👤 Eugene E Gussakovsky

BC200 is a noncoding RNA elevated in a broad spectrum of tumor cells that is critical for cell viability, invasion, and migration. Overexpression studies have implicated BC200 and the rodent analog BC1 as negative regulators of translation in both cell-based and in vitro translation assays. Although these studies are consistent, they have not been confirmed in knockdown studies and direct evidence for this function is lacking. Herein, we have demonstrated that BC200 knockdown is correlated with a decrease in global translation rates. As this conflicts with the hypothesis that BC200 is a translational suppressor, we overexpressed BC200 by transfection of in vitro transcribed RNA and transient expression from transfected plasmids. In this context BC200 suppressed translation; however, an innate immune response confounded the data. To overcome this, breast cancer cells stably overexpressing BC200 and various control RNAs were developed by selection for genomic incorporation of a plasmid coexpressing BC200 and the neomycin resistance gene. Stable overexpression of BC200 was associated with elevated translation levels in pooled stable cell lines and isolated single-cell clones. Cross-linking sucrose density gradient centrifugation demonstrated an association of BC200 and its reported binding partners SRP9/14, CSDE1, DHX36, and PABPC1 with both ribosomal subunits and polysomal RNA, an association not previously observed owing to the labile nature of the interactions. In summary, these data present a novel understanding of BC200 function as well as optimized methodology that has far reaching implications in the study of noncoding RNAs, particularly within the context of translational regulatory mechanisms. Show less

A subdomain of the human leptin receptor encoding part of the extracellular domain (amino acids 428 to 635) was subcloned, expressed in a prokaryotic host, and purified to homogeneity, as evidenced by SDS-PAGE, with over 95% monomeric protein. The purified leptin-binding domain (LBD) exhibited the predicted beta structure, was capable of binding human, ovine, and chicken leptins, and formed a stable 1:1 complex with all mammalian leptins. The binding kinetics, assayed by surface plasmon resonance methodology, showed respective k(on) and k(off) values (mean +/- S.E.) of 1.20 +/- 0.23 x 10(-5) mol(-1) s(-1) and 1.85 +/- 0.30 x 10(-3) s(-1) and a K(d) value of 1.54 x 10(-8) m. Similar results were achieved with conventional binding experiments. LBD blocked leptin-induced, but not interleukin-3-induced, proliferation of BAF/3 cells stably transfected with the long form of human leptin receptor. The modeled LBD structure and the known three-dimensional structure of human leptin were used to construct a model of 1:1 LBD.human leptin complex. Two main residues, Phe-500, located in loop L3, and Tyr-441, located in L1, are suggested to contribute to leptin binding. Show less