Abstract
Variations in a polymorphic (TG)m sequence near exon 9 of the human CFTR gene have been associated with variable proportions of exon skipping and occurrence of disease. We have recently identified nuclear factor TDP-43 as a novel splicing regulator capable of binding to this element in the CFTR pre-mRNA and inhibiting recognition of the neighboring exon. In this study we report the dissection of the RNA binding properties of TDP-43 and their functional implications in relationship with the splicing process. Our results show that this protein contains two fully functional RNA recognition motif (RRM) domains with distinct RNA/DNA binding characteristics. Interestingly, TDP-43 can bind a minimum number of six UG (or TG) single-stranded dinucleotide stretches, and binding affinity increases with the number of repeats. In particular, the highly conserved Phe residues in the first RRM region play a key role in nucleic acid recognition.
Highlights
The study of (UG)m elements can provide further insight concerning the mRNA splicing process in general because (UG)m sequences have been described to act as splicing regulatory sequences in different genomic contexts
This finding provided a functional basis that accounted for the ability of Nucleotide sequence (5Ј-3Ј)
TDP-43 to bind RNA sequences, and in this study we provide a detailed analysis of their functionality and importance
Summary
Plasmid Construction and Oligonucleotides—Plasmids pTCTT3 and pTG12 and minigenes lacking the (TG)m/T(n) elements were obtained as previously described [1]. UV Cross-linking Assay—Plasmids were linearized by digestion with HindIII and transcription was performed with T7 RNA polymerase (Stratagene) in the presence of labeled [␣-32P]UTP, DNase-treated according to standard protocols, and purified on a Nick column (Amersham Pharmacia Biotech) according to the manufacturer’s instructions. The UV cross-linking assay was performed by adding [␣-32P]UTP-labeled RNA probes (1 ϫ 106 cpm per incubation) in a water bath for 15 min at 30 ° C with 200 ng of each different purified protein in a 20-l final volume. Unbound RNA was digested with 30 g of RNase A (Sigma) and 6 units of RNase T1 (Sigma) by incubating at 37 °C for 30 min in a water bath and adding SDS-PAGE sample buffer. Each binding reaction was performed at room temperature for 15 min by mixing the purified protein with the labeled oligo (or RNA) in a 20-l final volume.
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