Results SRSF7 overexpression induces auto-regulation.To investigate the mechanisms of SRSF7 homeostasis, we generated cell lines SRSF7 is an essential RNA-binding protein whose misexpression promotes cancer. Here, we describe how SRSF7 maintains its protein homeostasis in murine P19 cells using an intricate negative feedback mechanism. SRSF7 binding to its premessenger RNA promotes inclusion of a poison cassette exon and transcript degradation via nonsense-mediated decay (NMD). However, elevated SRSF7 levels inhibit NMD and promote translation of two protein halves, termed Split-ORFs, from the bicistronic SRSF7-PCE transcript. The first half acts as dominant-negative isoform suppressing poison cassette exon inclusion and instead promoting the retention of flanking introns containing repeated SRSF7 binding sites. Massive SRSF7 binding to these sites and its oligomerization promote the assembly of large nuclear bodies, which sequester SRSF7 transcripts at their transcription site, preventing their export and restoring normal SRSF7 protein levels. We further show that hundreds of human and mouse NMD targets, especially RNA-binding proteins, encode potential Split-ORFs, some of which are expressed under specific cellular conditions. SRSF7 binding promotes splicing of NMD-sensitive and -resistant SRSF7 isoforms. To understand the mechanism of SRSF7 auto-regulation, we examined the binding of SRSF7 protein to SRSF7 transcripts using individual-nucleotide resolution ultraviolet (UV) crosslinking and immunoprecipitation (iCLIP). We used normalized significant crosslink events (X-links, false discovery rate (FDR) < 0.05) from SRSF3 and SRSF7 iCLIP datasets of P19 cell lines without OE 8 . Similar to SRSF3, which promotes the inclusion of the PCE in SRSF7 transcripts 17 , SRSF7 showed enriched crosslinks in an extended region encompassing the PCE, its flanking introns 3a and 3b, and exons 3, 4 and 5 ( Fig. 1d). Quantification revealed that SRSF7 binds ~50-fold more to SRSF7 transcripts than SRSF3 ( Supplementary Table 1), indicating that SRSF7 has an unusual preference for its own transcripts.RNA-seq followed by quantification of junction reads revealed that SRSF7 OE promotes inclusion of either the complete PCE (460 nucleotides (nt)) or a partial PCE (107 nt) in SRSF7 transcripts ( Fig. 1e). Additionally, both PCE-flanking introns (3a and 3b) and intron 5 displayed increased read coverage, indicating that they are partly retained upon SRSF7 OE. Semiquantitative reverse transcription PCR and sequencing confirmed that SRSF7 OE caused the appearance of transcripts containing the entire PCE (SRSF7-PCE, orange asterisk), the partial PCE (SRSF7-PCE 1/4 , yellow asterisk) or the PCE in combination with both flanking introns (SRSF7-I3a+b, red asterisk) or with only intron 3b (SRSF7-I3b, green asterisk; Fig. 1f and Extended Data Fig. 1b,c). Identical isoforms were detected for endogenous and SRSF7-GFP reporter transcripts, indicating that auto-regulation operates similarly on both.All these transcripts contain PTCs and should be s...

Oxaliplatin is a third-generation platinum-based anticancer drug that is widely used as first-line treatment for colorectal carcinoma. Patients treated with oxaliplatin develop an acute peripheral pain several hours after treatment, mostly characterized by cold allodynia as well as a long-term chronic neuropathy. These two phenomena seem to be causally connected. However, the underlying mechanisms that trigger the acute peripheral pain are still poorly understood. Here we show that the activity of the transient receptor potential melastatin 8 (TRPM8) channel but not the activity of any other member of the TRP channel family is transiently increased 1 h after oxaliplatin treatment and decreased 24 h after oxaliplatin treatment. Mechanistically, this is connected with activation of the phospholipase C (PLC) pathway and depletion of phosphatidylinositol 4,5-bisphosphate (PIP2) after oxaliplatin treatment. Inhibition of the PLC pathway can reverse the decreased TRPM8 activity as well as the decreased PIP2-concentrations after oxaliplatin treatment. In summary, these results point out transient changes in TRPM8 activity early after oxaliplatin treatment and a later occurring TRPM8 channel desensitization in primary sensory neurons. These mechanisms may explain the transient cold allodynia after oxaliplatin treatment and highlight an important role of TRPM8 in oxaliplatin-induced acute and neuropathic pain.

G-protein coupled receptor 40 (GPR40) is a promising target to support glucose-induced insulin release in patients with diabetes type 2. Here, we studied the role of GPR40 in the regulation of the blood-nerve-barrier integrity and its involvement in diabetes-induced neuropathies. Since GPR40 modulates insulin release, we used the streptozotocin-model for type 1 diabetes, since here GPR40 functions can be investigated independently of its effects on insulin release. Diabetic wildtype mice exhibited increased vascular endothelial permeability and showed epineural microlesions in sciatic nerves, which were also observed in naïve GPR40-/- mice. Fittingly, expression of VEGF-A, an inducer of vascular permeability, was increased in diabetic wildtype and naïve GPR40-/- mice. GPR40 antagonists increased VEGF-A expression in murine and human endothelial cells as well as permeability of transendothelial barriers. In contrast GPR40 agonists suppressed VEGF-A release and mRNA expression. The VEGF receptor inhibitor Axitinib prevented diabetes-induced hypersensitivities and reduced endothelial and epineural permeability. Importantly, the GPR40 agonist GW9508 reverted established diabetesinduced hypersensitivity, an effect which was blocked by VEGF-A administration. Thus, GPR40 activation suppresses VEGF-A expression thereby reducing diabetes-induced bloodnerve-barrier permeability and reverting diabetes-induced hypersensitivities.

G-protein coupled receptor 40 (GPR40) is a promising target to support glucose-induced insulin release in patients with diabetes type 2. Here, we studied the role of GPR40 in the regulation of the blood-nerve-barrier integrity and its involvement in diabetes-induced neuropathies. Since GPR40 modulates insulin release, we used the streptozotocin-model for type 1 diabetes, since here GPR40 functions can be investigated independently of its effects on insulin release. Diabetic wildtype mice exhibited increased vascular endothelial permeability and showed epineural microlesions in sciatic nerves, which were also observed in naïve GPR40^-/- mice. Fittingly, expression of VEGF-A, an inducer of vascular permeability, was increased in diabetic wildtype and naïve GPR40^-/-mice. GPR40 antagonists increased VEGF-A expression in murine and human endothelial cells as well as permeability of transendothelial barriers. In contrast GPR40 agonists suppressed VEGF-A release and mRNA expression. The VEGF receptor inhibitor Axitinib prevented diabetes-induced hypersensitivities and reduced endothelial and epineural permeability. Importantly, the GPR40 agonist GW9508 reverted established diabetes-induced hypersensitivity, an effect which was blocked by VEGF-A administration. Thus, GPR40 activation suppresses VEGF-A expression thereby reducing diabetes-induced blood-nerve-barrier permeability and reverting diabetes-induced hypersensitivities.

G-protein coupled receptor 40 (GPR40) is a promising target to support glucose-induced insulin release in patients with diabetes type 2. Here, we studied the role of GPR40 in the regulation of the blood-nerve-barrier integrity and its involvement in diabetes-induced neuropathies. Since GPR40 modulates insulin release, we used the streptozotocin-model for type 1 diabetes, since here GPR40 functions can be investigated independently of its effects on insulin release. Diabetic wildtype mice exhibited increased vascular endothelial permeability and showed epineural microlesions in sciatic nerves, which were also observed in naïve GPR40^-/- mice. Fittingly, expression of VEGF-A, an inducer of vascular permeability, was increased in diabetic wildtype and naïve GPR40^-/-mice. GPR40 antagonists increased VEGF-A expression in murine and human endothelial cells as well as permeability of transendothelial barriers. In contrast GPR40 agonists suppressed VEGF-A release and mRNA expression. The VEGF receptor inhibitor Axitinib prevented diabetes-induced hypersensitivities and reduced endothelial and epineural permeability. Importantly, the GPR40 agonist GW9508 reverted established diabetes-induced hypersensitivity, an effect which was blocked by VEGF-A administration. Thus, GPR40 activation suppresses VEGF-A expression thereby reducing diabetes-induced blood-nerve-barrier permeability and reverting diabetes-induced hypersensitivities.