TAR DNA-binding protein (TDP-43) is an evolutionarily conserved heterogeneous nuclear ribonucleoprotein (hnRNP) involved in RNA processing, whose abnormal cellular distribution and post-translational modification are key markers of certain neurodegenerative diseases, such as amyotrophic lateral sclerosis and frontotemporal lobar degeneration. We generated human cell lines expressing tagged forms of wild-type and mutant TDP-43 and observed that TDP-43 controls its own expression through a negative feedback loop. The RNA-binding properties of TDP-43 are essential for the autoregulatory activity through binding to 3 0 UTR sequences in its own mRNA. Our analysis indicated that the C-terminal region of TDP-43, which mediates TDP-43-hnRNP interactions, is also required for self-regulation. TDP-43 binding to its 3 0 UTR does not significantly change the pre-mRNA splicing pattern but promotes RNA instability. Moreover, blocking exosome-mediated degradation partially recovers TDP-43 levels. Our findings demonstrate that cellular TDP-43 levels are under tight control and it is likely that disease-associated TDP-43 aggregates disrupt TDP-43 self-regulation, thus contributing to pathogenesis.

One of the fundamental issues in RNA splicing research is represented by understanding how the spliceosome can successfully define exons and introns in a huge variety of pre-mRNA molecules with nucleotide-precision. Since its first description, researchers in this field have identified and characterized many fundamental elements and players capable of affecting the splicing process, both in a negative and positive manner. Indeed, it can be argued that today we know a great deal about the forces that make an exon, an exon and an intron, an intron. As will be discussed in this review, these decisions are a result of a complex combinatorial control resulting from many different factors/influences. Most importantly, these influences act across several levels of complexity starting from the relatively simple interaction between two consensus 5' and 3' splice sites to much more complex factors: such as the interplay between silencer or enhancer sequences, transcriptional processivity, genomic milieu, nucleosome positioning, and histone modifications at the chromatin level. Depending on local contexts, all these factors will act either antagonistically or synergistically to decide the exon/intron fate of any given RNA sequence. At present, however, what we still lack is a precise understanding of how all these processes add up to help the spliceosome reach a decision. Therefore, it is expected that future challenges in splicing research will be the careful characterization of all these influences to improve our ability to predict splicing choices in different organisms or in specific contexts.

Despite a growing number of splicing mutations found in hereditary diseases, utilization of aberrant splice sites and their effects on gene expression remain challenging to predict. We compiled sequences of 346 aberrant 5′splice sites (5′ss) that were activated by mutations in 166 human disease genes. Mutations within the 5′ss consensus accounted for 254 cryptic 5′ss and mutations elsewhere activated 92 de novo 5′ss. Point mutations leading to cryptic 5′ss activation were most common in the first intron nucleotide, followed by the fifth nucleotide. Substitutions at position +5 were exclusively G>A transitions, which was largely attributable to high mutability rates of C/G>T/A. However, the frequency of point mutations at position +5 was significantly higher than that observed in the Human Gene Mutation Database, suggesting that alterations of this position are particularly prone to aberrant splicing, possibly due to a requirement for sequential interactions with U1 and U6 snRNAs. Cryptic 5′ss were best predicted by computational algorithms that accommodate nucleotide dependencies and not by weight-matrix models. Discrimination of intronic 5′ss from their authentic counterparts was less effective than for exonic sites, as the former were intrinsically stronger than the latter. Computational prediction of exonic de novo 5′ss was poor, suggesting that their activation critically depends on exonic splicing enhancers or silencers. The authentic counterparts of aberrant 5′ss were significantly weaker than the average human 5′ss. The development of an online database of aberrant 5′ss will be useful for studying basic mechanisms of splice-site selection, identifying splicing mutations and optimizing splice-site prediction algorithms.

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.

SCN9A encodes the voltage-gated sodium channel Nav1.7, a protein highly expressed in pain-sensing neurons. Mutations in SCN9A cause three human pain disorders: bi-allelic loss of function mutations result in Channelopathy-associated Insensitivity to Pain (CIP), whereas activating mutations cause severe episodic pain in Paroxysmal Extreme Pain Disorder (PEPD) and Primary Erythermalgia (PE). To date, all mutations in SCN9A that cause a complete inability to experience pain are protein truncating and presumably lead to no protein being produced. Here, we describe the identification and functional characterization of two novel non-truncating mutations in families with CIP: a homozygously-inherited missense mutation found in a consanguineous Israeli Bedouin family (Nav1.7-R896Q) and a five amino acid in-frame deletion found in a sporadic compound heterozygote (Nav1.7-ΔR1370-L1374). Both of these mutations map to the pore region of the Nav1.7 sodium channel. Using transient transfection of PC12 cells we found a significant reduction in membrane localization of the mutant protein compared to the wild type. Furthermore, voltage clamp experiments of mutant-transfected HEK293 cells show a complete loss of function of the sodium channel, consistent with the absence of pain phenotype. In summary, this study has identified critical amino acids needed for the normal subcellular localization and function of Nav1.7. © 2010 Wiley-Liss, Inc.

N eurofibromatosis type 1 (NF1) is a common autosomal dominant genetic disease. In recent studies on the neurofibromatosis type 1 (NF1) gene neurofibromin, splicing abnormalities were seen in 30-50% of cases when RNA taken from cell lines was analysed.1 2 Unlike mutations that alter critical amino acids or generate premature stop codons, splicing abnormalities can be very hard to predict from sequence analysis alone. Apart from the two base pairs 5′ and 3′ of each exon, few of the nucleotides in regions critical for splicing are absolutely conserved. As a consequence, it can be very difficult to conclude that a sequence variation found in a patient will alter splicing and so represents a pathogenic mutation.This difficulty is well illustrated by a family with NF1 in which we recently identified a sequence variation. The three generation family is from the UK and meets NIH diagnostic criteria. The index case, at the age of 82, has classical features of NF1 including multiple café au lait macules, neurofibromas, and axillary and inguinal freckling. Her son was similarly affected and died in a road traffic accident. Her granddaughter additionally had macrocephaly and died aged 31 from a malignant nerve sheath tumour affecting the coeliac axis. Through sequencing all exons in the neurofibromin gene, as well as 50 base pairs of 5′ and 3′ intronic flanking sequence, only a single nucleotide change was found, consisting of a substitution of a cytidine for a guanosine 5 bp downstream of exon 3 (exon 3+5 G>C, fig 1). This variation was seen in the proband and the granddaughter and has not been found in 100 normal chromosomes (Mattocks et al, unpublished observations), consistent with it representing the disease causing mutation. The vicinity of the nucleotide change to the 5′ splice site of intervening sequence (IVS) 3 (figs 1 and 2A) suggests that it may interfere with the splicing of exon 3. However, there are examples of wild type sequences similar to the exon 3+5 G>C mutation in which the corresponding exon is spliced efficiently; for example within the normal NF1 gene IVS-1 and -7 share identical -1 to +5 sequence with the mutated IVS-3. We were therefore unable to predict on the basis of sequence analysis alone that this sequence change will Figure 1 Schematic representation of the α-globin fibronectin NF1 exon 3 minigene (black, shaded, and white boxes respectively, with intervening sequences (introns) shown as lines). The mutation is shown with exonic sequence in upper case and intronic sequence in lower case. Key points• Abnormalities of pre-mRNA splicing represent an important mechanism by which gene mutations cause disease.• Effects on splicing can be predicted from genomic DNA sequence analysis if mutations alter highly conserved canonical splicing signals. However, it is extremely difficult to predict the effects of changes in intronic and exonic sequences not obviously involved in the splicing process.• We present here an efficient and simple test using genomic DNA to construct a minigene and analyse the ef...

In recent times, high-throughput screening analyses have broadly defined the RNA cellular targets of TDP-43, a nuclear factor involved in neurodegeneration. A common outcome of all these studies is that changing the expression levels of this protein can alter the expression of several hundred RNAs within cells. What still remains to be clarified is which changes represent direct cellular targets of TDP-43 or just secondary variations due to the general role played by this protein in RNA metabolism. Using an HTS-based splicing junction analysis we identified at least six bona fide splicing events that are consistent with being controlled by TDP-43. Validation of the data, both in neuronal and non-neuronal cell lines demonstrated that TDP-43 substantially alters the levels of isoform expression in four genes potentially important for neuropathology: MADD/IG20, STAG2, FNIP1 and BRD8. For MADD/IG20 and STAG2, these changes could also be confirmed at the protein level. These alterations were also observed in a cellular model that successfully mimics TDP-43 loss of function effects following its aggregation. Most importantly, our study demonstrates that cell cycle alterations induced by TDP-43 knockdown can be recovered by restoring the STAG2, an important component of the cohesin complex, normal splicing profile.

We have recently reported a disease-causing substitution (+5G > C) at the donor site of NF-1 exon 3 that produces its skipping. We have now studied in detail the splicing mechanism involved in analyzing RNA-protein complexes at several 5' splice sites. Characteristic protein patterns were observed by pulldown and band-shift/super-shift analysis. Here, we show that hnRNP H binds specifically to the wild-type GGGgu donor sequence of the NF-1 exon 3. Depletion analyses shows that this protein restricts the accessibility of U1 small nuclear ribonucleoprotein (U1snRNA) to the donor site. In this context, the +5G > C mutation abolishes both U1snRNP base pairing and the 5' splice site (5'ss) function. However, exon recognition in the mutant can be rescued by disrupting the binding of hnRNP H, demonstrating that this protein enhances the effects of the +5G > C substitution. Significantly, a similar situation was found for a second disease-causing +5G > A substitution in the 5'ss of TSHbeta exon 2, which harbors a GGgu donor sequence. Thus, the reason why similar nucleotide substitutions can be either neutral or very disruptive of splicing function can be explained by the presence of specific binding signatures depending on local contexts.