Medulloblastomas are the most common malignant brain tumors in children1. Identifying and understanding the genetic events that drive these tumors is critical for the development of more effective diagnostic, prognostic and therapeutic strategies. Recently, our group and others described distinct molecular subtypes of medulloblastoma based on transcriptional and copy number profiles2–5. Here, we utilized whole exome hybrid capture and deep sequencing to identify somatic mutations across the coding regions of 92 primary medulloblastoma/normal pairs. Overall, medulloblastomas exhibit low mutation rates consistent with other pediatric tumors, with a median of 0.35 non-silent mutations per megabase. We identified twelve genes mutated at statistically significant frequencies, including previously known mutated genes in medulloblastoma such as CTNNB1, PTCH1, MLL2, SMARCA4 and TP53. Recurrent somatic mutations were identified in an RNA helicase gene, DDX3X, often concurrent with CTNNB1 mutations, and in the nuclear co-repressor (N-CoR) complex genes GPS2, BCOR, and LDB1, novel findings in medulloblastoma. We show that mutant DDX3X potentiates transactivation of a TCF promoter and enhances cell viability in combination with mutant but not wild type beta-catenin. Together, our study reveals the alteration of Wnt, Hedgehog, histone methyltransferase and now N-CoR pathways across medulloblastomas and within specific subtypes of this disease, and nominates the RNA helicase DDX3X as a component of pathogenic beta-catenin signaling in medulloblastoma.

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Complement is an important component of the innate immune system, which collaborates with the adaptive antibody immune system. It is activated through three different pathways, which all trigger cleavage of the three homolgous proteins C3, C4 and C5. The latter is cleaved into the small anaphylatoxin C5a and the large C5b fragment. C5a binds to two G-protein coupled receptors and thereby elicit chemotaxis, a respiratory burst, and release of proinflammatory mediators. C5b combine with four other complement proteins to form the membrane perforating membrane attack complex. To provide the structural basis of the functions of C5, we have determined the crystal structure of human C5 at 3.1 Å resolution [1]. In addition we have studied the interaction of C5 with inhibitors. The structure of C5 will be presented and the potential for using structural data in therautic approaches to diseases involving complement will be discussed.

Precursor-mRNA splicing is catalyzed by an extraordinarily large and highly dynamic macromolecular assemblage termed the spliceosome. Detailed biochemical and structural study of the spliceosome presents a formidable challenge, but there has recently been significant progress made on this front highlighted by the crystal structure of a 10-subunit human U1 snRNP. This review provides an overview of our current understanding of the architecture of the spliceosome and the RNA-protein complexes integral to its function, the U snRNPs.

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, but these had undesirable side effects in the nude mouse xenograft model system (4). 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. We have identified a benzodiazepine derivative, KRM-II-08, as a new potent inhibitor in several α5-GABAA receptor expressing tumor models. 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.

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Purpose Pediatric brain cancer medulloblastoma (MB) standard-of-care results in numerous comorbidities. MB is comprised of distinct molecular subgroups. Group 3 molecular subgroup patients have the highest relapse rates and after standard-of-care have a 20% survival. Group 3 tumors have high expression of GABRA5 , which codes for the α5 subunit of the γ-aminobutyric acid type A receptor (GABA A R). We are advancing a therapeutic approach for group 3 based on GABA A R modulation using benzodiazepine-derivatives. Methods We performed analysis of GABR and MYC expression in MB tumors and used molecular, cell biological, and whole-cell electrophysiology approaches to establish presence of a functional ‘druggable’ GABA A R in group 3 cells. Results Analysis of expression of 763 MB tumors reveals that group 3 tumors share high subgroup-specific and correlative expression of GABR genes, which code for GABA A R subunits α5, β3 and γ2 and 3. There are ~ 1000 functional α5-GABA A Rs per group 3 patient-derived cell that mediate a basal chloride-anion efflux of 2 × 10 9 ions/s. Benzodiazepines, designed to prefer α5-GABA A R, impair group 3 cell viability by enhancing chloride-anion efflux with subtle changes in their structure having significant impact on potency. A potent, non-toxic benzodiazepine (‘KRM-II-08’) binds to the α5-GABA A R (0.8 µM EC 50 ) enhancing a chloride-anion efflux that induces mitochondrial membrane depolarization and in response, TP53 upregulation and p53, constitutively phosphorylated at S392, cytoplasmic localization. This correlates with pro-apoptotic Bcl-2-associated death promoter protein localization. Conclusion GABRA5 expression can serve as a diagnostic biomarker for group 3 tumors, while α5-GABA A R is a therapeutic target for benzodiazepine binding, enhancing an ion imbalance that induces apoptosis. Electronic supplementary material The online version of this article (10.1007/s11060-019-03115-0) contains supplementary material, which is available to authorized users.

M1 RNA, the catalytic subunit of Escherichia coli RNase P, forms a secondary structure that includes five sequence variants of the tetraloop motif. Site-directed mutagenesis of the five tetraloops of M1 RNA, and subsequent steady-state kinetic analysis in vitro, with different substrates in the presence and absence of the protein cofactor, reveal that (i) certain mutants exhibit defects that vary in a substratedependent manner, and that (ii) the protein cofactor can correct the mutant phenotypes in vitro, a phenomenon that is also substrate dependent. Thermal denaturation curves of tetraloop mutants that exhibit kinetic defects differ from those of wild-type M1 RNA. Although the data collected in vitro underscore the importance of the tetraloop motif to M1 RNA function and structure, three of the five tetraloops we examined in vivo are essential for the function of E. coli RNase P. The kinetic data in vitro are not in total agreement with previous phylogenetic predictions but the data in vivo are, as only mutants in those tetraloops proposed to be involved in tertiary interactions fail to complement in vivo. Therefore, the tetraloop motif is critical for the stabilization of the structure of M1 RNA and essential to RNase P function in the cell.Escherichia coli RNase P is a ribonucleoprotein enzyme composed of a single RNA (M1 RNA) and a single protein (C5 protein) subunit. Both subunits are essential for RNase P function in vivo. However, in vitro, M1 RNA alone can catalyze the hydrolysis of all the known RNA substrates of RNase P (1, 2), including precursor tRNAs (ptRNAs) and precursors to 4.5S and 10Sa RNAs (2).Phylogenetic studies of ribosomal RNA led to the identification of recurring secondary structural motifs such as that of the tetraloop (loops of 4 nts) at the turn of an RNA duplex that has, primarily, one of two sequence variations: GNRA or UNCG (where, N ϭ A, C, G or U; R ϭ A or G) (3). The presence of a tetraloop can confer on short model helices an added thermodynamic stability and therefore one role of a tetraloop might be to stabilize an RNA duplex in a functional RNA molecule (4-6). The tetraloop also could serve to stabilize the tertiary structure of an RNA molecule by making specific contact(s) with a distal site in the molecule (an intramolecular interaction) (7-9). Tetraloops also can form intermolecular interactions, for example, by mediating formation of an RNA-protein complex (10).M1 RNA has several tetraloops in its proposed secondary structure, as do many other RNAs with identifiable function. Specifically, of the eight loops at the turn of RNA helices in M1 RNA, five are tetraloops (one has the sequence UNCG and four have the sequence GNRA; see Fig. 1). It is not clear what function all the tetraloops of M1 RNA serve or whether they are necessary for RNase P function in vivo. The M1 RNA tetraloops could, in principle, (i) mediate interactions between M1 RNA and the C5 protein, (ii) mediate the binding of M1 RNA to some or all of its RNA substrates, or (iii) stabilize the conform...