Cristiana Stuani

A new type of mutation causes a splicing defect in ATM.

Disease-causing splicing mutations described in the literature primarily produce changes in splice sites and, to a lesser extent, variations in exon-regulatory sequences such as the enhancer elements. The gene ATM is mutated in individuals with ataxia-telangiectasia; we have indentified the aberrant inclusion of a cryptic exon of 65 bp in one affected individual with a deletion of four nucleotides (GTAA) in intron 20. The deletion is located 12 bp downstream and 53 bp upstream from the 5′ and 3′ ends of the cryptic exon, respectively. Through analysis of the splicing defect using a hybrid minigene system, we identified a new intron-splicing processing element (ISPE) complementary to U1 snRNA, the RNA component of the U1 small nuclear ribonucleoprotein (snRNP). This element mediates accurate intron processing and interacts specifically with U1 snRNP particles. The 4-nt deletion completely abolished this interaction, causing activation of the cryptic exon. On the basis of this analysis, we describe a new type of U1 snRNP binding site in an intron that is essential for accurate intron removal. Deletion of this sequence is directly involved in the splicing processing defect.

Nuclear factor TDP-43 can affect selected microRNA levels.

TDP‐43 has recently been described as the major component of the inclusions found in the brain of patients with a variety of neurodegenerative diseases, such as frontotemporal lobar degeneration and amyotrophic lateral sclerosis. TDP‐43 is a ubiquitous protein whose specific functions are probably crucial to establishing its pathogenic role. Apart from its involvement in transcription, splicing and mRNA stability, TDP‐43 has also been described as a Drosha‐associated protein. However, our knowledge of the role of TDP‐43 in the microRNA (miRNA) synthesis pathway is limited to the association mentioned above. Here we report for the first time which changes occur in the total miRNA population following TDP‐43 knockdown in culture cells. In particular, we have observed that let‐7b and miR‐663 expression levels are down‐ and upregulated, respectively. Interestingly, both miRNAs are capable of binding directly to TDP‐43 in different positions: within the miRNA sequence itself (let‐7b) or in the hairpin precursor (miR‐663). Using microarray data and real‐time PCR we have also identified several candidate transcripts whose expression levels are selectively affected by these TDP‐43–miRNA interactions.

Functional mapping of the interaction between TDP-43 and hnRNP A2 in vivo

Nuclear factor TDP-43 has been reported to play multiple roles in transcription, pre-mRNA splicing, mRNA stability and mRNA transport. From a structural point of view, TDP-43 is a member of the hnRNP protein family whose structure includes two RRM domains flanked by the N-terminus and C-terminal regions. Like many members of this family, the C-terminal region can interact with cellular factors and thus serve to modulate its function. Previously, we have described that TDP-43 binds to several members of the hnRNP A/B family through this region. In this work, we set up a coupled minigene/siRNA cellular system that allows us to obtain in vivo data to address the functional significance of TDP-43-recruited hnRNP complex formation. Using this method, we have finely mapped the interaction between TDP-43 and the hnRNP A2 protein to the region comprised between amino acid residues 321 and 366. Our results provide novel details of protein–protein interactions in splicing regulation. In addition, we provide further insight on TDP-43 functional properties, particularly the lack of effects, as seen with our assays, of the disease-associated mutations that fall within the TDP-43 321-366 region: Q331K, M337V and G348C.

Molecular basis of UG-rich RNA recognition by the human splicing factor TDP-43

TDP-43 encodes an alternative-splicing regulator with tandem RNA-recognition motifs (RRMs). The protein regulates cystic fibrosis transmembrane regulator (CFTR) exon 9 splicing through binding to long UG-rich RNA sequences and is found in cytoplasmic inclusions of several neurodegenerative diseases. We solved the solution structure of the TDP-43 RRMs in complex with UG-rich RNA. Ten nucleotides are bound by both RRMs, and six are recognized sequence specifically. Among these, a central G interacts with both RRMs and stabilizes a new tandem RRM arrangement. Mutations that eliminate recognition of this key nucleotide or crucial inter-RRM interactions disrupt RNA binding and TDP-43–dependent splicing regulation. In contrast, point mutations that affect base-specific recognition in either RRM have weaker effects. Our findings reveal not only how TDP-43 recognizes UG repeats but also how RNA binding–dependent inter-RRM interactions are crucial for TDP-43 function.

Promoter Architecture Modulates CFTR Exon 9 Skipping

Using hybrid minigene experiments, we have investigated the role of the promoter architecture on the regulation of two alternative spliced exons, cystic fibrosis transmembrane regulator (CFTR) exon 9 and fibronectin extra domain-A (EDB). A specific alternative splicing pattern corresponded to each analyzed promoter. Promoter-dependent sensitivity to cotransfected regulatory splicing factor SF2/ASF was observed only for the CFTR exon 9, whereas that of the EDB was refractory to promoter-mediated regulation. Deletion in the CFTR minigene of the downstream intronic splicing silencer element binding SF2/ASF abolished the specific promoter-mediated response to this splicing factor. A systematic analysis of the regulatory cis-acting elements showed that in the presence of suboptimal splice sites or by deletion of exonic enhancer elements the promoter-dependent sensitivity to splicing factor-mediated inhibition was lost. However, the basal regulatory effect of each promoter was preserved. The complex relationships between the promoter-dependent sensitivity to SF2 modulated by the exon 9 definition suggest a kinetic model of promoter-dependent alternative splicing regulation that possibly involves differential RNA polymerase II elongation.

Cellular Model of TAR DNA-binding Protein 43 (TDP-43) Aggregation Based on Its C-terminal Gln/Asn-rich Region

Background: TDP-43 is the principal protein component of cellular inclusion in ALS and FTLD. Results: Tandem repetitions of TDP-43 residues 331–369 induce cellular aggregates that recruit endogenous TDP-43. Conclusion: Our results establish a cell-based TDP-43 aggregation model. Significance: This model will be useful to investigate TDP-43 aggregation and develop strategies/effectors able to prevent/reduce this phenomenon. TDP-43 is one of the major components of the neuronal and glial inclusions observed in several neurodegenerative diseases such as amyotrophic lateral sclerosis and frontotemporal lobar degeneration. These characteristic aggregates are a “landmark” of the disease, but their role in the pathogenesis is still obscure. In previous works, we have shown that the C-terminal Gln/Asn-rich region (residues 321–366) of TDP-43 is involved in the interaction of this protein with other members of the heterogeneous nuclear ribonucleoprotein protein family. Furthermore, we have shown that the interaction through this region is important for TDP-43 splicing inhibition of cystic fibrosis transmembrane regulator exon 9, and there were indications that it was involved in the aggregation process. Our experiments show that in cell lines and primary rat neuronal cultures, the introduction of tandem repeats carrying the 331–369-residue Gln/Asn region from TDP-43 can trigger the formation of phosphorylated and ubiquitinated aggregates that recapitulate many but not all the characteristics observed in patients. These results establish a much needed cell-based TDP-43 aggregation model useful to investigate the mechanisms involved in the formation of inclusions and the gain- and loss-of-function consequences of TDP-43 aggregation within cells. In addition, it will be a powerful tool to test novel therapeutic strategies/effectors aimed at preventing/reducing this phenomenon.

SR protein-mediated inhibition of CFTR exon 9 inclusion: molecular characterization of the intronic splicing silencer

The intronic splicing silencer (ISS) of CFTR exon 9 promotes exclusion of this exon from the mature mRNA. This negative influence has important consequences with regards to human pathologic events, as lack of exon 9 correlates well with the occurrence of monosymptomatic and full forms of CF disease. We have previously shown that the ISS element interacts with members of the SR protein family. In this work, we now provide the identification of SF2/ASF and SRp40 as the specific SR proteins binding to this element and map their precise binding sites in IVS9. We have also performed a functional analysis of the ISS element using a variety of unrelated SR-binding sequences and different splicing systems. Our results suggest that SR proteins mediate CFTR exon 9 exclusion by providing a ‘decoy’ sequence in the vicinity of its suboptimal donor site. The results of this study give an insight on intron ‘exonization’ mechanisms and provide useful indications for the development of novel therapeutic strategies aimed at the recovery of exon inclusion.

Misregulation of human sortilin splicing leads to the generation of a nonfunctional progranulin receptor.

Sortilin 1 regulates the levels of brain progranulin (PGRN), a neurotrophic growth factor that, when deficient, is linked to cases of frontotemporal lobar degeneration with TAR DNA-binding protein-43 (TDP-43)–positive inclusions (FTLD-TDP). We identified a specific splicing enhancer element that regulates the inclusion of a sortilin exon cassette (termed Ex17b) not normally present in the mature sortilin mRNA. This enhancer element is consistently present in sortilin RNA of mice and other species but absent in primates, which carry a premature stop codon within the Ex17b sequence. In the absence of TDP-43, which acts as a regulatory inhibitor, Ex17b is included in the sortilin mRNA. In humans, in contrast to mice, the inclusion of Ex17b in sortilin mRNA generates a truncated, nonfunctional, extracellularly released protein that binds to but does not internalize PGRN, essentially acting as a decoy receptor. Based on these results, we propose a potential mechanism linking misregulation of sortilin splicing with altered PGRN metabolism.

The TDP-43 N-terminal domain structure at high resolution.

Transactive response DNA‐binding protein 43 kDa (TDP‐43) is an RNA transporting and processing protein whose aberrant aggregates are implicated in neurodegenerative diseases. The C‐terminal domain of this protein plays a key role in mediating this process. However, the N‐terminal domain (residues 1–77) is needed to effectively recruit TDP‐43 monomers into this aggregate. In the present study, we report, for the first time, the essentially complete 1H, 15N and 13C NMR assignments and the structure of the N‐terminal domain determined on the basis of 26 hydrogen‐bond, 60 torsion angle and 1058 unambiguous NOE structural restraints. The structure consists of an α‐helix and six β‐strands. Two β‐strands form a β‐hairpin not seen in the ubiquitin fold. All Pro residues are in the trans conformer and the two Cys are reduced and distantly separated on the surface of the protein. The domain has a well defined hydrophobic core composed of F35, Y43, W68, Y73 and 17 aliphatic side chains. The fold is topologically similar to the reported structure of axin 1. The protein is stable and no denatured species are observed at pH 4 and 25 °C. At 4 kcal·mol−1, the conformational stability of the domain, as measured by hydrogen/deuterium exchange, is comparable to ubiquitin (6 kcal·mol−1). The β‐strands, α‐helix, and three of four turns are generally rigid, although the loop formed by residues 47–53 is mobile, as determined by model‐free analysis of the 15N{1H}NOE, as well as the translational and transversal relaxation rates.

Functional studies on the ATM intronic splicing processing element

In disease-associated genes, the understanding of the functional significance of deep intronic nucleotide variants may represent a difficult challenge. We have previously reported a new disease-causing mechanism that involves an intronic splicing processing element (ISPE) in ATM, composed of adjacent consensus 5′ and 3′ splice sites. A GTAA deletion within ISPE maintains potential adjacent splice sites, disrupts a non-canonical U1 snRNP interaction and activates an aberrant exon. In this paper, we demonstrate that binding of U1 snRNA through complementarity within a ∼40 nt window downstream of the ISPE prevents aberrant splicing. By selective mutagenesis at the adjacent consensus ISPE splice sites, we show that this effect is not due to a resplicing process occurring at the ISPE. Functional comparison of the ATM mouse counterpart and evaluation of the pre-mRNA splicing intermediates derived from affected cell lines and hybrid minigene assays indicate that U1 snRNP binding at the ISPE interferes with the cryptic acceptor site. Activation of this site results in a stringent 5′–3′ order of intron sequence removal around the cryptic exon. Artificial U1 snRNA loading by complementarity to heterologous exonic sequences represents a potential therapeutic method to prevent the usage of an aberrant CFTR cryptic exon. Our results suggest that ISPE-like intronic elements binding U1 snRNPs may regulate correct intron processing.