Characterization and Functional Implications of the RNA Binding Properties of Nuclear Factor TDP-43, a Novel Splicing Regulator ofCFTR Exon 9

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.We have recently reported the identification of TDP-43 as a splicing regulator that specifically binds the (UG)m-repeated polymorphic region near the 3Ј-splice site of CFTR exon 9 and down-regulates its recognition by the splicing machinery (1). This region, acting in concert with the adjacent (u)n element, is one of the key cis-acting sequences which regulate the proportion of exon 9 skipping in the mature CFTR mRNA transcript (1-3). Considering that exon 9 skipping produces a non-functional CFTR protein (4, 5) the study of the RNA binding properties of TDP-43 is of considerable importance to gain further insight concerning the potential disease-causing consequences of its binding in vivo. Indeed, the clinical relevance of these studies is highlighted by the existence of a clear association between certain (TG)m(T)n alleles with distinct forms of Cystic Fibrosis (1, 6 -9).In addition, 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. In fact, in addition to the CFTR gene, the presence of simple (UG)mrepeated sequences has been described to influence the splicing process of at least two other genes: the apolipoprotein AII gene (10) and the human cardiac Na ϩ /Ca 2ϩ exchanger (11). In the Apo AII gene the UG tract was shown to be functionally equivalent to a polypyrimidine tract and required for efficient splicing of Apo AII exon 2 (10) while in the human cardiac Na ϩ /Ca 2ϩ exchanger (11) it acts as a strong intronic splicing enhancer situated in intron 2. It should be noted that in contrast with these two genes, the CFTR (UG)m element was found to possess a strong inhibitory effect on CFTR exon 9 splicing, a property that may probably be linked to its peculiar evolutionary history. In fact, sequencing of the mouse CFTR exon 9 genomic region has shown that in the flanking introns, the (TG)m(T)n regulatory elements are absent and that the intron themselves are of very different length when compared with the human introns (...

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

“…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.…”

“…10,11 Previous studies have shown that mutations of TDP-43 are linked with neurodegenerative diseases, in particular, amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). [12][13][14][15][16][17] In addition, aberrant cleavage and cytoplasmic aggregation of TDP-43 are identified as molecular signatures for most forms of ALS and FTLD and contribute significantly to disease progression.…”

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

“…5a). GFP-tagged mTDP-43-FL and RNA-binding-deficient mutant, F147, 149L (RD) were transfected into 293T cells 36 . Compared with cells transfected with FL, mutant transfectants showed a 20-fold increase in granule formation (Fig.…”

Linking RNA Dysfunction and Neurodegeneration in Amyotrophic Lateral Sclerosis

“…The RNA-binding domains of TDP43 and FUS are essential for toxicity in ALS model systems, testifying to the importance of RNA dysregulation in ALS pathogenesis [34,[39][40][41]. Immunoprecipitation studies in murine brain [14], dissociated primary cortical neurons [37], and human brain [13] and cell lines [13,38] showed that TDP43 recognizes nearly one-thrid of all transcribed genes by binding to redundant GU-rich sequences [42]. While nuclear TDP43 binds preferentially to distal intronic sequences, cytoplasmic TDP43 recognizes more 3' untranslated region (UTR) binding sites [13].…”