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TDP-43 loss of cellular function through aggregation requires additional structural determinants beyond its C-terminal Q/N prion-like domain.

Budini M, Romano V, Quadri Z, Buratti E, Baralle FE - Hum. Mol. Genet. (2014)

Bottom Line: To our knowledge, this is the only system that achieves full functional TDP 43 depletion with effects similar to RNAi depletion or gene deletion.As a result, this model will prove useful to investigate the loss-of-function effects mediated by TDP-43 aggregation within cells without affecting the expression of the endogenous gene.These data show for the first time that cellular TDP-43 aggregation can lead to total loss of function and to defective splicing of TDP-43-dependent splicing events in endogenous genes.

View Article: PubMed Central - PubMed

Affiliation: International Centre for Genetic Engineering and Biotechnology (ICGEB), 34012 Trieste, Italy.

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Related in: MedlinePlus

(A) A schematic representation from constructs that include modifications in Flag-TDP-12xQ/N. TDP-12xQ/N F4/L (F147, 149, 229, 231/L); TDP-12xQ/N ▵RRM1/2 (deleted RMM1 and RMM2); TDP-12xQ/N ▵RRM1/2, ▵N (deleted RMM1, RMM2 and N-terminal portion); TDP-12xQ/N ▵RRM1/2, ▵C (deleted RMM1, RMM2 and C-terminal portion). In (B) is reported an RT-PCR indicating the splicing pattern of endogenous POLDIP3/SKAR gene (exon 3) after the induction or not of Flag-TDP-12xQ/N or its corresponding mutant proteins. (C) A western blot performed with an anti-SKAR antibody using proteins extracted from the same samples used in (B) The red boxes enclose the splicing and protein expression pattern of the mutant that does not induce loss of function.
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DDU415F4: (A) A schematic representation from constructs that include modifications in Flag-TDP-12xQ/N. TDP-12xQ/N F4/L (F147, 149, 229, 231/L); TDP-12xQ/N ▵RRM1/2 (deleted RMM1 and RMM2); TDP-12xQ/N ▵RRM1/2, ▵N (deleted RMM1, RMM2 and N-terminal portion); TDP-12xQ/N ▵RRM1/2, ▵C (deleted RMM1, RMM2 and C-terminal portion). In (B) is reported an RT-PCR indicating the splicing pattern of endogenous POLDIP3/SKAR gene (exon 3) after the induction or not of Flag-TDP-12xQ/N or its corresponding mutant proteins. (C) A western blot performed with an anti-SKAR antibody using proteins extracted from the same samples used in (B) The red boxes enclose the splicing and protein expression pattern of the mutant that does not induce loss of function.

Mentions: It was then of interest to use this system to investigate whether other sequences/domains of TDP-43 were contributing together with the 12xQ/N to these phenomena. To understand the contribution of the different domains of TDP-43 to induce aggregation and loss of function, we generated a variety of HEK293 stable cell lines expressing different mutants of Flag-TDP-12xQ/N (Fig. 4A). The expression of these proteins was induced by tetracycline and 72 h later TDP-43 functionality was tested by looking at the splicing of the endogenous POLDIP3/SKAR gene both at the mRNA and protein levels (Fig. 4B and C, respectively, indicated as variant 1 or variant 2).Figure 4.


TDP-43 loss of cellular function through aggregation requires additional structural determinants beyond its C-terminal Q/N prion-like domain.

Budini M, Romano V, Quadri Z, Buratti E, Baralle FE - Hum. Mol. Genet. (2014)

(A) A schematic representation from constructs that include modifications in Flag-TDP-12xQ/N. TDP-12xQ/N F4/L (F147, 149, 229, 231/L); TDP-12xQ/N ▵RRM1/2 (deleted RMM1 and RMM2); TDP-12xQ/N ▵RRM1/2, ▵N (deleted RMM1, RMM2 and N-terminal portion); TDP-12xQ/N ▵RRM1/2, ▵C (deleted RMM1, RMM2 and C-terminal portion). In (B) is reported an RT-PCR indicating the splicing pattern of endogenous POLDIP3/SKAR gene (exon 3) after the induction or not of Flag-TDP-12xQ/N or its corresponding mutant proteins. (C) A western blot performed with an anti-SKAR antibody using proteins extracted from the same samples used in (B) The red boxes enclose the splicing and protein expression pattern of the mutant that does not induce loss of function.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4262490&req=5

DDU415F4: (A) A schematic representation from constructs that include modifications in Flag-TDP-12xQ/N. TDP-12xQ/N F4/L (F147, 149, 229, 231/L); TDP-12xQ/N ▵RRM1/2 (deleted RMM1 and RMM2); TDP-12xQ/N ▵RRM1/2, ▵N (deleted RMM1, RMM2 and N-terminal portion); TDP-12xQ/N ▵RRM1/2, ▵C (deleted RMM1, RMM2 and C-terminal portion). In (B) is reported an RT-PCR indicating the splicing pattern of endogenous POLDIP3/SKAR gene (exon 3) after the induction or not of Flag-TDP-12xQ/N or its corresponding mutant proteins. (C) A western blot performed with an anti-SKAR antibody using proteins extracted from the same samples used in (B) The red boxes enclose the splicing and protein expression pattern of the mutant that does not induce loss of function.
Mentions: It was then of interest to use this system to investigate whether other sequences/domains of TDP-43 were contributing together with the 12xQ/N to these phenomena. To understand the contribution of the different domains of TDP-43 to induce aggregation and loss of function, we generated a variety of HEK293 stable cell lines expressing different mutants of Flag-TDP-12xQ/N (Fig. 4A). The expression of these proteins was induced by tetracycline and 72 h later TDP-43 functionality was tested by looking at the splicing of the endogenous POLDIP3/SKAR gene both at the mRNA and protein levels (Fig. 4B and C, respectively, indicated as variant 1 or variant 2).Figure 4.

Bottom Line: To our knowledge, this is the only system that achieves full functional TDP 43 depletion with effects similar to RNAi depletion or gene deletion.As a result, this model will prove useful to investigate the loss-of-function effects mediated by TDP-43 aggregation within cells without affecting the expression of the endogenous gene.These data show for the first time that cellular TDP-43 aggregation can lead to total loss of function and to defective splicing of TDP-43-dependent splicing events in endogenous genes.

View Article: PubMed Central - PubMed

Affiliation: International Centre for Genetic Engineering and Biotechnology (ICGEB), 34012 Trieste, Italy.

Show MeSH
Related in: MedlinePlus