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1.  Knockdown of MVK does not lead to changes in NALP3 expression or activation 
Mutations in the Mevalonate Kinase gene (MVK) are causes of a rare autoinflammatory disease: Mevalonate Kinase Deficiency and its more acute manifestation, Mevalonic Aciduria. The latter is characterized, among other features, by neuroinflammation, developmental delay and ataxia, due to failed cerebellar development or neuronal death through chronic inflammation. Pathogenesis of neuroinflammation in Mevalonate Kinase Deficiency and Mevalonic Aciduria has not yet been completely clarified, however different research groups have been suggesting the inflammasome complex as the key factor in the disease development. A strategy to mimic this disease is blocking the mevalonate pathway, using HMG-CoA reductase inhibitors (Statins), while knock-out mice for Mevalonate Kinase are non-vital and their hemyzygous (i.e only one copy of gene preserved) littermate display almost no pathological features.
We sought to generate a murine cellular model closely resembling the pathogenic conditions found in vivo, by direct silencing of Mevalonate Kinase gene. Knockdown of Mevalonate Kinase in a murine microglial cellular model (BV-2 cells) results in neither augmented NALP3 expression nor increase of apoptosis. On the contrary, statin treatment of BV-2 cells produces an increase both in Mevalonate Kinase and NALP3 expression.
MKD deficiency could be due or affected by protein accumulation leading to NALP3 activation, opening novel questions about strategies to tackle this disease.
Electronic supplementary material
The online version of this article (doi:10.1186/s12950-015-0048-5) contains supplementary material, which is available to authorized users.
PMCID: PMC4320511  PMID: 25663823
Immunology; Inflammation; Autoimmunity; Mevalonate kinase deficiency
2.  Role of Pseudoexons and Pseudointrons in Human Cancer 
In all eukaryotic organisms, pre-mRNA splicing and alternative splicing processes play an essential role in regulating the flow of information required to drive complex developmental and metabolic pathways. As a result, eukaryotic cells have developed a very efficient macromolecular machinery, called the spliceosome, to correctly recognize the pre-mRNA sequences that need to be inserted in a mature mRNA (exons) from those that should be removed (introns). In healthy individuals, alternative and constitutive splicing processes function with a high degree of precision and fidelity in order to ensure the correct working of this machinery. In recent years, however, medical research has shown that alterations at the splicing level play an increasingly important role in many human hereditary diseases, neurodegenerative processes, and especially in cancer origin and progression. In this minireview, we will focus on several genes whose association with cancer has been well established in previous studies, such as ATM, BRCA1/A2, and NF1. In particular, our objective will be to provide an overview of the known mechanisms underlying activation/repression of pseudoexons and pseudointrons; the possible utilization of these events as biomarkers of tumor staging/grading; and finally, the treatment options for reversing pathologic splicing events.
PMCID: PMC3800588  PMID: 24204383
3.  Drosophila Answers to TDP-43 Proteinopathies 
Journal of Amino Acids  2012;2012:356081.
Initially implicated in the pathogenesis of CFTR and HIV-1 transcription, nuclear factor TDP-43 was subsequently found to be involved in the origin and development of several neurodegenerative diseases. In 2006, in fact, it was reported for the first time the cytoplasmic accumulation of TDP-43 in ubiquitin-positive inclusions of ALS and FTLD patients, suggesting the presence of a shared underlying mechanism for these diseases. Today, different animal models of TDP-43 proteinopathies are available in rodents, nematodes, fishes, and flies. Although these models recapitulate several of the pathological features found in patients, the mechanisms underpinning the progressive neuronal loss observed in TDP-43 proteinopathies remain to be characterized. Compared to other models, Drosophila are appealing because they combine the presence of a sophisticated brain with the possibility to investigate quickly and massively phenotypic genetic modifiers as well as possible therapeutic strategies. At present, the development of TDP-43-related Drosophila models has further strengthened the hypothesis that both TDP-43 “loss-of-function” and “gain-of-function” mechanisms can contribute to disease. The aim of this paper is to describe and compare the results obtained in a series of transgenic and knockout flies, along with the information they have generated, towards a better understanding of the mechanisms underlying TDP-43 proteinopathies.
PMCID: PMC3337594  PMID: 22577517
4.  TDP-43 Regulates Drosophila Neuromuscular Junctions Growth by Modulating Futsch/MAP1B Levels and Synaptic Microtubules Organization 
PLoS ONE  2011;6(3):e17808.
TDP-43 is an evolutionarily conserved RNA binding protein recently associated with the pathogenesis of different neurological diseases. At the moment, neither its physiological role in vivo nor the mechanisms that may lead to neurodegeneration are well known. Previously, we have shown that TDP-43 mutant flies presented locomotive alterations and structural defects at the neuromuscular junctions. We have now investigated the functional mechanism leading to these phenotypes by screening several factors known to be important for synaptic growth or bouton formation. As a result we found that alterations in the organization of synaptic microtubules correlate with reduced protein levels in the microtubule associated protein futsch/MAP1B. Moreover, we observed that TDP-43 physically interacts with futsch mRNA and that its RNA binding capacity is required to prevent futsch down regulation and synaptic defects.
PMCID: PMC3055892  PMID: 21412434
5.  Functional mapping of the interaction between TDP-43 and hnRNP A2 in vivo 
Nucleic Acids Research  2009;37(12):4116-4126.
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.
PMCID: PMC2709582  PMID: 19429692
6.  Dissecting the splicing mechanism of the Drosophila editing enzyme; dADAR 
Nucleic Acids Research  2009;37(5):1663-1671.
In Drosophila melanogaster, the expression of adenosine deaminase acting on RNA is regulated by transcription and alternative splicing so that at least four different isoforms are generated that have a tissue-specific splicing pattern. Even though dAdar has been extensively studied, the complete adult expression pattern has yet to be elucidated. In the present study, we investigate mature transcripts of dAdar arising from different promoters. Two predominant isoforms of dAdar are expressed in gonads and dAdar is transcribed from both the embryonic and the adult promoters. Furthermore, full-length transcripts containing the alternatively spliced exon-1 are expressed in a tissue-specific manner. The splicing factor B52/SRp55 binds within the alternative spliced exon 3a and plays a role in this alternative splicing event.
PMCID: PMC2655694  PMID: 19153139
7.  Aberrant 5′ splice sites in human disease genes: mutation pattern, nucleotide structure and comparison of computational tools that predict their utilization 
Nucleic Acids Research  2007;35(13):4250-4263.
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.
PMCID: PMC1934990  PMID: 17576681
8.  Complex splicing control of the human Thrombopoietin gene by intronic G runs 
Nucleic Acids Research  2006;35(1):132-142.
The human thrombopoietin (THPO) gene displays a series of alternative splicing events that provide valuable models for studying splicing mechanisms. The THPO region spanning exon 1–4 presents both alternative splicing of exon 2 and partial intron 2 (IVS2) retention following the activation of a cryptic 3′ splice site 85 nt upstream of the authentic acceptor site. IVS2 is particularly rich in stretches of 3–5 guanosines (namely, G1–G10) and we have characterized the role of these elements in the processing of this intron. In vivo studies show that runs G7–G10 work in a combinatorial way to control the selection of the proper 3′ splice site. In particular, the G7 element behaves as the splicing hub of intron 2 and its interaction with hnRNP H1 is critical for the splicing process. Removal of hnRNP H1 by RNA interference promoted the usage of the cryptic 3′ splice site so providing functional evidence that this factor is involved in the selection of the authentic 3′ splice site of THPO IVS2.
PMCID: PMC1802585  PMID: 17158158
9.  Depletion of TDP 43 overrides the need for exonic and intronic splicing enhancers in the human apoA-II gene 
Nucleic Acids Research  2005;33(18):6000-6010.
Exon 3 of the human apolipoprotein A-II (apoA-II) gene is efficiently included in the mRNA although its acceptor site is significantly weak because of a peculiar (GU)16 tract instead of a canonical polypyrimidine tract within the intron 2/exon 3 junction. Our previous studies demonstrated that the SR proteins ASF/SF2 and SC35 bind specifically an exonic splicing enhancer (ESE) within exon 3 and promote exon 3 splicing. In the present study, we show that the ESE is necessary only in the proper context. In addition, we have characterized two novel sequences in the flanking introns that modulate apoA-II exon 3 splicing. There is a G-rich element in intron 2 that interacts with hnRNPH1 and inhibits exon 3 splicing. The second is a purine rich region in intron 3 that binds SRp40 and SRp55 and promotes exon 3 inclusion in mRNA. We have also found that the (GU) repeats in the apoA-II context bind the splicing factor TDP-43 and interfere with exon 3 definition. Significantly, blocking of TDP-43 expression by small interfering RNA overrides the need for all the other cis-acting elements making exon 3 inclusion constitutive even in the presence of disrupted exonic and intronic enhancers. Altogether, our results suggest that exonic and intronic enhancers have evolved to balance the negative effects of the two silencers located in intron 2 and hence rescue the constitutive exon 3 inclusion in apoA-II mRNA.
PMCID: PMC1270946  PMID: 16254078
10.  hnRNP H binding at the 5′ splice site correlates with the pathological effect of two intronic mutations in the NF-1 and TSHβ genes 
Nucleic Acids Research  2004;32(14):4224-4236.
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 TSHβ 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.
PMCID: PMC514374  PMID: 15299088
11.  Splicing of constitutive upstream introns is essential for the recognition of intra-exonic suboptimal splice sites in the thrombopoietin gene 
Nucleic Acids Research  2001;29(4):886-894.
The human thrombopoietin (TPO) gene, which codes for the principal cytokine involved in platelet maturation, shows a peculiar alternative splicing of its last exon, where an intra-exonic 116 nt alternative intron is spliced out in a fraction of its mRNA. To characterize the molecular mechanism underlying this alternative splicing, minigenes of TPO genomic constructs with variable exon–intron configurations or carrying exclusively the TPO cDNA were generated and transiently transfected in the Hep3B cell line. We have found that the final rate of the alternative intron splicing is determined by three elements: the presence of upstream constitutive introns, the suboptimal splice sites of the alternative intron and the length of the alternative intron itself. Our results indicate that the recognition of suboptimal intra-exonic splice junctions in the TPO gene is influenced by the assembly of the spliceosome complex on constitutive introns and by a qualitative scanning of the sequence by the transcriptional/splicing machinery complex primed by upstream splicing signals.
PMCID: PMC29620  PMID: 11160920

Results 1-11 (11)