Almost all polymerase II transcripts undergo alternative pre-mRNA splicing. Here, we review the functions of alternative splicing events that have been experimentally determined. The overall function of alternative splicing is to increase the diversity of mRNAs expressed from the genome. Alternative splicing changes proteins encoded by mRNAs, which has profound functional effects. Experimental analysis of these protein isoforms showed that alternative splicing regulates binding between proteins, between proteins and nucleic acids as well as between proteins and membranes. Alternative splicing regulates the localization of proteins, their enzymatic properties and their interaction with ligands. In most cases, changes caused by individual splicing isoforms are small. However, cells typically coordinate numerous changes in ‘splicing programs’, which can have strong effects on cell proliferation, cell survival and properties of the nervous system. Due to its widespread usage and molecular versatility, alternative splicing emerges as a central element in gene regulation that interferes with almost every biological function analyzed.

Resistance mechanisms against antiangiogenic drugs are unclear. Here, we correlated the antitumor and antivascular properties of five different antiangiogenic receptor tyrosine kinase inhibitors (RTKIs) (motesanib, pazopanib, sorafenib, sunitinib, vatalanib) with their intratumoral distribution data obtained by matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI). In the first mouse model, only sunitinib exhibited broad-spectrum antivascular and antitumor activities by simultaneously suppressing vascular endothelial growth factor receptor-2 (VEGFR2) and desmin expression, and by increasing intratumoral hypoxia and inhibiting both tumor growth and vascularisation significantly. Importantly, the highest and most homogeneous intratumoral drug concentrations have been found in sunitinib-treated animals. In another animal model, where - in contrast to the first model - vatalanib was detectable at homogeneously high intratumoral concentrations, the drug significantly reduced tumor growth and angiogenesis. In conclusion, the tumor tissue penetration and thus the antiangiogenic and antitumor potential of antiangiogenic RTKIs vary among the tumor models and our study demonstrates the potential of MALDI-MSI to predict the efficacy of unlabelled small molecule antiangiogenic drugs in malignant tissue. Our approach is thus a major technical and preclinical advance demonstrating that primary resistance to angiogenesis inhibitors involves limited tumor tissue drug penetration. We also conclude that MALDI-MSI may significantly contribute to the improvement of antivascular cancer therapies.

Alternative pre-mRNA processing is a central element of eukaryotic gene regulation. The cell frequently alters the use of alternative exons in response to physiological stimuli. Ceramides are lipid-signaling molecules composed of sphingosine and a fatty acid. Previously, water-insoluble ceramides were shown to change alternative splicing and decrease SR-protein phosphorylation by activating protein phosphatase-1 (PP1). To gain further mechanistical insight into ceramide-mediated alternative splicing, we analyzed the effect of C6 pyridinium ceramide (PyrCer) on alternative splice site selection. PyrCer is a water-soluble ceramide analog that is under investigation as a cancer drug. We found that PyrCer binds to the PP1 catalytic subunit and inhibits the dephosphorylation of several splicing regulatory proteins containing the evolutionarily conserved RVxF PP1-binding motif (including PSF/SFPQ, Tra2-beta1 and SF2/ASF). In contrast to natural ceramides, PyrCer promotes phosphorylation of splicing factors. Exons that are regulated by PyrCer have in common suboptimal splice sites, are unusually short and share two 4-nt motifs, GAAR and CAAG. They are dependent on PSF/SFPQ, whose phosphorylation is regulated by PyrCer. Our results indicate that lipids can influence pre-mRNA processing by regulating the phosphorylation status of specific regulatory factors, which is mediated by protein phosphatase activity.

BackgroundFetal akinesia (FA) results in variable clinical presentations and has been associated with more than 166 different disease loci. However, the underlying molecular cause remains unclear in many individuals. We aimed to further define the set of genes involved.MethodsWe performed in-depth clinical characterisation and exome sequencing on a cohort of 23 FA index cases sharing arthrogryposis as a common feature.ResultsWe identified likely pathogenic or pathogenic variants in 12 different established disease genes explaining the disease phenotype in 13 index cases and report 12 novel variants. In the unsolved families, a search for recessive-type variants affecting the same gene was performed; and in five affected fetuses of two unrelated families, a homozygous loss-of-function variant in the kinesin family member 21A gene (KIF21A) was found.ConclusionOur study underlines the broad locus heterogeneity of FA with well-established and atypical genotype–phenotype associations. We describe KIF21A as a new factor implicated in the pathogenesis of severe neurogenic FA sequence with arthrogryposis of multiple joints, pulmonary hypoplasia and facial dysmorphisms. This hypothesis is further corroborated by a recent report on overlapping phenotypes observed in Kif21a null piglets.

Alternative pre-mRNA splicing is a central element of eukaryotic gene expression. Its deregulation can lead to disease, and methods to change splice site selection are developed as potential therapies. Spinal muscular atrophy is caused by the loss of the SMN1 (survival of motoneuron 1) gene. A therapeutic avenue for spinal muscular atrophy treatment is to promote exon 7 inclusion of the almost identical SMN2 (survival of motoneuron 2) gene. The splicing factor tra2-beta1 promotes inclusion of this exon and is antagonized by protein phosphatase (PP) 1. To identify new compounds that promote exon 7 inclusion, we synthesized analogs of cantharidin, an inhibitor of PP1, and PP2A. Three classes of compounds emerged from these studies. The first class blocks PP1 and PP2A activity, blocks constitutive splicing in vitro, and promotes exon 7 inclusion in vivo. The second class has no measurable effect on PP1 activity but activates PP2A. This class represents the first compounds described with these properties. These compounds cause a dephosphorylation of Thr-33 of tra2-beta1, which promotes exon 7 inclusion. The third class had no detectable effect on phosphatase activity and could promote exon 7 via allosteric effects. Our data show that subtle changes in similar compounds can turn a phosphatase inhibitor into an activator. These chemically related compounds influence alternative splicing by distinct mechanisms.An estimated 95% of human multi-exon genes undergo alternative pre-mRNA splicing (1, 2). Unlike promoter activity that regulates the abundance of transcripts, alternative splicing influences the structure of the mRNAs and their encoded proteins (reviewed in Ref. 3). The high incidence of alternative splicing and its ability to increase the coding capacity of the genome makes it a central element in eukaryotic gene expression.Despite its importance, we do not fully understand how splice sites, especially the alternative ones, are selected. The accurate recognition of splice sites in vivo is the result of a combinatorial mechanism (4 -7) where protein complexes assemble on the nascent pre-mRNA, because of the combination of weak RNA/RNA, RNA/protein, and protein/protein interactions. Transient interaction between the spliceosome and regulatory proteins assembling on the pre-mRNA help in the recognition of splice sites (8). There is increasing evidence that the phosphorylation state of splicing regulatory proteins is a crucial element for this recognition process (reviewed in Ref. 9), because phosphorylation/dephosphorylation often changes the affinity between proteins in these complexes. The spliceosome acts in a stepwise fashion on the pre-mRNA, and four distinct splicing complexes, the H, A, B and C complexes, have been identified (7). The H and E complexes contain the pre-mRNA imbedded with various proteins, among them hnRNPs and SR proteins as well as the U1 snRNP bound to the 5Ј splice site. The entry of U2 snRNP defines the branch point and leads to the formation of the A complex. The entry of the U4/5/6 snRN...