Nuclear factor TDP-43 and SR proteins promote in vitro and in vivo CFTR exon 9 skipping

Abstract

Alternative splicing of human cystic ®brosis transmembrane conductance regulator (CFTR) exon 9 is regulated by a combination of cis-acting elements distributed through the exon and both¯anking introns (IVS8 and IVS9). Several studies have identi®ed in the IVS8 intron 3¢ splice site a regulatory element that is composed of a polymorphic (TG)m(T)n repeated sequence. At present, no cellular factors have been identi®ed that recognize this element. We have identi®ed TDP-43, a nuclear protein not previously described to bind RNA, as the factor binding speci®c-ally to the (TG)m sequence. Transient TDP-43 overexpression in Hep3B cells results in an increase in exon 9 skipping. This effect is more pronounced with concomitant overexpression of SR proteins. Antisense inhibition of endogenous TDP-43 expression results in increased inclusion of exon 9, providing a new therapeutic target to correct aberrant splicing of exon 9 in CF patients. The clinical and biological relevance of this ®nding in vivo is demonstrated by our characterization of a CF patient carrying a TG10T9(DF508)/ TG13T3(wt) genotype leading to a disease-causing high proportion of exon 9 skipping.

Cytoplasmic translocation, aggregation, and cleavage of TDP-43 by enteroviral proteases modulate viral pathogenesis

“…TDP-43 functional activity was evaluated through measuring exon 9 splicing of cystic fibrosis transmembrane conductance regulator (CFTR) gene as previously documented. 17,34 Briefly, HeLa cells were transiently cotransfected with a CFTR minigene reporter construct (a generous gift from Dr. E Buratti from the International Centre for Genetic Engineering and Biotechnology, Trieste, Italy) and various TDP-43 constructs for 24 h. Total RNA was extracted by QIAGEN RNeasy kit (#74104) and equal amounts of RNA (500 ng) were used for RT-PCR for the detection of exon 9 splicing using primers a2-3 and Bra2 as previously described. 17,34 PCR products were visualized on a 1.5% agarose gel and quantified using ImageJ software.…”

“…TDP-43 plays an important role in the regulation of alternative splicing. [34][35][36] To determine whether TDP-43-N has an impact on the function of native TDP-43 in RNA splicing, we utilized a well-established technique, CFTR exon 9 skipping. 17,34 TDP-43 facilitates exon 9 skipping by interacting with UG repeats in intron 8 of CFTR pre-mRNA, thus generating an exon 9-deficient transcript at resting states.…”

“…17,34 TDP-43 facilitates exon 9 skipping by interacting with UG repeats in intron 8 of CFTR pre-mRNA, thus generating an exon 9-deficient transcript at resting states. 10,11,34 HeLa cells were transiently transfected with a CFTR minigene reporter construct (TG (13) T (5)), 17,34 together with empty vector, full-length TDP-43, or TDP-43-N. Transfection was carried out for 24 h to achieve desired expression levels of TDP-43-N similar to those observed in virus-infected cells. RT-PCR was performed to detect the transcripts of CFTR reporter plasmid.…”

The self-interaction of native TDP-43 C terminus inhibits its degradation and contributes to early proteinopathies

“…Among mammals, zebrafish and flies, the protein sequence, biochemical properties and biological functions of TDP-43 are well conserved. In the nucleus, TDP-43 forms visible 50-250-nm granules that serve as a scaffold to link other functionally related sub-compartments, and participates in transcriptional repression as well as alternative splicing [9][10][11][12] . TDP-43 specifically binds to the UG repeat sequences in RNA, altering pre-mRNA splicing of cystic fibrosis transmembrane conductance regulator (CFTR) and may associate with long non-coding RNA to regulate nuclear speckle or paraspeckle 11,13 .…”

“…In the nucleus, TDP-43 forms visible 50-250-nm granules that serve as a scaffold to link other functionally related sub-compartments, and participates in transcriptional repression as well as alternative splicing [9][10][11][12] . TDP-43 specifically binds to the UG repeat sequences in RNA, altering pre-mRNA splicing of cystic fibrosis transmembrane conductance regulator (CFTR) and may associate with long non-coding RNA to regulate nuclear speckle or paraspeckle 11,13 . TDP-43 mutants lacking a C-terminal glycine-rich region fail to excise exon 9 in the CFTR gene 9 , but how TDP-43 C terminus governs exon 9 skipping of CFTR, as well as its role in pathogenesis of TDP-43 proteinopathies, is still unknown.…”