RnaseIII and T4 Polynucleotide Kinase Sequence Biases and Solutions During RNA-Seq Library Construction

Background: RNA-seq is a next generation sequencing method with a wide range of applications including single nucleotide polymorphism (SNP) detection, splice junction identification, and gene expression level measurement. However, the RNA-seq sequence data can be biased during library constructions resulting in incorrect data for SNP, splice junction, and gene expression studies. Here, we developed new library preparation methods to limit such biases. Results: A whole transcriptome library prepared for the SOLiD system displayed numerous read duplications (pile-ups) and gaps in known exons. The pile-ups and gaps of the whole transcriptome library caused a loss of SNP and splice junction information and reduced the quality of gene expression results. Further, we found clear sequence biases for both 5' and 3' end reads in the whole transcriptome library. To remove this bias, RNaseIII fragmentation was replaced with heat fragmentation. For adaptor ligation, T4 Polynucleotide Kinase (T4PNK) was used following heat fragmentation. However, its kinase and phosphatase activities introduced additional sequence biases. To minimize them, we used OptiKinase before T4PNK. Our study further revealed the specific target sequences of RNaseIII and T4PNK. Conclusions: Our results suggest that the heat fragmentation removed the RNaseIII sequence bias and significantly reduced the pile-ups and gaps. OptiKinase minimized the T4PNK sequence biases and removed most of the remaining pile-ups and gaps, thus maximizing the quality of RNA-seq data.National Institute on Alcohol Abuse and Alcoholism (NIAAA) AA12404, AA019382, AA020926, AA016648National Institutes of Health (NIH) R01 GM088344Waggoner Center for Alcohol and Addiction Researc

Inter- and Intra-Subunit Butanol/Isoflurane Sites of Action in the Human Glycine Receptor

Glycine receptors (GlyRs) mediate inhibitory neurotransmission and are targets for alcohols and anesthetics in brain. GlyR transmembrane (TM) domains contain critical residues for alcohol/anesthetic action: amino acid A288 in TM3 forms crosslinks with TM1 (I229) in the adjacent subunit as well as TM2 (S267) and TM4 (Y406, W407, I409, Y410) in the same subunit. We hypothesized that these residues may participate in intra-subunit and inter-subunit sites of alcohol/anesthetic action. The following double and triple mutants of GLRA1 cDNA (encoding human glycine receptor alpha 1 subunit) were injected into Xenopus laevis oocytes: I229C/A288C, I229C/A288C/C290S, A288C/Y406C, A288C/W407C, A288C/I409C, and A288C/Y410C along with the corresponding single mutants and wild-type GLRA1. Butanol (22 mM) or isoflurane (0.6 mM) potentiation of GlyR-mediated currents before and after application of the cysteine crosslinking agent HgCl(2) (10 μM) was measured using two-electrode voltage clamp electrophysiology. Crosslinking nearly abolished butanol and isoflurane potentiation in the I229C/A288C and I229C/A288C/C290S mutants but had no effect in single mutants or wild-type. Crosslinking also inhibited butanol and isoflurane potentiation in the TM3-4 mutants (A288C/Y406C, A288C/W407C, A288C/I409C, A288C/Y410C) with no effect in single mutants or wild-type. We extracted proteins from oocytes expressing I229C/288C, A288C/Y410C, or wild-type GlyRs, used mass spectrometry to verify their expression and possible inter-subunit dimerization, plus immunoblotting to investigate the biochemical features of proposed crosslinks. Wild-type GlyR subunits measured about 50 kDa; after crosslinking, the dimeric/monomeric 100:50 kDa band ratio was significantly increased in I229C/288C but not A288C/Y410C mutants or wild-type, providing support for TM1-3 inter-subunit and TM3-4 intra-subunit crosslinking. A GlyR homology model based on the GluCl template provides further evidence for a multi-site model for alcohol/anesthetic interaction with human GLRA1

Intron 4 Containing Novel GABAB1 Isoforms Impair GABAB Receptor Function

Background -- Gamma-aminobutyric acid type B (GABAB) receptors decrease neural activity through G protein signaling. There are two subunits, GABAB1 and GABAB2. Alternative splicing provides GABAB1 with structural and functional diversity. cDNA microarrays showed strong signals from human brain RNA using GABAB1 intron 4 region probes. Therefore, we predicted the existence of novel splice variants. Methodology/Principal Findings -- Based on the probe sequence analysis, we proposed two possible splice variants, GABAB1j and GABAB1k. The existence of human GABAB1j was verified by quantitative real-time PCR, and mouse GABAB1j was found from a microarray probe set based on human GABAB1j sequence. GABAB1j open reading frames (ORF) and expression patterns are not conserved across species, and they do not have any important functional domains except sushi domains. Thus, we focused on another possible splice variant, GABAB1k. After obtaining PCR evidence for GABAB1k existence from human, mouse, and rat, it was cloned from human and mouse by PCR along with three additional isoforms, GABAB1l, GABAB1m, and GABAB1n. Their expression levels by quantitative real-time PCR are relatively low in brain although they may be expressed in specific cell types. GABAB1l and GABAB1m inhibit GABAB receptor-induced G protein-activated inwardly rectifying K+ channel (GIRK) currents at Xenopus oocyte two-electrode voltage clamp system. Conclusions/Significance -- This study supports previous suggestions that intron 4 of GABAB1 gene is a frequent splicing spot across species. Like GABAB1e, GABAB1l and GABAB1m do not have transmembrane domains but have a dimerization motif. So, they also could be secreted and bind GABAB2 dominantly instead of GABAB1a. However, only GABAB1l and GABAB1m are N- and C-terminal truncated splicing variants and impair receptor function. This suggests that the intron 4 containing N-terminal truncation is necessary for the inhibitory action of the new splice variants.This study was supported by National Institute on Alcohol Abuse and Alcoholism (NIAAA) grant AA06399 and by the NIAAA Integrative Neuroscience Initiative on Alcoholism (INIA)-West consortium. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Cellular and Molecular Biolog

Structural Basis for Potentiation by Alcohols and Anaesthetics in a Ligand-gated Ion Channel

Ethanol alters nerve signalling by interacting with proteins in the central nervous system, particularly pentameric ligand-gated ion channels. A recent series of mutagenesis experiments on Gloeobacter violaceus ligand-gated ion channel, a prokaryotic member of this family, identified a single-site variant that is potentiated by pharmacologically relevant concentrations of ethanol. Here we determine crystal structures of the ethanol-sensitized variant in the absence and presence of ethanol and related modulators, which bind in a transmembrane cavity between channel subunits and may stabilize the open form of the channel. Structural and mutagenesis studies defined overlapping mechanisms of potentiation by alcohols and anaesthetics via the inter-subunit cavity. Furthermore, homology modelling show this cavity to be conserved in human ethanol-sensitive glycine and GABA(A) receptors, and to involve residues previously shown to influence alcohol and anaesthetic action on these proteins. These results suggest a common structural basis for ethanol potentiation of an important class of targets for neurological actions of ethanol

CNS cell-type localization and LPS response of TLR signaling pathways [version 1; referees: 2 approved]

Background: Innate immune signaling in the brain has emerged as a contributor to many central nervous system (CNS) pathologies, including mood disorders, neurodegenerative disorders, neurodevelopmental disorders, and addiction. Toll-like receptors (TLRs), a key component of the innate immune response, are particularly implicated in neuroimmune dysfunction. However, most of our understanding about TLR signaling comes from the peripheral immune response, and it is becoming clear that the CNS immune response is unique. One controversial aspect of neuroimmune signaling is which CNS cell types are involved. While microglia are the CNS cell-type derived from a myeloid lineage, studies suggest that other glial cell types and even neurons express TLRs, although this idea is controversial. Furthermore, recent work suggests a discrepancy between RNA and protein expression within the CNS. Methods: To elucidate the CNS cell-type localization of TLRs and their downstream signaling molecules, we isolated microglia and astrocytes from the brain of adult mice treated with saline or the TLR4 ligand lipopolysaccharide (LPS). Glial mRNA and protein expression was compared to a cellular-admixture to determine cell-type enrichment. Results: Enrichment analysis revealed that most of the TLR pathway genes are localized in microglia and changed in microglia following immune challenge. However, expression of Tlr3 was enriched in astrocytes, where it increased in response to LPS. Furthermore, attempts to determine protein cell-type localization revealed that many antibodies are non-specific and that antibody differences are contributing to conflicting localization results. Conclusions: Together these results highlight the cell types that should be looked at when studying TLR signaling gene expression and suggest that non-antibody approaches need to be used to accurately evaluate protein expression

Interacting amino acid replacements allow poison frogs to evolve epibatidine resistance

Animals that wield toxins face self-intoxication. Poison frogs have a diverse arsenal of defensive alkaloids that target the nervous system. Among them is epibatidine, a nicotinic acetylcholine receptor (nAChR) agonist that is lethal at microgram doses. Epibatidine shares a highly conserved binding site with acetylcholine, making it difficult to evolve resistance yet maintain nAChR function. Electrophysiological assays of human and frog nAChR revealed that one amino acid replacement, which evolved three times in poison frogs, decreased epibatidine sensitivity but at a cost of acetylcholine sensitivity. However, receptor functionality was rescued by additional amino acid replacements that differed among poison frog lineages. Our results demonstrate how resistance to agonist toxins can evolve and that such genetic changes propel organisms toward an adaptive peak of chemical defense

Peroxisome Proliferator Activated Receptor Agonists Modulate Transposable Element Expression in Brain and Liver

Peroxisome proliferator activated receptors (PPARs) are nuclear hormone receptors that act as transcription factors in response to endogenous lipid messengers. The fibrates and thiazolidinediones are synthetic PPAR agonists used clinically to treat dyslipidemia and Type 2 Diabetes Mellitus, respectively, but also improve symptoms of several other diseases. Transposable elements (TEs), repetitive sequences in mammalian genomes, are implicated in many of the same conditions for which PPAR agonists are therapeutic, including neurodegeneration, schizophrenia, and drug addiction. We tested the hypothesis that there is a link between actions of PPAR agonists and TE expression. We developed an innovative application of microarray data by mapping Illumina mouse WG-6 microarray probes to areas of the mouse genome that contain TEs. Using this information, we assessed the effects of systemic administration of three PPAR agonists with different PPAR subtype selectivity: fenofibrate, tesaglitazar, and bezafibrate, on TE probe expression in mouse brain [prefrontal cortex (PFC) and amygdala] and liver. We found that fenofibrate, and bezafibrate to a lesser extent, up-regulated probes mapped to retrotransposons: Short-Interspersed Elements (SINEs) and Long-Interspersed Elements (LINEs), in the PFC. Conversely, all PPAR agonists down-regulated LINEs and tesaglitazar and bezafibrate also down-regulated SINEs in liver. We built gene coexpression networks that partitioned the diverse transcriptional response to PPAR agonists into groups of probes with highly correlated expression patterns (modules). Most of the differentially expressed retrotransposons were within the same module, suggesting coordinated regulation of their expression, possibly by PPAR signaling. One TE module was conserved across tissues and was enriched with genes whose products participate in epigenetic regulation, suggesting that PPAR agonists affect TE expression via epigenetic mechanisms. Other enriched functional categories included phenotypes related to embryonic development and learning and memory, suggesting functional links between these biological processes and TE expression. In summary, these findings suggest mechanistic relationships between retrotransposons and PPAR agonists and provide a basis for future exploration of their functional roles in brain and liver

How Should Addiction-Related Research at the National Institutes of Health be Reorganized?

The decades-old debate about the optimum organizational structure of the National Institute on Alcohol Abuse and Alcoholism (NIAAA) and National Institute on Drug Abuse (NIDA) has reached a crescendo with the recent deliberations of the Scientific Management Review Board, which, despite the lack of a crisis, proposed a structural reorganization that would dissolve the two institutes and create a new institute for substance use, abuse, and addiction, in hope of new scientific and public health advances (Collins, 2010). For a new institute to succeed, a multitude of potential challenges need to be negotiated effectivel

GABAA Receptors Containing ρ1 Subunits Contribute to In Vivo Effects of Ethanol in Mice

Yuri A. Blednov, Jillian M. Benavidez, Mendy Black, Courtney R. Leiter, Elizabeth Osterndorff-Kahanek, David Johnson, Cecilia M. Borghese, R. Adron Harris, Waggoner Center for Alcohol and Addiction Research, The University of Texas at Austin, Austin, Texas, United States of AmericaJane R. Hanrahan, Mary Chebib, Faculty of Pharmacy, The University of Sydney, Sydney NSW, AustraliaGraham A. R. Johnston, Department of Pharmacology, The University of Sydney, Sydney NSW, AustraliaGABAA receptors consisting of ρ1, ρ2, or ρ3 subunits in homo- or hetero-pentamers have been studied mainly in retina but are detected in many brain regions. Receptors formed from ρ1 are inhibited by low ethanol concentrations, and family-based association analyses have linked ρ subunit genes with alcohol dependence. We determined if genetic deletion of ρ1 in mice altered in vivo ethanol effects. Null mutant male mice showed reduced ethanol consumption and preference in a two-bottle choice test with no differences in preference for saccharin or quinine. Null mutant mice of both sexes demonstrated longer duration of ethanol-induced loss of righting reflex (LORR), and males were more sensitive to ethanol-induced motor sedation. In contrast, ρ1 null mice showed faster recovery from acute motor incoordination produced by ethanol. Null mutant females were less sensitive to ethanol-induced development of conditioned taste aversion. Measurement of mRNA levels in cerebellum showed that deletion of ρ1 did not change expression of ρ2, α2, or α6 GABAA receptor subunits. (S)-4-amino-cyclopent-1-enyl butylphosphinic acid (“ρ1” antagonist), when administered to wild type mice, mimicked the changes that ethanol induced in ρ1 null mice (LORR and rotarod tests), but the ρ1 antagonist did not produce these effects in ρ1 null mice. In contrast, (R)-4-amino-cyclopent-1-enyl butylphosphinic acid (“ρ2” antagonist) did not change ethanol actions in wild type but produced effects in mice lacking ρ1 that were opposite of the effects of deleting (or inhibiting) ρ1. These results suggest that ρ1 has a predominant role in two in vivo effects of ethanol, and a role for ρ2 may be revealed when ρ1 is deleted. We also found that ethanol produces similar inhibition of function of recombinant ρ1 and ρ2 receptors. These data indicate that ethanol action on GABAA receptors containing ρ1/ρ2 subunits may be important for specific effects of ethanol in vivo.This work was supported by NIH grants AA013520 and AA06399. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Waggoner Center for Alcohol and Addiction ResearchEmail: [email protected]