Regulation of 3' splice site selection after step 1 of splicing by spliceosomal C* proteins.

Alternative precursor messenger RNA splicing is instrumental in expanding the proteome of higher eukaryotes, and changes in 3' splice site (3'ss) usage contribute to human disease. We demonstrate by small interfering RNA-mediated knockdowns, followed by RNA sequencing, that many proteins first recruited to human C* spliceosomes, which catalyze step 2 of splicing, regulate alternative splicing, including the selection of alternatively spliced NAGNAG 3'ss. Cryo-electron microscopy and protein cross-linking reveal the molecular architecture of these proteins in C* spliceosomes, providing mechanistic and structural insights into how they influence 3'ss usage. They further elucidate the path of the 3' region of the intron, allowing a structure-based model for how the C* spliceosome potentially scans for the proximal 3'ss. By combining biochemical and structural approaches with genome-wide functional analyses, our studies reveal widespread regulation of alternative 3'ss usage after step 1 of splicing and the likely mechanisms whereby C* proteins influence NAGNAG 3'ss choices.

Inactivation of Coronaviruses during Sample Preparation for Proteomics Experiments.

Mass spectrometry-based proteomics is applied in SARS-CoV-2 research and is, moreover, being discussed as a novel method for SARS-CoV-2 diagnostics. However, the safe inactivation of coronaviruses by proteomics lysis buffers has not been systematically analyzed yet. Hence, for safety reasons a heating step prior to sample preparation is often performed. This step could be omitted once the safe inactivation with the typical buffers is proven. Here we test five different proteomics lysis buffers-4% SDS, 1% SDC, TFA, 6 M GdmCl, and 8 M urea-for their inactivation capacity of coronaviruses. Two representative human coronaviruses, namely HCoV-229E and HCoV-OC43, were used as surrogate for SARS-CoV-2. Lysis was performed at room temperature and at 95 °C for 5 min. Inactivation was confirmed by the absence of a cytopathic effect in MRC-5 cells, and equivocal results were further confirmed by serial passaging and quantitative real-time PCR. While at room temperature SDS, SDC, and TFA inactivated both coronaviruses, and GdmCl and urea resulted in partially incomplete inactivation. This demonstrates that care should be taken when choosing lysis buffers for proteomics analysis of coronaviruses, because some buffers do not ensure inactivation and, hence, biosafety during the further sample preparation.