In vivo characterization of the bacterial intramembrane-cleaving protease RseP using the heme binding tag-based assay iCliPSpy.

Regulated intramembrane proteolysis (RIP) describes the protease-dependent cleavage of transmembrane proteins within the hydrophobic core of cellular membranes. Intramembrane-cleaving proteases (I-CliPs) that catalyze these reactions are found in all kingdoms of life and are involved in a wide range of cellular processes, including signaling and protein homeostasis. I-CLiPs are multispanning membrane proteins and represent challenging targets in structural and enzyme biology.

CAPRI enables comparison of evolutionarily conserved RNA interacting regions.

RNA-protein complexes play essential regulatory roles at nearly all levels of gene expression. Using in vivo crosslinking and RNA capture, we report a comprehensive RNA-protein interactome in a metazoan at four levels of resolution: single amino acids, domains, proteins and multisubunit complexes. We devise CAPRI, a method to map RNA-binding domains (RBDs) by simultaneous identification of RNA interacting crosslinked peptides and peptides adjacent to such crosslinked sites.

Cellular differentiation state modulates the mRNA export activity of SR proteins.

SR proteins function in nuclear pre-mRNA processing, mRNA export, and translation. To investigate their cellular dynamics, we developed a quantitative assay, which detects differences in nucleocytoplasmic shuttling among seven canonical SR protein family members. As expected, SRSF2 and SRSF5 shuttle poorly in HeLa cells but surprisingly display considerable shuttling in pluripotent murine P19 cells.

Long Noncoding RNA MANTIS Facilitates Endothelial Angiogenic Function.

Background: The angiogenic function of endothelial cells is regulated by numerous mechanisms, but the impact of long noncoding RNAs (lncRNAs) has hardly been studied. We set out to identify novel and functionally important endothelial lncRNAs.

Methods: Epigenetically controlled lncRNAs in human umbilical vein endothelial cells were searched by exon-array analysis after knockdown of the histone demethylase JARID1B.

sST2 translation is regulated by FGF2 via an hnRNP A1-mediated IRES-dependent mechanism.

Translation is an energy-intensive process and tightly regulated. Generally, translation is initiated in a cap-dependent manner. Under stress conditions, typically found within the tumor microenvironment in association with e.

Dithiothreitol (DTT) Acts as a Specific, UV-inducible Cross-linker in Elucidation of Protein-RNA Interactions.

Protein-RNA cross-linking by UV irradiation at 254 nm wavelength has been established as an unbiased method to identify proteins in direct contact with RNA, and has been successfully applied to investigate the spatial arrangement of protein and RNA in large macromolecular assemblies, e.g. ribonucleoprotein-complex particles (RNPs).

Acetylation of histone H3 at lysine 64 regulates nucleosome dynamics and facilitates transcription.

Post-translational modifications of proteins have emerged as a major mechanism for regulating gene expression. However, our understanding of how histone modifications directly affect chromatin function remains limited. In this study, we investigate acetylation of histone H3 at lysine 64 (H3K64ac), a previously uncharacterized acetylation on the lateral surface of the histone octamer.

Prdm3 and Prdm16 are H3K9me1 methyltransferases required for mammalian heterochromatin integrity.

Heterochromatin serves important functions, protecting genome integrity and stabilizing gene expression programs. Although the Suv39h methyltransferases (KMTs) are known to ensure pericentric H3K9me3 methylation, the mechanisms that initiate and maintain mammalian heterochromatin organization remain elusive. We developed a biochemical assay and used in vivo analyses in mouse embryonic fibroblasts to identify Prdm3 and Prdm16 as redundant H3K9me1-specific KMTs that direct cytoplasmic H3K9me1 methylation.

Merging molecular electron microscopy and mass spectrometry by carbon film-assisted endoproteinase digestion.

Many fundamental processes in the cell are performed by complex macromolecular assemblies that comprise a large number of proteins. Numerous macromolecular assemblies are structurally rather fragile and may suffer during purification, resulting in the partial dissociation of the complexes. These limitations can be overcome by chemical fixation of the assemblies, and recently introduced protocols such as gradient fixation during ultracentrifugation (GraFix) offer advantages for the analysis of fragile macromolecular assemblies.

Mapping the binding site of snurportin 1 on native U1 snRNP by cross-linking and mass spectrometry.

Mass spectrometry allows the elucidation of molecular details of the interaction domains of the individual components in macromolecular complexes subsequent to cross-linking of the individual components. Here, we applied chemical and UV cross-linking combined with tandem mass-spectrometric analysis to identify contact sites of the nuclear import adaptor snurportin 1 to the small ribonucleoprotein particle U1 snRNP in addition to the known interaction of m(3)G cap and snurportin 1. We were able to define previously unknown sites of protein-protein and protein-RNA interactions on the molecular level within U1 snRNP.

Enrichment of protein-RNA crosslinks from crude UV-irradiated mixtures for MS analysis by on-line chromatography using titanium dioxide columns.

UV crosslinking is an appropriate method to identify proteins that directly contact nucleic acid, e.g., RNA.