FAX-RIC enables robust profiling of dynamic RNP complex formation in multicellular organisms in vivo.

RNA-protein interaction is central to post-transcriptional gene regulation. Identification of RNA-binding proteins relies mainly on UV-induced crosslinking (UVX) followed by the enrichment of RNA-protein conjugates and LC-MS/MS analysis. However, UVX has limited applicability in tissues of multicellular organisms due to its low penetration depth. Here, we introduce formaldehyde crosslinking (FAX) as an alternative chemical crosslinking for RNA interactome capture (RIC). Mild FAX captures RNA-protein interaction with high specificity and efficiency in cell culture. Unlike UVX-RIC, FAX-RIC robustly detects proteins that bind to structured RNAs or uracil-poor RNAs (e.g. AGO1, STAU1, UPF1, NCBP2, EIF4E, YTHDF proteins and PABP), broadening the coverage. Applied to Xenopus laevis oocytes and embryos, FAX-RIC provided comprehensive and unbiased RNA interactome, revealing dynamic remodeling of RNA-protein complexes. Notably, translation machinery changes during oocyte-to-embryo transition, for instance, from canonical eIF4E to noncanonical eIF4E3. Furthermore, using Mus musculus liver, we demonstrate that FAX-RIC is applicable to mammalian tissue samples. Taken together, we report that FAX can extend the RNA interactome profiling into multicellular organisms.

Purification of an Intact Human Protein Overexpressed from Its Endogenous Locus via Direct Genome Engineering.

The overproduction and purification of human proteins is a requisite of both basic and medical research. Although many recombinant human proteins have been purified, current protein production methods have several limitations; recombinant proteins are frequently truncated, fail to fold properly, and/or lack appropriate post-translational modifications. In addition, such methods require subcloning of the target gene into relevant plasmids, which can be difficult for long proteins with repeated domains. Here we devised a novel method for target protein production by introduction of a strong promoter for overexpression and an epitope tag for purification in front of the endogenous human gene, in a sense performing molecular cloning directly in the human genome, which does not require cloning of the target gene. As a proof of concept, we successfully purified intact human Reelin protein, which is lengthy (3460 amino acids) and contains repeating domains, and confirmed that it was biologically functional.

Development of Multiplexed Immuno-N-Terminomics to Reveal the Landscape of Proteolytic Processing in Early Embryogenesis of Drosophila melanogaster.

Protein expression levels are regulated through both translation and degradation mechanisms. Levels of degradation intermediates, that is, partially degraded proteins, cannot be distinguished from those of intact proteins by global proteomics analysis, which quantify total protein abundance levels. This study aimed to develop a tool for assessing the aspects of degradation regulation via proteolytic processing through a new multiplexed N-terminomics method involving selective isobaric labeling of protein N-termini and immunoaffinity capture of the labeled N-terminal peptides. Our method allows for not only identification of proteolytic cleavage sites, but also highly multiplexed quantification of proteolytic processing. We profiled a number of potential cleavage sites by signal peptidase and provided experimental confirmation of predicted cleavage sites of signal peptide. Furthermore, the present method uniquely represents the landscape of proteomic proteolytic processing rate during early embryogenesis in Drosophila melanogaster, revealing the underlying mechanism of stringent decay regulation of zygotically expressed proteins during early stages of embryogenesis.

Stichoposide C induces apoptosis through the generation of ceramide in leukemia and colorectal cancer cells and shows in vivo antitumor activity.

PURPOSE: Marine triterpene glycosides that are physiologically active natural compounds isolated from sea cucumbers (holothurians) and sponges have antifungal, cytotoxic, and antitumor activities, whose specific molecular mechanisms remain to be elucidated. In this study, we examined if and through which mechanisms stichoposide C (STC) from Thelenota anax (family Stichopodidae) induces apoptosis in leukemia and colorectal cancer cells. EXPERIMENTAL DESIGN: We examined STC-induced apoptosis in human leukemia and colorectal cancer cells in the context of mitochondrial injury and signaling pathway disturbances, and investigated the antitumor effect of STC in mouse CT-26 subcutaneous tumor and HL-60 leukemia xenograft models. RESULTS: We found that STC induces apoptosis in these cells in a dose-dependent manner and leads to the activation of Fas and caspase-8, cleavage of Bid, mitochondrial damage, and activation of caspase-3. STC activates acid sphingomyelinase (SMase) and neutral SMase, which resulted in the generation of ceramide. Specific inhibition of acid SMase or neutral SMase and siRNA knockdown experiments partially blocked STC-induced apoptosis. Moreover, STC markedly reduced tumor growth of HL-60 xenograft and CT-26 subcutaneous tumors and increased ceramide generation in vivo. CONCLUSIONS: Ceramide generation by STC, through activation of acid and neutral SMase, may in part contribute to STC-induced apoptosis and antitumor activity. Thus, STC may have therapeutic relevance for human leukemia and colorectal cancer.