A p53-dependent translational program directs tissue-selective phenotypes in a model of ribosomopathies.

In ribosomopathies, perturbed expression of ribosome components leads to tissue-specific phenotypes. What accounts for such tissue-selective manifestations as a result of mutations in the ribosome, a ubiquitous cellular machine, has remained a mystery. Combining mouse genetics and in vivo ribosome profiling, we observe limb-patterning phenotypes in ribosomal protein (RP) haploinsufficient embryos, and we uncover selective translational changes of transcripts that controlling limb development. Surprisingly, both loss of p53, which is activated by RP haploinsufficiency, and augmented protein synthesis rescue these phenotypes. These findings are explained by the finding that p53 functions as a master regulator of protein synthesis, at least in part, through transcriptional activation of 4E-BP1. 4E-BP1, a key translational regulator, in turn, facilitates selective changes in the translatome downstream of p53, and this thereby explains how RP haploinsufficiency may elicit specificity to gene expression. These results provide an integrative model to help understand how in vivo tissue-specific phenotypes emerge in ribosomopathies.

Rapid, Refined, and Robust Method for Expression, Purification, and Characterization of Recombinant Human Amyloid beta 1-42.

Amyloid plaques found in the brains of Alzheimer's disease patients primarily consists of amyloid beta 1-42 (Aß42). Commercially, Aß42 is synthesized using high-throughput peptide synthesizers resulting in the presence of impurities and the racemization of amino acids that affects its aggregation properties. Furthermore, the repeated purchase of even a small quantity (~1 mg) of commercial Aß42 can be expensive for academic researchers. Here, we describe a detailed methodology for robust expression of recombinant human Aß(M1-42) in Rosetta(DE3)pLysS and BL21(DE3)pLysS competent E. coli using standard molecular biology techniques with refined and rapid one-step analytical purification techniques. The peptide is isolated and purified from transformed cells using an optimized reverse-phase high-performance liquid chromatography (HPLC) protocol with commonly available C18 columns, yielding high amounts of peptide (~15-20 mg per 1 L culture) within a short period of time. The recombinant human Aß(M1-42) forms characteristic aggregates similar to synthetic Aß42 aggregates as verified by western blotting and atomic force microscopy to warrant future biological use. Our rapid, refined, and robust technique produces pure recombinant human Aß(M1-42) that may be used to synthesize chemical probes and in several downstream in vitro and in vivo assays to facilitate Alzheimer's disease research.