Catherine C. L. Wong

Structure of a yeast spliceosome at 3.6-angstrom resolution

Structure and function of the spliceosome When RNA is transcribed from DNA in the eukaryotic cell nucleus, the initial transcript includes noncoding introns that must be spliced out. This splicing is done by a complex macromolecular machine, the spliceosome, which comprises five small nuclear RNAs and more than 100 associated proteins. Now, two papers reveal insights into the structure and function of the yeast spliceosome. Yan et al. describe a high-resolution structure determined by electron microscopy of a spliceosome complex comprising four RNAs and 37 proteins. Hang et al. focus on the catalytic site and show how protein components anchor the transcribed RNA and allow sufficient flexibility to deliver RNA components involved in catalyzing the splicing reaction. Science, this issue pp. 1182 and 1191 A high-resolution structure determined by electron microscopy provides insight into how the spliceosome functions. Splicing of precursor messenger RNA (pre-mRNA) in yeast is executed by the spliceosome, which consists of five small nuclear ribonucleoproteins (snRNPs), NTC (nineteen complex), NTC-related proteins (NTR), and a number of associated enzymes and cofactors. Here, we report the three-dimensional structure of a Schizosaccharomyces pombe spliceosome at 3.6-angstrom resolution, revealed by means of single-particle cryogenic electron microscopy. This spliceosome contains U2 and U5 snRNPs, NTC, NTR, U6 small nuclear RNA, and an RNA intron lariat. The atomic model includes 10,574 amino acids from 37 proteins and four RNA molecules, with a combined molecular mass of approximately 1.3 megadaltons. Spp42 (Prp8 in Saccharomyces cerevisiae), the key protein component of the U5 snRNP, forms a central scaffold and anchors the catalytic center. Both the morphology and the placement of protein components appear to have evolved to facilitate the dynamic process of pre-mRNA splicing. Our near-atomic-resolution structure of a central spliceosome provides a molecular framework for mechanistic understanding of pre-mRNA splicing.

βCaMKII in Lateral Habenula Mediates Core Symptoms of Depression

Depression and the Habenula The lateral habenula (LHb) appears to have a role in depression. However, the underlying mechanisms are poorly understood, and by using multiple rodent models of depression, Li et al. (p. 1016) identified a signaling pathway and associated neuronal adaptations in which the enzyme βCaMKII was selectively up-regulated in the LHb. Manipulations that enhanced βCaMKII levels increased depression-related phenotypes, and RNA interference of CaMKIIb blunted depression. Enhanced βCaMKII levels in the habenula promoted excitatory synaptic transmission on these neurons and increased action potential firing mediated by an up-regulation of a specific subtype of glutamate receptors. In a rodent model of depression, the increase of a protein kinase is accompanied by an up-regulation of GluR1 receptors. The lateral habenula (LHb) has recently emerged as a key brain region in the pathophysiology of depression. However, the molecular mechanism by which LHb becomes hyperactive in depression remains unknown. Through a quantitative proteomic screen, we found that expression of the β form of calcium/calmodulin-dependent protein kinase type II (βCaMΚΙΙ) was significantly up-regulated in the LHb of animal models of depression and down-regulated by antidepressants. Increasing β-, but not α-, CaMKII in the LHb strongly enhanced the synaptic efficacy and spike output of LHb neurons and was sufficient to produce profound depressive symptoms, including anhedonia and behavioral despair. Down-regulation of βCaMKII levels, blocking its activity or its target molecule the glutamate receptor GluR1 reversed the depressive symptoms. These results identify βCaMKII as a powerful regulator of LHb neuron function and a key molecular determinant of depression.

Extensive translation of circular RNAs driven by N 6 -methyladenosine

Extensive pre-mRNA back-splicing generates numerous circular RNAs (circRNAs) in human transcriptome. However, the biological functions of these circRNAs remain largely unclear. Here we report that N6-methyladenosine (m6A), the most abundant base modification of RNA, promotes efficient initiation of protein translation from circRNAs in human cells. We discover that consensus m6A motifs are enriched in circRNAs and a single m6A site is sufficient to drive translation initiation. This m6A-driven translation requires initiation factor eIF4G2 and m6A reader YTHDF3, and is enhanced by methyltransferase METTL3/14, inhibited by demethylase FTO, and upregulated upon heat shock. Further analyses through polysome profiling, computational prediction and mass spectrometry reveal that m6A-driven translation of circRNAs is widespread, with hundreds of endogenous circRNAs having translation potential. Our study expands the coding landscape of human transcriptome, and suggests a role of circRNA-derived proteins in cellular responses to environmental stress.

Transnitrosylation of XIAP Regulates Caspase-Dependent Neuronal Cell Death

X-linked inhibitor of apoptosis (XIAP) is a potent antagonist of caspase apoptotic activity. XIAP also functions as an E3 ubiquitin ligase, targeting caspases for degradation. However, molecular pathways controlling XIAP activities remain unclear. Here, we report that nitric oxide (NO) reacts with XIAP by S-nitrosylating its RING domain (forming SNO-XIAP), thereby inhibiting E3 ligase and antiapoptotic activity. NO-mediated neurotoxicity and caspase activation have been linked to several neurodegenerative disorders, including Alzheimers, Parkinsons, and Huntingtons diseases. We find significant SNO-XIAP formation in brains of patients with these diseases, implicating this reaction in the etiology of neuronal damage. Conversely, S-nitrosylation of caspases is known to inhibit apoptotic activity. Unexpectedly, we find that SNO-caspase transnitrosylates (transfers its NO group) to XIAP, forming SNO-XIAP, and thus promotes cell injury and death. These findings provide insights into the regulation of caspase activation in neurodegenerative disorders mediated, at least in part, by nitrosative stress.

Calcium Signaling through CaMKII Regulates Hepatic Glucose Production in Fasting and Obesity

Hepatic glucose production (HGP) is crucial for glucose homeostasis, but the underlying mechanisms have not been fully elucidated. Here, we show that a calcium-sensing enzyme, CaMKII, is activated in a calcium- and IP3R-dependent manner by cAMP and glucagon in primary hepatocytes and by glucagon and fasting in vivo. Genetic deficiency or inhibition of CaMKII blocks nuclear translocation of FoxO1 by affecting its phosphorylation, impairs fasting- and glucagon/cAMP-induced glycogenolysis and gluconeogenesis, and lowers blood glucose levels, while constitutively active CaMKII has the opposite effects. Importantly, the suppressive effect of CaMKII deficiency on glucose metabolism is abrogated by transduction with constitutively nuclear FoxO1, indicating that the effect of CaMKII deficiency requires nuclear exclusion of FoxO1. This same pathway is also involved in excessive HGP in the setting of obesity. These results reveal a calcium-mediated signaling pathway involved in FoxO1 nuclear localization and hepatic glucose homeostasis.

ProLuCID: An improved SEQUEST-like algorithm with enhanced sensitivity and specificity

ProLuCID, a new algorithm for peptide identification using tandem mass spectrometry and protein sequence databases has been developed. This algorithm uses a three tier scoring scheme. First, a binomial probability is used as a preliminary scoring scheme to select candidate peptides. The binomial probability scores generated by ProLuCID minimize molecular weight bias and are independent of database size. A modified cross-correlation score is calculated for each candidate peptide identified by the binomial probability. This cross-correlation scoring function models the isotopic distributions of fragment ions of candidate peptides which ultimately results in higher sensitivity and specificity than that obtained with the SEQUEST XCorr. Finally, ProLuCID uses the distribution of XCorr values for all of the selected candidate peptides to compute a Z score for the peptide hit with the highest XCorr. The ProLuCID Z score combines the discriminative power of XCorr and DeltaCN, the standard parameters for assessing the quality of the peptide identification using SEQUEST, and displays significant improvement in specificity over ProLuCID XCorr alone. ProLuCID is also able to take advantage of high resolution MS/MS spectra leading to further improvements in specificity when compared to low resolution tandem MS data. A comparison of filtered data searched with SEQUEST and ProLuCID using the same false discovery rate as estimated by a target-decoy database strategy, shows that ProLuCID was able to identify as many as 25% more proteins than SEQUEST. ProLuCID is implemented in Java and can be easily installed on a single computer or a computer cluster. This article is part of a Special Issue entitled: Computational Proteomics.

The Interaction of CtIP and Nbs1 Connects CDK and ATM to Regulate HR–Mediated Double-Strand Break Repair

CtIP plays an important role in homologous recombination (HR)–mediated DNA double-stranded break (DSB) repair and interacts with Nbs1 and BRCA1, which are linked to Nijmegen breakage syndrome (NBS) and familial breast cancer, respectively. We identified new CDK phosphorylation sites on CtIP and found that phosphorylation of these newly identified CDK sites induces association of CtIP with the N-terminus FHA and BRCT domains of Nbs1. We further showed that these CDK-dependent phosphorylation events are a prerequisite for ATM to phosphorylate CtIP upon DNA damage, which is important for end resection to activate HR by promoting recruitment of BLM and Exo1 to DSBs. Most notably, this CDK-dependent CtIP and Nbs1 interaction facilitates ATM to phosphorylate CtIP in a substrate-specific manner. These studies reveal one important mechanism to regulate cell-cycle-dependent activation of HR upon DNA damage by coupling CDK- and ATM-mediated phosphorylation of CtIP through modulating the interaction of CtIP with Nbs1, which significantly helps to understand how DSB repair is regulated in mammalian cells to maintain genome stability.

Improving the comprehensiveness and sensitivity of sheathless capillary electrophoresis-tandem mass spectrometry for proteomic analysis.

We describe a solid phase microextraction (SPME), multistep elution, transient isotachophoresis (tITP) capillary electrophoresis-tandem mass spectrometry (CE-MS/MS) procedure which employs a high sensitivity porous electrospray ionization (ESI) sprayer for the proteomic analysis of a moderately complex protein mixture. In order to improve comprehensiveness and sensitivity over a previously reported proteomic application of the ESI sprayer, we evaluated preconcentration with SPME and multistep elution prior to tITP stacking and CE separation. To maximize separation efficiency, we primarily employed electrokinetic methods for elution and separation after loading the sample by application of pressure. Conditions were developed for optimum simultaneous electrokinetic elution and sample stacking using a tryptic digest of 16 proteins to maximize peptide identifications and minimize band broadening. We performed comparative proteomic analysis of a dilution series using CE and nanoflow liquid chromatography (nLC). We found complementary peptide and protein identifications with larger quantities (100 ng) of a Pyrococcus furiosus tryptic digest, but with mass-limited amounts (5 ng) CE was 3 times more effective at identifying proteins. We attribute these gains in sensitivity to lower noise levels with the porous CE sprayer, illustrated by better signal-to-noise ratios of peptide precursor ions and associated higher XCorr values of identified peptides when compared directly to nLC. From comparative analysis of SPME-tITP-CE with direct injection CE, the SPME-tITP process improved comprehensiveness and sensitivity.

Protein cysteine phosphorylation of SarA/MgrA family transcriptional regulators mediates bacterial virulence and antibiotic resistance

Protein posttranslational modifications (PTMs), particularly phosphorylation, dramatically expand the complexity of cellular regulatory networks. Although cysteine (Cys) in various proteins can be subject to multiple PTMs, its phosphorylation was previously considered a rare PTM with almost no regulatory role assigned. We report here that phosphorylation occurs to a reactive cysteine residue conserved in the staphylococcal accessary regulator A (SarA)/MarR family global transcriptional regulator A (MgrA) family of proteins, and is mediated by the eukaryotic-like kinase-phosphatase pair Stk1-Stp1 in Staphylococcus aureus. Cys-phosphorylation is crucial in regulating virulence determinant production and bacterial resistance to vancomycin. Cell wall-targeting antibiotics, such as vancomycin and ceftriaxone, inhibit the kinase activity of Stk1 and lead to decreased Cys-phosphorylation of SarA and MgrA. An in vivo mouse model of infection established that the absence of stp1, which results in elevated protein Cys-phosphorylation, significantly reduces staphylococcal virulence. Our data indicate that Cys-phosphorylation is a unique PTM that can play crucial roles in bacterial signaling and regulation.

Structure‐Based Discovery of Natural‐Product‐like TNF‐α Inhibitors

Small but effective: two natural-product-like inhibitors of tumor necrosis factor α (TNF-α; represented in green in the picture) have been identified using structure-based virtual screening. These compounds represent only the third and fourth examples of direct targeting of TNF-α by a small molecule, and display potency comparable to that of the strongest TNF-α inhibitor reported to date.