for ALS. To test this hypothesis, we first analysed if TDP-43 associated with U snRNPs. TDP-43 distribution was similar to U snRNPs, which were marked by the anti-dimethylated Sm proteins antibody (Y12), and both TDP-43 and U snRNPs were concentrated to same nuclear bodies in Hela cells and primary cultured mouse hippocampal neurons (Fig 4A, arrows), suggesting the interaction of TDP-43 and snRNPs. Since C-terminus of TDP-43 is required for TDP-43-containing foci in nuclei (Fig 2B, Supporting Information Fig S3C), we identified proteins interacting with C-terminus of TDP-43. Comparison of proteins immunoprecipitated with wild type FLAG-tagged TDP-43 or deletion mutant of C-terminal domain, followed by LC–MS/MS, revealed many proteins associated with a TDP-43 C-terminus including U snRNP components such as PRPF3 (Supporting Information Fig S4A and B). The association of TDP-43 with U snRNP components PRPF3 and U1-70K was confirmed by IP-Western blotting (Supporting Information Fig S4C). To investigate the association between U snRNPs and TDP-43, U snRNPs were immunoprecipitated with the anti-Sm proteins antibody (Y12) (Supporting Information Fig S4D). Subsequent immunoblotting confirmed that TDP-43 was co-immunoprecipitated with U snRNPs although at a relatively low level. These results suggest a possible involvement of TDP-43 in maintaining the integrity of U snRNPs.

Bottom Line: We also show that in ALS, Gems are lost, U snRNA levels are up-regulated and spliceosomal U snRNPs abnormally and extensively accumulate in motor neuron nuclei, but not in the temporal lobe of FTLD with TDP-43 pathology.This aberrant accumulation of U snRNAs in ALS motor neurons is in direct contrast to SMA motor neurons, which show reduced amounts of U snRNAs, while both have defects in the spliceosome.These findings indicate that a profound loss of spliceosome integrity is a critical mechanism common to neurodegeneration in ALS and SMA, and may explain cell-type specific vulnerability of motor neurons.

View Article: PubMed Central - PubMed

Affiliation: Laboratory for Motor Neuron Disease, RIKEN Brain Science Institute, Wako, Saitama, Japan. hitomitsuiji@brain.riken.jp

Show MeSH
Related in: MedlinePlus