Reduced splicing efficiency induced by synonymous substitutions may generate a substrate for natural selection of new splicing isoforms: the case of CFTR exon 12

Alternative splicing has been associated with increased evolutionary changes and with recent exon creation or loss. The addition of a new exon can be explained by its inclusion in only a fraction of the transcripts leaving the original form intact and giving to the new form the possibility to evolve independently but the exon loss phenomenon is less clear. To explore the mechanism that could be involved in CFTR exon 12 lower splicing efficiency in primates, we have analyzed the effect of multiple synonymous variations. Random patterns of synonymous variations were created in CFTR exon12 and the majority of them induced exon inclusion, suggesting a suboptimal splicing efficiency of the human gene. In addition, the effect of each single synonymous substitution on splicing is strongly dependent on the exonic context and does not correlate with available in silico exon splicing prediction programs. We propose that casual synonymous substitutions may lead to a reduced splicing efficiency that can result in a variable proportion of exon loss. If this phenomenon happens in in-frame exons and to an extent tolerated by the cells it can have an important evolutionary effect since it may generate a substrate for natural selection of new splicing isoforms

Direct evidence of dust growth in L183 from MIR light scattering

Theoretical arguments suggest that dust grains should grow in the dense cold parts of molecular clouds. Evidence of larger grains has so far been gathered in near/mid infrared extinction and millimeter observations. Interpreting the data is, however, aggravated by the complex interplay of density and dust properties (as well as temperature for thermal emission). We present new Spitzer data of L183 in bands that are sensitive and insensitive to PAHs. The visual extinction AV map derived in a former paper was fitted by a series of 3D Gaussian distributions. For different dust models, we calculate the scattered MIR radiation images of structures that agree agree with the AV map and compare them to the Spitzer data. The Spitzer data of L183 show emission in the 3.6 and 4.5 micron bands, while the 5.8 micron band shows slight absorption. The emission layer of stochastically heated particles should coincide with the layer of strongest scattering of optical interstellar radiation, which is seen as an outer surface on I band images different from the emission region seen in the Spitzer images. Moreover, PAH emission is expected to strongly increase from 4.5 to 5.8 micron, which is not seen. Hence, we interpret this emission to be MIR cloudshine. Scattered light modeling when assuming interstellar medium dust grains without growth does not reproduce flux measurable by Spitzer. In contrast, models with grains growing with density yield images with a flux and pattern comparable to the Spitzer images in the bands 3.6, 4.5, and 8.0 micron.Comment: 13 pages, 11 figures, accepted for publication in Astronomy and Astrophysic

Inguinocrural dermolipectomy A retrospective study

Inguinocrural dermolipectomy is a constantly increasing surgical procedure, especially for postbariatric patients with significant weight loss. The "crescent medial" and "vertical medial" thigh lifting techniques (CMTL/VMTL) are the most effective type of surgery to treat laxity and excess of skin and soft tissues in the medial region of the thighs Aim of this article is to suggest which patients may be eligible for a surgical thighplasty with an acceptable risk of postoperative complications

Missense, Nonsense, and Neutral Mutations Define Juxtaposed Regulatory Elements of Splicing in Cystic Fibrosis Transmembrane Regulator Exon 9

Exonic sequence variations may induce exon inclusion or exclusion from the mature mRNA by disrupting exonic regulatory elements and/or by affecting a nuclear reading frame scanning mechanism. We have carried out a systematic study of the effect on cystic fibrosis transmembrane regulator exon 9 splicing of natural and site-directed sequence mutations. We have observed that changes in the splicing pattern were not related to the creation of premature termination codons, a fact that indicates the lack of a significant nuclear check of the reading frame in this system. In addition, the splice pattern could not be predicted by available Ser/Arg protein matrices score analysis. An extensive site-directed mutagenesis of the 3' portion of the exon has identified two juxtaposed splicing enhancer and silencer elements. The study of double mutants at these regulatory elements showed a complex regulatory activity. For example, one natural mutation (146C) enhances exon inclusion and overrides all of the downstream silencing mutations except for a C to G transversion (155G). This unusual effect is explained by the creation of a specific binding site for the inhibitory splicing factor hnRNPH. In fact, on the double mutant 146C-155G, the silencing effect is dominant. These results indicate a strict dependence between the two juxtaposed enhancer and silencer sequences and show that many point mutations in these elements cause changes in splicing efficiency by different mechanisms

An Intronic Polypyrimidine-rich Element Downstream of the Donor Site Modulates Cystic Fibrosis Transmembrane Conductance Regulator Exon 9 Alternative Splicing *

Two intronic elements, a polymorphic TGmTn locus at the end of intron 8 and an intronic splicing silencer in intron 9, regulate aberrant splicing of human cystic fibrosis transmembrane conductance regulator (CFTR) exon 9. Previous studies (Pagani, F., Buratti, E., Stuani, C., Romano, M., Zuccato, E., Niksic, M., Giglio, L., Faraguna, D., and Baralle, F. E. (2000) J. Biol. Chem. 275, 21041–21047 and Buratti, E., Dork, T., Zuccato, E., Pagani, F., Romano, M., and Baralle, F. E. (2001) Embo J. 20, 1774–1784) have demonstrated that trans-acting factors that bind to these sequences, TDP43 and Ser/Arg-rich proteins, respectively, mediate splicing inhibition. Here, we report the identification of two polypyrimidine-binding proteins, TIA-1 and polypyrimidine tract-binding protein (PTB), as novel players in the regulation of CFTR exon 9 splicing. In hybrid minigene experiments, TIA-1 induced exon inclusion, whereas PTB induced exon skipping. TIA-1 bound specifically to a polypyrimidine-rich controlling element (PCE) located between the weak 5′-splice site (ss) and the intronic splicing silencer. Mutants of the PCE polypyrimidine motifs did not bind TIA-1 and, in a splicing assay, did not respond to TIA-1 splicing enhancement. PTB antagonized in vitro TIA-1 binding to the PCE, but its splicing inhibition was independent of its binding to the PCE. Recruitment of U1 small nuclear RNA to the weak 5′-ss by complementarity also induced exon 9 inclusion, consistent with the facilitating role of TIA-1 in weak 5′-ss recognition by U1 small nuclear ribonucleoprotein. Interestingly, in the presence of a high number of TG repeats and a low number of T repeats in the TGmTn locus, TIA-1 activated a cryptic exonic 3′-ss. This effect was independent of both TIA-1 binding to the PCE and U1 small nuclear RNA recruitment to the 5′-ss. Moreover, it was abolished by deletion of either the TG or T sequence. These data indicate that, in CFTR exon 9, TIA-1 binding to the PCE recruits U1 small nuclear ribonucleoprotein to the weak 5′-ss and induces exon inclusion. The TIA-1-mediated alternative usage of the 3′-splice sites, which depends on the composition of the unusual TGmTn element, represents a new mechanism of splicing regulation by TIA-1

Biharmonic Conformal Field Theories

The main result of this paper is the construction of a conformally covariant operator in two dimensions acting on scalar fields and containing fourth order derivatives. In this way it is possible to derive a class of Lagrangians invariant under conformal transformations. They define conformal field theories satisfying equations of the biharmonic type. Two kinds of these biharmonic field theories are distinguished, characterized by the possibility or not of the scalar fields to transform non-trivially under Weyl transformations. Both cases are relevant for string theory and two dimensional gravity. The biharmonic conformal field theories provide higher order corrections to the equations of motion of the metric and give a possibility of adding new terms to the Polyakov action.Comment: 11 pages, LaTeX, no figure

New lysosomal acid lipase gene mutants explain the phenotype of Wolman disease and cholesteryl ester storage disease.

Deficiency of lysosomal acid lipase (LAL) leads to either Wolman disease(WD) or the more benign cholesteryl ester storage disease (CESD). To identifythe molecular basis of the different phenotypes we have characterised the LALgene mutations in three new patients with LAL deficiency. A patient with WD washomozygote for a null allele Y303X. The other two patients, with CESD, presentedeither homozygosity for T267I or compound heterozygosity consisting of Q64R andan exon 8 donor splice site substitution (G→A in position–1). The mutants T267I and Q64R and the previously reported L273S, G66V,and H274Y CESD substitutions, overexpressed in stable clones, were found to befully glycosylated and show an enzymatic activity of 3–8% of that ofnormal LAL. On the other hand, the Δ254–277 mutant proteinderived from exon 8 skipping and the Y303X protein were totally inactive. Bytransient transfection of hybrid minigene constructs, the CESD G→A(–1) substitution resulted in partial exon inclusion, thus allowing theproduction of a small amount of normal LAL mRNA and hence of a functionalenzyme. In contrast, a G→Asubstitution observed in WD at position +1 of the same exon 8 donor siteresulted in complete exon skipping and the sole production of an inactiveΔ254–277 protein.In conclusion,LAL genotypes determine the level of residual enzymatic activity, thusexplaining the severity of the phenotype.—Pagani, F., R. Pariyarath, R.Garcia, C. Stuani, A. B. Burlina, G. Ruotolo, M. Rabusin, and F. E. Baralle. Newlysosomal acid lipase gene mutants explain the phenotype of Wolman disease andcholesteryl ester storage disease. J. Lipid Res. 1998. 39:1382–1388

An exon-specific U1 small nuclear RNA (snRNA) strategy to correct splicing defects

A significant proportion of disease-causing mutations affect precursor-mRNA splicing, inducing skipping of the exon from the mature transcript. Using F9 exon 5, CFTR exon 12 and SMN2 exon 7 models, we characterized natural mutations associated to exon skipping in Haemophilia B, cystic fibrosis and spinal muscular atrophy (SMA), respectively, and the therapeutic splicing rescue by using U1 small nuclear RNA (snRNA). In minigene expression systems, loading of U1 snRNA by complementarity to the normal or mutated donor splice sites (5′ss) corrected the exon skipping caused by mutations at the polypyrimidine tract of the acceptor splice site, at the consensus 5′ss or at exonic regulatory elements. To improve specificity and reduce potential off-target effects, we developed U1 snRNA variants targeting non-conserved intronic sequences downstream of the 5′ss. For each gene system, we identified an exon-specific U1 snRNA (ExSpeU1) able to rescue splicing impaired by the different types of mutations. Through splicing-competent cDNA constructs, we demonstrated that the ExSpeU1-mediated splicing correction of several F9 mutations results in complete restoration of secreted functional factor IX levels. Furthermore, two ExSpeU1s for SMA improved SMN exon 7 splicing in the chromosomal context of normal cells. We propose ExSpeU1s as a novel therapeutic strategy to correct, in several human disorders, different types of splicing mutations associated with defective exon definition

Splicing Factors Induce Cystic Fibrosis Transmembrane Regulator Exon 9 Skipping through a Nonevolutionary Conserved Intronic Element

In monosymptomatic forms of cystic fibrosis such as congenital bilateral absence of vas deferens, variations in the TG(m) and T(n) polymorphic repeats at the 3' end of intron 8 of the cystic fibrosis transmembrane regulator (CFTR) gene are associated with the alternative splicing of exon 9, which results in a nonfunctional CFTR protein. Using a minigene model system, we have previously shown a direct relationship between the TG(m)T(n) polymorphism and exon 9 splicing. We have now evaluated the role of splicing factors in the regulation of the alternative splicing of this exon. Serine-arginine-rich proteins and the heterogeneous nuclear ribonucleoprotein A1 induced exon skipping in the human gene but not in its mouse counterpart. The effect of these proteins on exon 9 exclusion was strictly dependent on the composition of the TG(m) and T(n) polymorphic repeats. The comparative and functional analysis of the human and mouse CFTR genes showed that a region of about 150 nucleotides, present only in the human intron 9, mediates the exon 9 splicing inhibition in association with exonic regulatory elements. This region, defined as the CFTR exon 9 intronic splicing silencer, is a target for serine-arginine-rich protein interactions. Thus, the nonevolutionary conserved CFTR exon 9 alternative splicing is modulated by the TG(m) and T(n) polymorphism at the 3' splice region, enhancer and silencer exonic elements, and the intronic splicing silencer in the proximal 5' intronic region. Tissue levels and individual variability of splicing factors would determine the penetrance of the TG(m)T(n) locus in monosymptomatic forms of cystic fibrosis