The molecular mechanisms of exon definition and back-splicing are fundamental unanswered questions in pre-mRNA splicing. Here we report cryoEM structures of the yeast E complex assembled on introns, providing the first view of the earliest event in the splicing cycle that commits pre-mRNAs to splicing. The E complex architecture suggests that the same spliceosome can assemble across an exon, which either remodels to span an intron for canonical linear splicing (typically on short exons) or catalyzes back-splicing generating circRNA (on long exons). The model is supported by our experiments demonstrating that E complex assembled on the yeast EFM5 or HMRA1 middle exon can be chased into circRNA when the exon is sufficiently long. Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

BackgroundGalloway-Mowat syndrome (GAMOS) is characterized by neurodevelopmental defects and a progressive nephropathy, which typically manifests as steroid-resistant nephrotic syndrome. The prognosis of GAMOS is poor, and the majority of children progress to renal failure. The discovery of monogenic causes of GAMOS has uncovered molecular pathways involved in the pathogenesis of disease.MethodsHomozygosity mapping, whole-exome sequencing, and linkage analysis were used to identify mutations in four families with a GAMOS-like phenotype, and high-throughput PCR technology was applied to 91 individuals with GAMOS and 816 individuals with isolated nephrotic syndrome. In vitro and in vivo studies determined the functional significance of the mutations identified.ResultsThree biallelic variants of the transcriptional regulator PRDM15 were detected in six families with proteinuric kidney disease. Four families with a variant in the protein’s zinc-finger (ZNF) domain have additional GAMOS-like features, including brain anomalies, cardiac defects, and skeletal defects. All variants destabilize the PRDM15 protein, and the ZNF variant additionally interferes with transcriptional activation. Morpholino oligonucleotide-mediated knockdown of Prdm15 in Xenopus embryos disrupted pronephric development. Human wild-type PRDM15 RNA rescued the disruption, but the three PRDM15 variants did not. Finally, CRISPR-mediated knockout of PRDM15 in human podocytes led to dysregulation of several renal developmental genes.ConclusionsVariants in PRDM15 can cause either isolated nephrotic syndrome or a GAMOS-type syndrome on an allelic basis. PRDM15 regulates multiple developmental kidney genes, and is likely to play an essential role in renal development in humans.

Liver cancer stem cells (LCSCs) play a critical role in hepatocellular carcinoma (HCC) by virtue of their aggressive behaviour and association with poor prognoses. Aquaporin-9 (AQP9) is a transmembrane protein that transports water and reportedly transports H 2 O 2 . Recent studies have shown that AQP9 expression has a negative effect on HCC cell invasion by inhibiting the epithelial-to-mesenchymal transition. However, the role of AQP9 in LCSCs remains obscure. We performed spheroid formation assay and flow cytometric analysis to investigate LCSCs stemness. CD133 + and CD133cells were isolated by flow cytometry. Real-time quantitative PCR (RT-qPCR), western blot and immunofluorescence assay were used to estimate gene expression.The protein association of β-catenin with TCF4 and the interaction of β-catenin with FOXO3a were detected by immunoprecipitation (IP). Here, we found that AQP9 was preferentially decreased in LCSCs. Upregulated AQP9 significantly suppressed LCSCs stemness. In contrast, the inhibition of AQP9 had the opposite effect. Mechanistically, AQP9 was shown to be downregulated by insulin-like growth factor 2 (IGF2), which was widely reported to contribute to maintaining CSCs stemness. Further, AQP9 overexpression was found to result in reactive oxygen species (ROS) accumulation, which inhibited β-catenin activity by attenuating the interaction of β-catenin with TCF4 while concurrently enhancing the association of β-catenin with FOXO3a, ultimately inhibiting LCSCs stemness. Our study implies that stimulation of the AQP9 on August 31, 2021.

Recently identified broadly neutralizing antibodies (bnAbs) show great potential for clinical interventions against HIV-1 infection. However, resistant strains may impose substantial challenges. Here, we report on the identification and characterization of a panel of HIV-1 strains with broad and potent resistance against a large number of bnAbs, particularly those targeting the CD4-binding site (CD4bs). Site-directed mutagenesis revealed that several key epitope mutations facilitate resistance and are located in the inner domain, loop D, and β23/loop V5/β24 of HIV-1 gp120. The resistance is largely correlated with binding affinity of antibodies to the envelope trimers expressed on the cell surface. Our results therefore demonstrate the existence of broadly resistant HIV-1 strains against CD4bs neutralizing antibodies. Treatment strategies based on the CD4bs bnAbs must overcome such resistance to achieve optimal clinical outcomes.

Current cancer treatment is partly limited by chemotherapy-induced vascular toxicity associated with damage to vascular endothelial cells. In this study, the cytotoxicity of farrerol against SGC7901 gastric cancer cells and human umbilical vein endothelial cells (HUVECs) in vitro was investigated along with the underlying mechanisms of its growth-inhibitory effect against SGC7901 cells. MTT assays showed that farrerol inhibited SGC7901 cell growth, but exerted no cytotoxicity against HUVECs. Flow cytometry showed that treatment of SGC7901 cells with farrerol (5, 40, or 160 μmol/l) for 24 h caused G0/G1 cell cycle arrest in a concentration-dependent manner. Western blotting indicated that exposure of SGC7901 cells to farrerol resulted in significant upregulation of p27KIP1 (p27), accompanied by sustained activation of ERK1/2 and p38 MAPK instead of JNK. Farrerol-stimulated p27 expression, p38 MAPK activation, and cell growth inhibition were attenuated by pretreatment with U0126, an MEK1/2 inhibitor. In conclusion, this study indicates the selective cytotoxicity of farrerol against SGC7901 cells, but not HUVECs. Furthermore, it provides the first evidence that farrerol could induce cancer cell growth inhibition by G0/G1-phase cell-cycle arrest mediated by sustained ERK activation. The findings show the potential of farrerol as a chemotherapeutic agent without vascular toxicity for use against gastric cancer.