Hot off the presses! Oct 14 Cell

The Oct 14 issue of the Cell is now up on Pubget (About Cell): if you're at a subscribing institution, just click the link in the latest link at the home page. (Note you'll only be able to get all the PDFs in the issue if your institution subscribes to Pubget.)

Latest Articles Include:

• In This Issue
  - Cell 147(2):249,251 (2011)
  
• Targeted Therapeutics
  - Cell 147(2):253,255 (2011)
  Deciphering the molecular underpinnings of a disease is often only the first step toward developing an effective treatment. The path from target identification to a successful therapeutic can be filled with numerous hurdles that call for creative strategies. This Select highlights recent advances in using gene therapy and engineered cells to treat cancers and chronic heart disease. Their success in preliminary trials suggests that these unconventional strategies are close to becoming mainstream options for refractory diseases.
• Is iNOS Beginning to Smoke?
  - Cell 147(2):257-258 (2011)
  Inducible nitric oxide synthase (iNOS) helps drive numerous inflammatory disorders, but its inhibition has not had therapeutic success. Now make a case for inhibiting iNOS in an effort to treat one of the world's leading causes of deathâ€"chronic obstructive pulmonary disease.
• The Powerstroke and Camshaft of the RIG-I Antiviral RNA Detection Machine
  - Cell 147(2):259-261 (2011)
  The innate immune sensor RIG-I responds to infection by binding to viral double-stranded RNA (dsRNA). In this issue of Cell, and reveal the structure of RIG-I, and in combination with functional analyses, they show how RIG-I recognizes viral RNA to initiate signaling and a type I interferon response.
• Why Does Morphine Make You Itch?
  - Cell 147(2):261-262 (2011)
  Opioids such as morphine numb pain but often concomitantly induce itch. now separate the sensation of itch from opioid-induced analgesia, showing that in a subset of spinal neurons, morphine directly induces itch by signaling through a heteromer of opioid- and itch-mediating G protein-coupled receptors.
• Genome-wide "Re"-Modeling of Nucleosome Positions
  - Cell 147(2):263-266 (2011)
  Two recent studies mapped nucleosomes across the yeast and human genomes, teasing apart the relative contributions of DNA sequence and chromatin remodelers to nucleosome organization. These data suggest two emerging models: chromatin remodelers position nucleosomes around transcriptional start sites in yeast, and a few "locked" nucleosomes may serve as barriers from which nucleosome arrays emanate in human genomes.
• The Impressionistic Landscape of Meiotic Recombination
  - Cell 147(2):267-270 (2011)
  Two high-resolution maps of meiotic recombination initiation sites across the genomes of budding yeast and mice illuminate broad similarities in the control of meiotic recombination in these diverse species but also highlight key differences. These studies offer new insights into the relationships between recombination, chromosome structure, and genome evolution.
• Hiding in Plain Sight: How HIV Evades Innate Immune Responses
  - Cell 147(2):271-274 (2011)
  Two groups have identified SAMHD1, a protein encoded by an Aicardi-Goutières Syndrome susceptibility gene, as the factor that restricts infection of macrophages and dendritic cells with HIV-1. Here we discuss implications of this discovery for induction of antiviral protective immunity.
• Tumor Metastasis: Molecular Insights and Evolving Paradigms
  - Cell 147(2):275-292 (2011)
  Metastases represent the end products of a multistep cell-biological process termed the invasion-metastasis cascade, which involves dissemination of cancer cells to anatomically distant organ sites and their subsequent adaptation to foreign tissue microenvironments. Each of these events is driven by the acquisition of genetic and/or epigenetic alterations within tumor cells and the co-option of nonneoplastic stromal cells, which together endow incipient metastatic cells with traits needed to generate macroscopic metastases. Recent advances provide provocative insights into these cell-biological and molecular changes, which have implications regarding the steps of the invasion-metastasis cascade that appear amenable to therapeutic targeting.
• Inducible NOS Inhibition Reverses Tobacco-Smoke-Induced Emphysema and Pulmonary Hypertension in Mice
  - Cell 147(2):293-305 (2011)
  Chronic obstructive pulmonary disease (COPD) is one of the most common causes of death worldwide. We report in an emphysema model of mice chronically exposed to tobacco smoke that pulmonary vascular dysfunction, vascular remodeling, and pulmonary hypertension (PH) precede development of alveolar destruction. We provide evidence for a causative role of inducible nitric oxide synthase (iNOS) and peroxynitrite in this context. Mice lacking iNOS were protected against emphysema and PH. Treatment of wild-type mice with the iNOS inhibitor N6-(1-iminoethyl)-L-lysine (L-NIL) prevented structural and functional alterations of both the lung vasculature and alveoli and also reversed established disease. In chimeric mice lacking iNOS in bone marrow (BM)-derived cells, PH was dependent on iNOS from BM-derived cells, whereas emphysema development was dependent on iNOS from non-BM-derived cells. Similar regulatory and structural alterations as seen in mouse lungs were found in lung t! issue from humans with end-stage COPD.
• Targeting the SH2-Kinase Interface in Bcr-Abl Inhibits Leukemogenesis
  - Cell 147(2):306-319 (2011)
  Chronic myelogenous leukemia (CML) is caused by the constitutively active tyrosine kinase Bcr-Abl and treated with the tyrosine kinase inhibitor (TKI) imatinib. However, emerging TKI resistance prevents complete cure. Therefore, alternative strategies targeting regulatory modules of Bcr-Abl in addition to the kinase active site are strongly desirable. Here, we show that an intramolecular interaction between the SH2 and kinase domains in Bcr-Abl is both necessary and sufficient for high catalytic activity of the enzyme. Disruption of this interface led to inhibition of downstream events critical for CML signaling and, importantly, completely abolished leukemia formation in mice. Furthermore, disruption of the SH2-kinase interface increased sensitivity of imatinib-resistant Bcr-Abl mutants to TKI inhibition. An engineered Abl SH2-binding fibronectin type III monobody inhibited Bcr-Abl kinase activity both in vitro and in primary CML cells, where it induced apoptosis. Thi! s work validates the SH2-kinase interface as an allosteric target for therapeutic intervention. PaperFlick To view the video inline, enable JavaScript on your browser. However, you can download and view the video by clicking on the icon below Download this Video (11038K) View Within Article
• Introns within Ribosomal Protein Genes Regulate the Production and Function of Yeast Ribosomes
  - Cell 147(2):320-331 (2011)
  In budding yeast, the most abundantly spliced pre-mRNAs encode ribosomal proteins (RPs). To investigate the contribution of splicing to ribosome production and function, we systematically eliminated introns from all RP genes to evaluate their impact on RNA expression, pre-rRNA processing, cell growth, and response to stress. The majority of introns were required for optimal cell fitness or growth under stress. Most introns are found in duplicated RP genes, and surprisingly, in the majority of cases, deleting the intron from one gene copy affected the expression of the other in a nonreciprocal manner. Consistently, 70% of all duplicated genes were asymmetrically expressed, and both introns and gene deletions displayed copy-specific phenotypic effects. Together, our results indicate that splicing in yeast RP genes mediates intergene regulation and implicate the expression ratio of duplicated RP genes in modulating ribosome function. PaperClip View Within Article
• Two Forms of Loops Generate the Chromatin Conformation of the Immunoglobulin Heavy-Chain Gene Locus
  - Cell 147(2):332-343 (2011)
  The immunoglobulin heavy-chain (IgH) gene locus undergoes radial repositioning within the nucleus and locus contraction in preparation for gene recombination. We demonstrate that IgH locus conformation involves two levels of chromosomal compaction. At the first level, the locus folds into several multilooped domains. One such domain at the 3′ end of the locus requires an enhancer, Eμ; two other domains at the 5′ end are Eμ independent. At the second level, these domains are brought into spatial proximity by Eμ-dependent interactions with specific sites within the VH region. Eμ is also required for radial repositioning of IgH alleles, indicating its essential role in large-scale chromosomal movements in developing lymphocytes. Our observations provide a comprehensive view of the conformation of IgH alleles in pro-B cells and the mechanisms by which it is established.
• Coding-Independent Regulation of the Tumor Suppressor PTEN by Competing Endogenous mRNAs
  - Cell 147(2):344-357 (2011)
  Here, we demonstrate that protein-coding RNA transcripts can crosstalk by competing for common microRNAs, with microRNA response elements as the foundation of this interaction. We have termed such RNA transcripts as competing endogenous RNAs (ceRNAs). We tested this hypothesis in the context of PTEN, a key tumor suppressor whose abundance determines critical outcomes in tumorigenesis. By a combined computational and experimental approach, we identified and validated endogenous protein-coding transcripts that regulate PTEN, antagonize PI3K/AKT signaling, and possess growth- and tumor-suppressive properties. Notably, we also show that these genes display concordant expression patterns with PTEN and copy number loss in cancers. Our study presents a road map for the prediction and validation of ceRNA activity and networks and thus imparts a trans-regulatory function to protein-coding mRNAs.
• A Long Noncoding RNA Controls Muscle Differentiation by Functioning as a Competing Endogenous RNA
  - Cell 147(2):358-369 (2011)
  Recently, a new regulatory circuitry has been identified in which RNAs can crosstalk with each other by competing for shared microRNAs. Such competing endogenous RNAs (ceRNAs) regulate the distribution of miRNA molecules on their targets and thereby impose an additional level of post-transcriptional regulation. Here we identify a muscle-specific long noncoding RNA, linc-MD1, which governs the time of muscle differentiation by acting as a ceRNA in mouse and human myoblasts. Downregulation or overexpression of linc-MD1 correlate with retardation or anticipation of the muscle differentiation program, respectively. We show that linc-MD1 "sponges" miR-133 and miR-135 to regulate the expression of MAML1 and MEF2C, transcription factors that activate muscle-specific gene expression. Finally, we demonstrate that linc-MD1 exerts the same control over differentiation timing in human myoblasts, and that its levels are strongly reduced in Duchenne muscle cells. We conclude tha! t the ceRNA network plays an important role in muscle differentiation.
• An Extensive MicroRNA-Mediated Network of RNA-RNA Interactions Regulates Established Oncogenic Pathways in Glioblastoma
  - Cell 147(2):370-381 (2011)
  By analyzing gene expression data in glioblastoma in combination with matched microRNA profiles, we have uncovered a posttranscriptional regulation layer of surprising magnitude, comprising more than 248,000 microRNA (miR)-mediated interactions. These include ∼7,000 genes whose transcripts act as miR "sponges" and 148 genes that act through alternative, nonsponge interactions. Biochemical analyses in cell lines confirmed that this network regulates established drivers of tumor initiation and subtype implementation, including PTEN, PDGFRA, RB1, VEGFA, STAT3, and RUNX1, suggesting that these interactions mediate crosstalk between canonical oncogenic pathways. siRNA silencing of 13 miR-mediated PTEN regulators, whose locus deletions are predictive of PTEN expression variability, was sufficient to downregulate PTEN in a 3′UTR-dependent manner and to increase tumor cell growth rates. Thus, miR-mediated interactions provide a mechanistic, experimentally validated rat! ionale for the loss of PTEN expression in a large number of glioma samples with an intact PTEN locus.
• In Vivo Identification of Tumor- Suppressive PTEN ceRNAs in an Oncogenic BRAF-Induced Mouse Model of Melanoma
  - Cell 147(2):382-395 (2011)
  We recently proposed that competitive endogenous RNAs (ceRNAs) sequester microRNAs to regulate mRNA transcripts containing common microRNA recognition elements (MREs). However, the functional role of ceRNAs in cancer remains unknown. Loss of PTEN, a tumor suppressor regulated by ceRNA activity, frequently occurs in melanoma. Here, we report the discovery of significant enrichment of putative PTEN ceRNAs among genes whose loss accelerates tumorigenesis following Sleeping Beauty insertional mutagenesis in a mouse model of melanoma. We validated several putative PTEN ceRNAs and further characterized one, the ZEB2 transcript. We show that ZEB2 modulates PTEN protein levels in a microRNA-dependent, protein coding-independent manner. Attenuation of ZEB2 expression activates the PI3K/AKT pathway, enhances cell transformation, and commonly occurs in human melanomas and other cancers expressing low PTEN levels. Our study genetically identifies multiple putative microRNA decoys ! for PTEN, validates ZEB2 mRNA as a bona fide PTEN ceRNA, and demonstrates that abrogated ZEB2 expression cooperates with BRAFV600E to promote melanomagenesis.
• A Primary Role for Release Factor 3 in Quality Control during Translation Elongation in Escherichia coli
  - Cell 147(2):396-408 (2011)
  Release factor 3 (RF3) is a GTPase found in a broad range of bacteria where it is thought to play a critical "recycling" role in translation by facilitating the removal of class 1 release factors (RF1 and RF2) from the ribosome following peptide release. More recently, RF3 was shown in vitro to stimulate a retrospective editing reaction on the bacterial ribosome wherein peptides carrying mistakes are prematurely terminated during protein synthesis. Here, we examine the role of RF3 in the bacterial cell and show that the deletion of this gene sensitizes cells to other perturbations that reduce the overall fidelity of protein synthesis. We further document substantial effects on mRNA stability and protein expression using reporter systems, native mRNAs and proteins. We conclude that RF3 plays a primary role in vivo in specifying the fidelity of protein synthesis thus impacting overall protein quantity and quality.
• Structural Insights into RNA Recognition by RIG-I
  - Cell 147(2):409-422 (2011)
  Intracellular RIG-I-like receptors (RLRs, including RIG-I, MDA-5, and LGP2) recognize viral RNAs as pathogen-associated molecular patterns (PAMPs) and initiate an antiviral immune response. To understand the molecular basis of this process, we determined the crystal structure of RIG-I in complex with double-stranded RNA (dsRNA). The dsRNA is sheathed within a network of protein domains that include a conserved "helicase" domain (regions HEL1 and HEL2), a specialized insertion domain (HEL2i), and a C-terminal regulatory domain (CTD). A V-shaped pincer connects HEL2 and the CTD by gripping an α-helical shaft that extends from HEL1. In this way, the pincer coordinates functions of all the domains and couples RNA binding with ATP hydrolysis. RIG-I falls within the Dicer-RIG-I clade of the superfamily 2 helicases, and this structure reveals complex interplay between motor domains, accessory mechanical domains, and RNA that has implications for understanding the nanomec! hanical function of this protein family and other ATPases more broadly.
• Structural Basis for the Activation of Innate Immune Pattern-Recognition Receptor RIG-I by Viral RNA
  - Cell 147(2):423-435 (2011)
  RIG-I is a key innate immune pattern-recognition receptor that triggers interferon expression upon detection of intracellular 5′triphosphate double-stranded RNA (5′ppp-dsRNA) of viral origin. RIG-I comprises N-terminal caspase activation and recruitment domains (CARDs), a DECH helicase, and a C-terminal domain (CTD). We present crystal structures of the ligand-free, autorepressed, and RNA-bound, activated states of RIG-I. Inactive RIG-I has an open conformation with the CARDs sequestered by a helical domain inserted between the two helicase moieties. ATP and dsRNA binding induce a major rearrangement to a closed conformation in which the helicase and CTD bind the blunt end 5′ppp-dsRNA with perfect complementarity but incompatibly with continued CARD binding. We propose that after initial binding of 5′ppp-dsRNA to the flexibly linked CTD, co-operative tight binding of ATP and RNA to the helicase domain liberates the CARDs for downstream signaling. These findings! significantly advance our molecular understanding of the activation of innate immune signaling helicases.
• Activation of STAT6 by STING Is Critical for Antiviral Innate Immunity
  - Cell 147(2):436-446 (2011)
  STAT6 plays a prominent role in adaptive immunity by transducing signals from extracellular cytokines. We now show that STAT6 is required for innate immune signaling in response to virus infection. Viruses or cytoplasmic nucleic acids trigger STING (also named MITA/ERIS) to recruit STAT6 to the endoplasmic reticulum, leading to STAT6 phosphorylation on Ser407 by TBK1 and Tyr641, independent of JAKs. Phosphorylated STAT6 then dimerizes and translocates to the nucleus to induce specific target genes responsible for immune cell homing. Virus-induced STAT6 activation is detected in all cell-types tested, in contrast to the cell-type specific role of STAT6 in cytokine signaling, and Stat6–/– mice are susceptible to virus infection. Thus, STAT6 mediates immune signaling in response to both cytokines at the plasma membrane, and virus infection at the endoplasmic reticulum.
• Unidirectional Cross-Activation of GRPR by MOR1D Uncouples Itch and Analgesia Induced by Opioids
  - Cell 147(2):447-458 (2011)
  Spinal opioid-induced itch, a prevalent side effect of pain management, has been proposed to result from pain inhibition. We now report that the μ-opioid receptor (MOR) isoform MOR1D is essential for morphine-induced scratching (MIS), whereas the isoform MOR1 is required only for morphine-induced analgesia (MIA). MOR1D heterodimerizes with gastrin-releasing peptide receptor (GRPR) in the spinal cord, relaying itch information. We show that morphine triggers internalization of both GRPR and MOR1D, whereas GRP specifically triggers GRPR internalization and morphine-independent scratching. Providing potential insight into opioid-induced itch prevention, we demonstrate that molecular and pharmacologic inhibition of PLCβ3 and IP3R3, downstream effectors of GRPR, specifically block MIS but not MIA. In addition, blocking MOR1D-GRPR association attenuates MIS but not MIA. Together, these data suggest that opioid-induced itch is an active process concomitant with but independ! ent of opioid analgesia, occurring via the unidirectional cross-activation of GRPR signaling by MOR1D heterodimerization.
• Global Identification of Modular Cullin-RING Ligase Substrates
  - Cell 147(2):459-474 (2011)
  Cullin-RING ligases (CRLs) represent the largest E3 ubiquitin ligase family in eukaryotes, and the identification of their substrates is critical to understanding regulation of the proteome. Using genetic and pharmacologic Cullin inactivation coupled with genetic (GPS) and proteomic (QUAINT) assays, we have identified hundreds of proteins whose stabilities or ubiquitylation status are regulated by CRLs. Together, these approaches yielded many known CRL substrates as well as a multitude of previously unknown putative substrates. We demonstrate that one substrate, NUSAP1, is an SCFCyclin F substrate during S and G2 phases of the cell cycle and is also degraded in response to DNA damage. This collection of regulated substrates is highly enriched for nodes in protein interaction networks, representing critical connections between regulatory pathways. This demonstrates the broad role of CRL ubiquitylation in all aspects of cellular biology and provides a set of proteins lik! ely to be key indicators of cellular physiology.
• SnapShot: Caspases
  - Cell 147(2):476-476.e1 (2011)