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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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Determination of domains required for association of TDP-43, FUS/TLS and SMN complexA. A schematic diagram of C-terminal 3XFLAG-tagged expression constructs for TDP-43 used in this study.B. The latter half of the C-terminal glycine-rich region of TDP-43 was important for the proper localization to Gems. Hela cells were transfected with TDP-43-3XFLAG or indicated mutants, and stained with anti-SMN and anti-FLAG antibodies. Co-localization of TDP-43 and SMN was assessed by confocal microscope, numbers of TDP-43-positive Gems and -negative Gems were counted. More than 100 Gems were counted for each construct, and the localization to Gem (%) was defined as TDP-43-postive Gems per total Gems (%). To eliminate variation in the number of Gems per nucleus, cloned Hela cells were used. The average and error bars from three independent experiments were plotted.C,D. The SMN/Gemin8/FUS interactions with TDP-43 were dependent on the TDP-43 C-terminus. TDP-43-3xFLAG mutants were expressed in Hela cells, and the TDP-43 interacting proteins were immunoprecipitated using an anti-FLAG antibody and identified by Western blot analysis using the specific antibodies as indicated. Asterisks indicate degraded FLAG-tagged TDP-43 proteins.E. A schematic diagram of N-terminal 3XFLAG-tagged expression constructs for FUS/TLS used in this study.F. 3xFLAG-FUS/TLS mutants were expressed in Hela cells, and the FUS/TLS interacting proteins were immunoprecipitated using an anti-FLAG antibody and identified by Western blot analysis using the specific antibodies as indicated.G. Mutations in Tudor domain of SMN1 decreased association of TDP-43 and FUS/TLS. HA-tagged human SMN1 and its mutants were expressed in Hela cells, and SMN interacting proteins were immunoprecipitated using an anti-HA antibody and identified by Western blot analysis using the specific antibodies as indicated.
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fig02: Determination of domains required for association of TDP-43, FUS/TLS and SMN complexA. A schematic diagram of C-terminal 3XFLAG-tagged expression constructs for TDP-43 used in this study.B. The latter half of the C-terminal glycine-rich region of TDP-43 was important for the proper localization to Gems. Hela cells were transfected with TDP-43-3XFLAG or indicated mutants, and stained with anti-SMN and anti-FLAG antibodies. Co-localization of TDP-43 and SMN was assessed by confocal microscope, numbers of TDP-43-positive Gems and -negative Gems were counted. More than 100 Gems were counted for each construct, and the localization to Gem (%) was defined as TDP-43-postive Gems per total Gems (%). To eliminate variation in the number of Gems per nucleus, cloned Hela cells were used. The average and error bars from three independent experiments were plotted.C,D. The SMN/Gemin8/FUS interactions with TDP-43 were dependent on the TDP-43 C-terminus. TDP-43-3xFLAG mutants were expressed in Hela cells, and the TDP-43 interacting proteins were immunoprecipitated using an anti-FLAG antibody and identified by Western blot analysis using the specific antibodies as indicated. Asterisks indicate degraded FLAG-tagged TDP-43 proteins.E. A schematic diagram of N-terminal 3XFLAG-tagged expression constructs for FUS/TLS used in this study.F. 3xFLAG-FUS/TLS mutants were expressed in Hela cells, and the FUS/TLS interacting proteins were immunoprecipitated using an anti-FLAG antibody and identified by Western blot analysis using the specific antibodies as indicated.G. Mutations in Tudor domain of SMN1 decreased association of TDP-43 and FUS/TLS. HA-tagged human SMN1 and its mutants were expressed in Hela cells, and SMN interacting proteins were immunoprecipitated using an anti-HA antibody and identified by Western blot analysis using the specific antibodies as indicated.

Mentions: To identify the region within TDP-43 that is required for localization to Gems, we performed domain analysis using deletion mutants of TDP-43 (Fig 2A). Our analysis revealed that the C-terminus of TDP-43, where several known ALS-linked mutations are located, was responsible for the proper localization to Gems (Fig 2B and Supporting Information Fig S3). More specifically, amino acids 321–366 of the C-terminus, which interact with hnRNP A2 (D'Ambrogio et al, 2009), were important for localization to Gems (Fig 2B). Furthermore, the RNA-binding activity of TDP-43 is partially required, as both RNA-recognition motif 1 (RRM1) deletion mutants and F147L/F149L double point mutants had reduced localizations to Gems (Fig 2B). These results indicate that protein–protein interactions mediated by the latter half of the C-terminal region and the RNA binding activity play critical roles in the proper localization of TDP-43 to Gems.


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)

Determination of domains required for association of TDP-43, FUS/TLS and SMN complexA. A schematic diagram of C-terminal 3XFLAG-tagged expression constructs for TDP-43 used in this study.B. The latter half of the C-terminal glycine-rich region of TDP-43 was important for the proper localization to Gems. Hela cells were transfected with TDP-43-3XFLAG or indicated mutants, and stained with anti-SMN and anti-FLAG antibodies. Co-localization of TDP-43 and SMN was assessed by confocal microscope, numbers of TDP-43-positive Gems and -negative Gems were counted. More than 100 Gems were counted for each construct, and the localization to Gem (%) was defined as TDP-43-postive Gems per total Gems (%). To eliminate variation in the number of Gems per nucleus, cloned Hela cells were used. The average and error bars from three independent experiments were plotted.C,D. The SMN/Gemin8/FUS interactions with TDP-43 were dependent on the TDP-43 C-terminus. TDP-43-3xFLAG mutants were expressed in Hela cells, and the TDP-43 interacting proteins were immunoprecipitated using an anti-FLAG antibody and identified by Western blot analysis using the specific antibodies as indicated. Asterisks indicate degraded FLAG-tagged TDP-43 proteins.E. A schematic diagram of N-terminal 3XFLAG-tagged expression constructs for FUS/TLS used in this study.F. 3xFLAG-FUS/TLS mutants were expressed in Hela cells, and the FUS/TLS interacting proteins were immunoprecipitated using an anti-FLAG antibody and identified by Western blot analysis using the specific antibodies as indicated.G. Mutations in Tudor domain of SMN1 decreased association of TDP-43 and FUS/TLS. HA-tagged human SMN1 and its mutants were expressed in Hela cells, and SMN interacting proteins were immunoprecipitated using an anti-HA antibody and identified by Western blot analysis using the specific antibodies as indicated.
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Related In: Results  -  Collection

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fig02: Determination of domains required for association of TDP-43, FUS/TLS and SMN complexA. A schematic diagram of C-terminal 3XFLAG-tagged expression constructs for TDP-43 used in this study.B. The latter half of the C-terminal glycine-rich region of TDP-43 was important for the proper localization to Gems. Hela cells were transfected with TDP-43-3XFLAG or indicated mutants, and stained with anti-SMN and anti-FLAG antibodies. Co-localization of TDP-43 and SMN was assessed by confocal microscope, numbers of TDP-43-positive Gems and -negative Gems were counted. More than 100 Gems were counted for each construct, and the localization to Gem (%) was defined as TDP-43-postive Gems per total Gems (%). To eliminate variation in the number of Gems per nucleus, cloned Hela cells were used. The average and error bars from three independent experiments were plotted.C,D. The SMN/Gemin8/FUS interactions with TDP-43 were dependent on the TDP-43 C-terminus. TDP-43-3xFLAG mutants were expressed in Hela cells, and the TDP-43 interacting proteins were immunoprecipitated using an anti-FLAG antibody and identified by Western blot analysis using the specific antibodies as indicated. Asterisks indicate degraded FLAG-tagged TDP-43 proteins.E. A schematic diagram of N-terminal 3XFLAG-tagged expression constructs for FUS/TLS used in this study.F. 3xFLAG-FUS/TLS mutants were expressed in Hela cells, and the FUS/TLS interacting proteins were immunoprecipitated using an anti-FLAG antibody and identified by Western blot analysis using the specific antibodies as indicated.G. Mutations in Tudor domain of SMN1 decreased association of TDP-43 and FUS/TLS. HA-tagged human SMN1 and its mutants were expressed in Hela cells, and SMN interacting proteins were immunoprecipitated using an anti-HA antibody and identified by Western blot analysis using the specific antibodies as indicated.
Mentions: To identify the region within TDP-43 that is required for localization to Gems, we performed domain analysis using deletion mutants of TDP-43 (Fig 2A). Our analysis revealed that the C-terminus of TDP-43, where several known ALS-linked mutations are located, was responsible for the proper localization to Gems (Fig 2B and Supporting Information Fig S3). More specifically, amino acids 321–366 of the C-terminus, which interact with hnRNP A2 (D'Ambrogio et al, 2009), were important for localization to Gems (Fig 2B). Furthermore, the RNA-binding activity of TDP-43 is partially required, as both RNA-recognition motif 1 (RRM1) deletion mutants and F147L/F149L double point mutants had reduced localizations to Gems (Fig 2B). These results indicate that protein–protein interactions mediated by the latter half of the C-terminal region and the RNA binding activity play critical roles in the proper localization of TDP-43 to Gems.

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