A Small-Molecule Inhibitor of Lin28.

New discoveries in RNA biology underscore a need for chemical tools to clarify their roles in pathophysiological mechanisms. In certain cancers, synthesis of the let-7 microRNA tumor suppressor is blocked by an RNA binding protein (RBP) Lin28, which docks onto a conserved sequence in let-7 precursor RNA molecules and prevents their maturation. Thus, the Lin28/let-7 interaction might be an attractive drug target, if not for the well-known difficulty in targeting RNA-protein interactions with drugs.

Properties of short double-stranded RNAs carrying randomized base pairs: toward better controls for RNAi experiments.

Short interfering RNAs (siRNAs) are mediators of RNA interference (RNAi), a commonly used technique for selective down-regulation of target gene expression. Using an equimolar mixture of A, G, C, and U phosphoramidites during solid-phase synthesis, we introduced degenerate positions into RNA guide and passenger strands so that, when annealed, a large pool of distinct siRNA duplexes with randomized base pairs at defined sites was created. We assessed the randomization efficiency by deep sequencing one of the RNAs.

Blotting from Immobilized pH Gradient Gels: Application to Total Cell Lysates.

Isoelectric focusing as used in the first dimension of two-dimensional gel electrophoresis separates protein isoforms such as those due to phosphorylation and acetylation. The immunoblotting method described here reveals this diversity by a one-dimensional separation. Using commercially available immobilized pH gradient plates or strips, the resolved proteins are transferred to PVDF membranes by diffusion and are probed with protein-specific antibody.

From little helpers to automation.

Western blot technology has continually evolved to enhance sensitivity, speed, and ease of operation. For enhancing awareness to these developments, this brief review outlines a representative selection of methods and devices, many of which are commercial products. In particular, the steps taken towards partial and full automation of western blotting are addressed.

Origins of protein blotting.

The development of protein blotting in its early days is recounted as arising from the need to tackle a specific analytical problem. Combining diverse elements of common methods and simple lab equipment resulted in a procedure of general utility. The expansion of the idea of carrying out immunoassays on membranes as predecessors of microarrays is briefly touched upon.

Short loop-targeting oligoribonucleotides antagonize Lin28 and enable pre-let-7 processing and suppression of cell growth in let-7-deficient cancer cells.

MicroRNAs (miRNAs) originate from stem-loop-containing precursors (pre-miRNAs, pri-miRNAs) and mature by means of the Drosha and Dicer endonucleases and their associated factors. The let-7 miRNAs have prominent roles in developmental differentiation and in regulating cell proliferation. In cancer, the tumor suppressor function of let-7 is abrogated by overexpression of Lin28, one of several RNA-binding proteins that regulate let-7 biogenesis by interacting with conserved motifs in let-7 precursors close to the Dicer cleavage site.

Systematic screens of proteins binding to synthetic microRNA precursors.

We describe a new, broadly applicable methodology for screening in parallel interactions of RNA-binding proteins (RBPs) with large numbers of microRNA (miRNA) precursors and for determining their affinities in native form in the presence of cellular factors. The assays aim at identifying pre-miRNAs that are potentially affected by the selected RBP during their biogenesis. The assays are carried out in microtiter plates and use chemiluminescent readouts.

Structural basis of pre-let-7 miRNA recognition by the zinc knuckles of pluripotency factor Lin28.

Lin28 inhibits the biogenesis of let-7 miRNAs through a direct interaction with the terminal loop of pre-let-7. This interaction requires the zinc-knuckle domains of Lin28. We show that the zinc knuckle domains of Lin28 are sufficient to provide binding selectivity for pre-let-7 miRNAs and present the NMR structure of human Lin28 zinc knuckles bound to the short sequence 5'-AGGAGAU-3'.

Suppression of latent transforming growth factor (TGF)-beta1 restores growth inhibitory TGF-beta signaling through microRNAs.

Cancer cells secreting excess latent TGF-β are often resistant to TGF-β induced growth inhibition. We observed that RNAi against TGF-β1 led to apoptotic death in such cell lines with features that were, paradoxically, reminiscent of TGF-β signaling activity and that included transiently enhanced SMAD2 and AKT phosphorylation. A comprehensive search in Hela cells for potential microRNA drivers of this mechanism revealed that RNAi against TGF-β1 led to induction of pro-apoptotic miR-34a and to a globally decreased oncomir expression.

The potent protein kinase C-selective inhibitor AEB071 (sotrastaurin) represents a new class of immunosuppressive agents affecting early T-cell activation.

There is a pressing need for immunosuppressants with an improved safety profile. The search for novel approaches to blocking T-cell activation led to the development of the selective protein kinase C (PKC) inhibitor AEB071 (sotrastaurin). In cell-free kinase assays AEB071 inhibited PKC, with K(i) values in the subnanomolar to low nanomolar range.

Blotting from immobilized pH gradient gels: application to total cell lysates.

Isoelectric focusing as used in the first dimension of two-dimensional gel electrophoresis separates protein isoforms such as those due to phosphorylation and acetylation. The immunoblotting method described here reveals this diversity by a one-dimensional separation. Using commercially available immobilized pH gradient plates or strips, the resolved proteins are transferred to PVDF membranes by diffusion and are probed with protein specific antibody.

Origins of protein blotting.

The development of protein blotting in its early days is recounted as arising from the need to tackle a specific analytical problem. Combining diverse elements of common methods and simple lab equipment resulted in a procedure of general utility. The expansion of the idea of carrying out immunoassays on membranes as predecessors of microarrays is briefly touched upon.

Analysis of dynamic changes in post-translational modifications of human histones during cell cycle by mass spectrometry.

The N-terminal tails of the four core histones are subject to several types of covalent post-translational modifications that have specific roles in regulating chromatin structure and function. Here we present an extensive analysis of the core histone modifications occurring through the cell cycle. Our MS experiments characterized the modification patterns of histones from HeLa cells arrested in phase G1, S, and G2/M.

Characterization of histone H2A and H2B variants and their post-translational modifications by mass spectrometry.

The nucleosome, the fundamental structural unit of chromatin, contains an octamer of core histones H3, H4, H2A, and H2B. Incorporation of histone variants alters the functional properties of chromatin. To understand the global dynamics of chromatin structure and function, analysis of histone variants incorporated into the nucleosome and their covalent modifications is required.

Proteomics-based target identification: bengamides as a new class of methionine aminopeptidase inhibitors.

LAF389 is a synthetic analogue of bengamides, a class of marine natural products that produce inhibitory effects on tumor growth in vitro and in vivo. A proteomics-based approach has been used to identify signaling pathways affected by bengamides. LAF389 treatment of cells resulted in altered mobility of a subset of proteins on two-dimensional gel electrophoresis.

Gene expression profiling of epothilone A-resistant cells.

In the current study, we isolated sublines of the human breast adenocarcinoma cell line MDA 435 that exhibited increasing resistance to epothilone A, a microtubule-stabilizing cytotoxic agent. The resistant cells did not express P glycoprotein or multidrug resistance-associated protein (MRP) which are known mediators of multidrug resistance (MDR). Two groups of epothilone A-resistant cells were selected: cells which exhibited low resistance to both epothilone A and Taxol, and cells which exhibit low resistance to Taxol but high resistance to epothilone A.