Thermophile 90S Pre-ribosome Structures Reveal the Reverse Order of Co-transcriptional 18S rRNA Subdomain Integration.

Eukaryotic ribosome biogenesis involves RNA folding and processing that depend on assembly factors and small nucleolar RNAs (snoRNAs). The 90S (SSU-processome) is the earliest pre-ribosome structurally analyzed, which was suggested to assemble stepwise along the growing pre-rRNA from 5' > 3', but this directionality may not be accurate. Here, by analyzing the structure of a series of 90S assembly intermediates from Chaetomium thermophilum, we discover a reverse order of 18S rRNA subdomain incorporation. Large parts of the 18S rRNA 3' and central domains assemble first into the 90S before the 5' domain is integrated. This final incorporation depends on a contact between a heterotrimer Enp2-Bfr2-Lcp5 recruited to the flexible 5' domain and Kre33, which reconstitutes the Kre33-Enp-Brf2-Lcp5 module on the compacted 90S. Keeping the 5' domain temporarily segregated from the 90S scaffold could provide extra time to complete the multifaceted 5' domain folding, which depends on a distinct set of snoRNAs and processing factors.

Structural insights into coordinating 5S RNP rotation with ITS2 pre-RNA processing during ribosome formation.

The rixosome defined in Schizosaccharomyces pombe and humans performs diverse roles in pre-ribosomal RNA processing and gene silencing. Here, we isolate and describe the conserved rixosome from Chaetomium thermophilum, which consists of two sub-modules, the sphere-like Rix1-Ipi3-Ipi1 and the butterfly-like Las1-Grc3 complex, connected by a flexible linker. The Rix1 complex of the rixosome utilizes Sda1 as landing platform on nucleoplasmic pre-60S particles to wedge between the 5S rRNA tip and L1-stalk, thereby facilitating the 180° rotation of the immature 5S RNP towards its mature conformation. Upon rixosome positioning, the other sub-module with Las1 endonuclease and Grc3 polynucleotide-kinase can reach a strategic position at the pre-60S foot to cleave and 5' phosphorylate the nearby ITS2 pre-rRNA. Finally, inward movement of the L1 stalk permits the flexible Nop53 N-terminus with its AIM motif to become positioned at the base of the L1-stalk to facilitate Mtr4 helicase-exosome participation for completing ITS2 removal. Thus, the rixosome structure elucidates the coordination of two central ribosome biogenesis events, but its role in gene silencing may adapt similar strategies.

Translation of the cell adhesion molecule ALCAM in axonal growth cones - regulation and functional importance.

ALCAM is a cell adhesion molecule that is present on extending axons and has been shown to be crucial for elongation and navigation of retinal ganglion cell (RGC) axons. In the present study, we show that ALCAM mRNA is present in axonal growth cones of RGCs in vivo and in vitro, and that translation of ALCAM occurs in RGC growth cones separated from their soma. This growth cone translation is regulated by the 3'-untranslated region (3'-UTR) of ALCAM and depends on the activity of the kinases ERK and TOR (target of rapamycin). We also investigated the impact of the growth cone translation of ALCAM on axonal functions. Growth cone translation of ALCAM is crucial for the enhanced elongation of axons extending in contact with ALCAM protein. The local translation of ALCAM in the growth cone is able to rapidly counterbalance experimentally induced ALCAM internalization, thereby contributing to the maintenance of constant ALCAM levels in the plasma membrane. Assays where RGC axons have the choice to grow on laminin or both ALCAM and laminin - as is the case in the developing retina - reveal that the axonal preference for ALCAM-containing lanes depends on translation of ALCAM in growth cones. Taken together, these results show for the first time that translation of a cell adhesion molecule in growth cones, as well as the impact of this local translation on the behavior of axon and growth cone.

Development of a high throughput oxidation profiling strategy for monoclonal antibody products.

Oxidation is one of the most common degradation pathways of biopharmaceutics, potentially leading to altered product stability, pharmacokinetics, reduced biological activity and/or an increased immunogenicity. However, it is often insufficiently assessed in early development stages, leaving potential molecule liabilities undiscovered. Aim of the present work was the development of a high throughput oxidation profiling strategy, applicable throughout various stages of biopharmaceutical development. The study demonstrates that the combination of multiple stress assays, including peroxide-based, visible light, and metal-catalyzed oxidation (MCO), enables a comprehensive understanding of a mAb's oxidation susceptibility. The most effective parameters to evaluate oxidation in a high-throughput screening workflow are aggregation, tryptophan oxidation and changes in the hydrophobicity profile of the Fc and Fab subunit measured via Size Exclusion Chromatography, Intrinsic Tryptophan Fluorescence Emission spectroscopy and Reversed-Phase Chromatography subunit analysis, respectively. This oxidation profiling approach is valuable tool to systematically characterize the oxidation susceptibility under relevant conditions, time effective and with minimal sample consumption.

Application of tiny-TIM as a mechanistic tool to investigate the in vitro performance of different itraconazole formulations under physiologically relevant conditions.

The increasing number of poorly water-soluble compounds in drug development is one of the major challenges in oral drug delivery nowadays. For rational formulation development, biopharmaceutical tools are needed that closely simulate the conditions present within the human gastrointestinal (GI) tract in order to early predict the potential effect of important factors like meal intake or acid-reducing agents on oral bioavailability. The tiny-TIM system equipped with the advanced gastric compartment is one of the most realistic in vitro models for the simulation of the physiological processes occurring in human stomach and small intestine. In the present study, this model was applied to study the in vitro performance of an ASD-based formulation of itraconazole under different clinically relevant conditions. Apart from the assessment of the bioaccessible fraction (i.e., the fraction available for drug absorption), the implementation of two additional sampling ports enabled the measurement of intraluminal concentration profiles. Along with solubility experiments in biorelevant media, deeper mechanistic insights into drug product performance in different prandial states as well as in case of gastric pH modification could be generated. The comparison of the in vitro data with published in vivo data revealed that the model successfully predicted the effect of food intake as well as of modified gastric pH conditions on the bioavailability of itraconazole from this formulation. In contrast, the negative food effect observed for an oral solution formulation could not be predicted. For this cyclodextrin-based formulation, the formulation effect on permeation needs to be considered. Nonetheless, the data presented in this study showed that tiny-TIM is an interesting tool to mechanistically study the impact of different physiological conditions on drug release from oral drug products.