TDP-43 loss of cellular function through aggregation requires additional structural determinants beyond its C-terminal Q/N prion-like domain.
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.
Affiliation: International Centre for Genetic Engineering and Biotechnology (ICGEB), 34012 Trieste, Italy.Show MeSH
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Mentions: As previously described, loss of TDP-43 function (e.g. by siRNA TDP-43 downregulation) can be easily observed using a minigene system based on the inclusion/skipping of CFTR exon 9 (13,35). Therefore, cells expressing Flag-TDP-12xQ/N and Flag-TDP-43 WT (used as a control) were transfected with this minigene and after 24 h post-transfection the splicing pattern of exon 9 was evaluated by RT-PCR. In order to have a positive control of TDP-43 loss of function, we knocked down TDP-43 by transfecting siTDP-43 against this protein. Knockdown of TDP-43 resulted in the complete inclusion of exon 9 (Fig. 3A, compare lines 1 and 2). In keeping with expectations, the overexpression of Flag-TDP-43 WT repressed the inclusion of exon 9 (Fig. 3A, compare lines 3 and 4), while the overexpression of Flag-TDP-12xQ/N induced the inclusion of this exon in a similar way as the siTDP-43 treatment (Fig. 3A, compare lines 5, 6 with 1, 2). This last observation is compatible with loss of active nuclear TDP-43 following its aggregation in the nucleus/cytoplasm.Figure 3.
Affiliation: International Centre for Genetic Engineering and Biotechnology (ICGEB), 34012 Trieste, Italy.