Engineering Crystal Packing in RNA-Protein Complexes II: A Historical Perspective from the Structural Studies of the Spliceosome.

Cryo-electron microscopy has greatly advanced our understanding of how the spliceosome cycles through different conformational states to conduct the chemical reactions that remove introns from pre-mRNA transcripts. The Cryo-EM structures were built upon decades of crystallographic studies of various spliceosomal RNA-protein complexes. In this review we give an overview of the crystal structures solved in the Nagai group, utilizing many of the strategies to design crystal packing as described in the accompanying paper.

First In Vivo Testing of Compounds Targeting Group 3 Medulloblastomas Using an Implantable Microdevice as a New Paradigm for Drug Development.

Medulloblastoma is the most common childhood malignant brain tumor. The most lethal medulloblastoma subtype exhibits a high expression of the GABAA receptor α5 subunit gene and MYC amplification. New benzodiazepines have been synthesized to function as α5-GABAA receptor ligands. To compare their efficacy with that of standard-of-care treatments, we have employed a newly developed microscale implantable device that allows for high-throughput localized intratumor drug delivery and efficacy testing. Microdoses of each drug were delivered into small distinct regions of tumors, as confirmed by tissue mass spectrometry, and the local drug effect was determined by immunohistochemistry. We have identified a benzodiazepine derivative, KRM-II-08, as a new potent inhibitor in several α5-GABAA receptor expressing tumor models. This is the first instance of in vivo testing of several benzodiazepine derivatives and standard chemotherapeutic drugs within the same tumor. Obtaining high-throughput drug efficacy data within a native tumor microenvironment as detailed herein, prior to pharmacological optimization for bioavailability or safety and without systemic exposure or toxicity, may allow for rapid prioritization of drug candidates for further pharmacological optimization.

Interpreting a low resolution map of human U1 snRNP using anomalous scatterers.

We recently determined the crystal structure of the functional core of human U1 snRNP, consisting of nine proteins and one RNA, based on a 5.5 A resolution electron density map. At 5-7 A resolution, alpha helices and beta sheets appear as rods and slabs, respectively, hence it is not possible to determine protein fold de novo. Using inverse beam geometry, accurate anomalous signals were obtained from weakly diffracting and radiation sensitive P1 crystals. We were able to locate anomalous scatterers with positional errors below 2 A. This enabled us not only to place protein domains of known structure accurately into the map but also to trace an extended polypeptide chain, of previously undetermined structure, using selenomethionine derivatives of single methionine mutants spaced along the sequence. This method of Se-Met scanning, in combination with structure prediction, is a powerful tool for building a protein of unknown fold into a low resolution electron density map.